Development of Luminescence Based Biosensors for Dengue Diagnostics

Thesis submitted in partial fulfillment of the requirements for the degree of

“DOCTOR OF PHILOSOPHY”

by

Danit Atias

Submitted to the Senate of Ben-Gurion University

of the Negev

January 2010

Beer-Sheva

II

Development of Luminescence Based Biosensors for Dengue Diagnostics

Thesis submitted in partial fulfillment

of the requirements for the degree of

“DOCTOR OF PHILOSOPHY”

by

Danit Atias

Submitted to the Senate of Ben-Gurion University

of the Negev

Approved by the advisor: Prof. Robert S. Marks ____________________________________________

Dr. Leslie Lobel ____________________________________________

Approved by the Dean of the Kreitman

School of Advanced Graduate Studies _____________________________________

January 2010

Beer-Sheva

III

This work was carried out under the supervision of

Prof. Robert S. Marks

Dr. Leslie Lobel

In the Virology Department

Faculty of Health Sciences

and the Department of Biotechnology Engineering

Faculty of Engineering Sciences

IV

AAcckknnoowwlleeddggmmeennttss

First and foremost, I would like to thank my supervisors Prof. Robert S. Marks and Dr.

Leslie Lobel for their guidance, understanding, patience and support, in sharing their knowledge and

ideas, for the many opportunities to collaborate with scientists from different institutes, and the scientific

freedom to expand my research into new areas, especially for making the last four years a lovely and

memorable experience. I appreciate the advice and support of Profs. Angel Porgador and Maureen

Friedman in their roles on my advisory committee.

My gratitude is extended also to members of both Robert and Leslie groups for their friendship,

support, and advice - in particular, Dr. Khalil Abu-Rabeah, Dr. Michal Shani-Sekler, Dr. Sebastian

Herrmann, Liron Amir, Avraham Ashkenazi, Ariel Sobarzo, Ariel Hanemann, Pavel Naumenko, Daria

Prilutsky, Evgeni Eltzov, Julia Frenkel, Itai Nuri, Amit Elbaz and Karin Golberg. I was very lucky to

work with such wonderful and helpful friends. You were always willing to assist, support and provide

good advice. Thank you for making the lab a place to which it was a pleasure to arrive every day. A

Special thanks to Gina Hughes for her expert editorial help, as and when needed.

I am also grateful to my friends outside the lab: Smadar Kleiman, Regina Pavlovich, Ohad

Bukelman, Tamar Levav, Shirly Amar, Shira Tamir, Ayelet Zukerman, Micha Avitan and Liron Varon,

for knowing how to say the right word at the right moment, for support and encouragement, each in his/

her own special way that I love.

I would like to thank you my partner in life, Ido Yosef, for supporting me in difficult times and

for being next to me in happy times. Your love, encouragement, and understanding enabled the

submission of this thesis.

Finally, I would like to thank you my family - my parents Naomi and Avi, my sisters Ketty and

Michal, their husbands Itzik and Roby, and my nephews Shlomo-Haim and Isaac-Itamar. You are my

solid ground. From your endless love and trust, my goals and dreams were encouraged and carried out.

Thank you for the patience, support and listening to small matters, as also to big ones. To my nephews,

for the joy you have brought into my life.

V

I dedicate this work to the memory of my late grandparents Rabbi Isaac and Yakutt Benizri,

Pinhas and Donna Attias for their endless love, encouragement and for instilling in me the

values of higher education.

VI

Author’s contribution to publications

To Whom It May Concern

All research , scientific results, ideas, new techniques and technologies were developed in our

laboratory in addition the publications derived from this thesis are original work from our

laboratory, with Danit Atias as the principle scientist behind the research. Our main collaborators

in these efforts were Dr. Phillipe Dussart, Dr. Vered Chalifa-Caspi, Prof. Serge Cosnier, Dr.

Marko Virta and Prof. H. Bedouelle. Therefore, Danit has our permission to enter all data and

results from these manuscripts in her doctoral thesis as they have been satellite and

complementary research projects exclusively based on her own research.

Prof. Robert S. Marks and Dr. Leslie Lobel

1

Table of contents

TTAABBLLEE OOFF CCOO33TTEE33TTSS........................................................................................................................................................................................................................................................................................ 11

LLIISSTT OOFF FFIIGGUURREESS ........................................................................................................................................................................................................................................................................................................ 33

LLIISSTT OOFF TTAABBLLEESS ............................................................................................................................................................................................................................................................................................................ 55

LLIISSTT OOFF AABBBBRREEVVIIAATTIIOO33SS.......................................................................................................................................................................................................................................................................... 66

AABBSSTTRRAACCTT.................................................................................................................................................................................................................................................................................................................................. 88

1. I3TRODUCTIO3 ........................................................................................................................................... 10

1.1. DENGUE VIRUSES ....................................................................................................................................... 10

1.1.1. EMERGING FLAVIVIRUS DISEASES.............................................................................................................. 10

1.1.2. EPIDEMIOLOGY AND PUBLIC HEALTH CONCERNS ....................................................................................... 12

1.1.3. DENGUE VIRUS STRAINS AND 'IMMUNE PROTECTION' ................................................................................. 14

1.1.4. VIRUS STRUCTURE ..................................................................................................................................... 17

1.2. DIAGNOSTIC TOOLS FOR DENGUE............................................................................................................... 19

1.2.1. LABORATORY DIAGNOSIS .......................................................................................................................... 19

1.2.1.1. SEROLOGICAL DIAGNOSES ......................................................................................................................... 22

1.3. THE BIOSENSOR TECHNOLOGY ................................................................................................................... 27

1.3.1. CHEMILUMINESCENCE BASED BIOSENSORS ................................................................................................ 28

1.3.1.1. THE BIOCHEMISTRY OF CHEMILUMINESCENCE ........................................................................................... 29

1.3.1.2. ENZYMES USED IN CHEMILUMINESCENT REACTIONS.................................................................................. 30

1.3.1.3. CHEMILUMINESCENCE BASED OPTICAL FIBER IMMUNOSENSORS................................................................ 30

1.3.1.4. DETECTION OF CHEMILUMINESCENCE: PHOTON-COUNTING SYSTEM.......................................................... 33

1.3.2. ELECTROCHEMICAL BIOSENSOR................................................................................................................. 34

1.3.2.1. INTRODUCTION TO ELECTROCHEMISTRY .................................................................................................... 34

1.3.2.1.1. CYCLIC VOLTAMMETRY ......................................................................................................................... 34

1.3.2.1.2. ELECTROCHEMICAL IMPEDANCE SPECTROSCOPY ................................................................................... 35

1.3.2.1.3. ELECTROPOLYMERIZATION ..................................................................................................................... 35

1.3.2.2. ELECTROCHEMICAL BIOSENSOR................................................................................................................. 35

1.3.2.3. AMPEROMETRIC BIOSENSORS .................................................................................................................... 36

1.4. ALTERNATIVE FIBER FOR OPTIC BIOSENSORS ............................................................................................. 36

1.5. ALTERNATIVE ANTIGEN ............................................................................................................................. 37

1.5.1. RECOMBINANT PROTEIN AND EPITOPES..................................................................................................... 37

2. AIMS A3D EXPECTED SIG3IFICA3CE OF THE RESEARCH ........................................................... 38

3. THE LOGICAL STRUCTURE A3D ORDER OF PUBLISHED ARTICLES WITHI3 THE THESIS39

2

4. LIST OF ORIGI3AL PUBLICATIO3S....................................................................................................... 41

4.1. ARTICLE NO. 1:.......................................................................................................................................... 41

4.2. ARTICLE NO. 2:.......................................................................................................................................... 52

4.3. ARTICLE NO. 3:.......................................................................................................................................... 57

5. SUMMARY OF MATERIALS A3D METHODS 3OT FOU3D I3 PUBLISHED ARTICLES. ........... 63

5.1. DETECTION OF ANTI-DENV IGM ANTIBODIES USING ED3 MAC ELISA .................................................. 63

5.1.1. CHEMICALS AND BIOCHEMICALS ............................................................................................................... 63

5.1.2. ED3-PHOA AND ED3 MAC-ELISA .......................................................................................................... 63

5.2. HUMAN SERA ............................................................................................................................................. 64

5.2.1. INTERNAL PANEL OF REFERENCE SERA....................................................................................................... 64

5.2.2. HUMAN SERA SAMPLES .............................................................................................................................. 65

5.2.3. DETECTION OF ANTI-DENV IGM ANTIBODIES USING THE ROUTINE MAC-ELISA .................................... 65

5.2.4. DETECTION OF ANTI-DENV IGM ANTIBODIES USING ED3 MAC-ELISA.................................................. 66

5.2.4.1. COLORIMETRIC DETECTION........................................................................................................................ 66

5.2.4.2. LUMINESCENT DETECTION ......................................................................................................................... 67

5.2.4.3. STATISTICAL ANALYSIS ............................................................................................................................. 67

6. SUMMARY OF RESULTS 3OT FOU3D I3 PUBLISHED ARTICLES................................................. 68

6.1. DETECTION OF ANTI-DENV IGM ANTIBODIES USING ED3 MAC-ELISA.................................................. 68

6.1.1. SCREENING OF PANEL REFERENCE SERA .................................................................................................... 68

6.1.2. SCREENING OF HUMAN SERA...................................................................................................................... 71

7. OVERALL DISCUSSIO3.............................................................................................................................. 74

7.1. DEVELOPMENT OF CHEMILUMINESCENT OPTICAL FIBER IMMUNOSENSOR FOR THE DETECTION OF IGM

ANTIBODY TO DENGUE VIRUS IN HUMANS ................................................................................................. 74

7.2. DEVELOPMENT AND CHARACTERIZATION OF POLY (METHYL METACRYLATE) CONDUCTIVE FIBER OPTIC

TRANSDUCERS AS DUAL BIOSENSOR PLATFORMS ....................................................................................... 75

7.3. ALTERNATIVE ANTIGEN ............................................................................................................................. 76

8. CO3CLUDI3G REMARKS A3D FUTURE PERSPECTIVES ................................................................ 77

9. REFERE3CES ................................................................................................................................................ 79

3

List of figures

Figure 1. Flavivirus classification

11

Figure 2. Transmission of dengue viruses

12

Figure 3. The global resurgence of dengue and dengue hemorrhagic fever

13

Figure 4. Dengue: its current distribution, and countries with Aedes aegypti and at risk introduction

14

Figure 5. The range of dengue disease

15

Figure 6. Model for antibody-dependent enhancement of dengue virus replication

17

Figure 7. Dengue virus structure

19

Figure 8. Biosensor elements

28

Figure 9. Chemiluminescent reaction

29

Figure 10. Descriptive scheme of fiber optic based biosensor with the setups for dengue virus detection

32

Figure 11. Comparison between MAC-ELISA to ED3 MAC-ELISA

64

Figure 12. Colorimetric ED3 MAC-ELISA

69

4

Figure 13. Screening of panel reference sera

70

Figure 14. Signal to noise ratio (S/N) of panel reference sera

70

Figure 15. Comparison between colorimetric DENV1-4 ED3 MAC-ELISA to

Luminescence DENV1-4 ED3 MAC-ELISA

73

5

List of tables

Table 1 Advantages and disadvantages of the main laboratories use of serological and

virological dengue diagnostic assays

24

Table 2 Commercial kits for anti DENV detection 26

Table 3 Screening of human sera using ED3 MAC-ELISA, DENV1-4 ED3 72

6

List of abbreviations

A Ampere

Ab Antibody

ADE Antibody dependent enhancement

ANOVA Analysis of variance AP Alkaline phosphatase APD Avalanche photodiodes

ATP Adenosine triphosphate

Bz Benzophenone

CV Cyclic voltammetry

DENV Dengue virus

DF Dengue fever

DHF Dengue hemorrhagic fever

DNA Deoxyribonucleic acid

DSS Dengue shock syndrome

E Envelope protein

ED3 Ectodomain 3

EIS Electrochemical impedance spectroscopy

ELISA Enzyme-Linked ImmunoSorbent Assay

EMCS 6-Maleimidohexanoic acid n-hydroxysuccinimide ester

ELISA Enzyme-Linked ImmunoSorbent Assay

FcγR Fcγ receptors

G Gram

GAC ELISA IgG capture, enzyme-linked immunosorbent assay gpE Envelope glycoprotein

HI Hemagglutination inhibition

H2O2 Hydrogen peroxide

HRP Horseradish peroxidase I Current

IgG Immunoglobulin G

IgM Immunoglobulin M

JEV Japanese encephalitis virus

kΩ Kilo-ohm

L Liter

M Molar

mAb Monoclonal antibody

MAC-ELISA IgM capture, enzyme-linked immunosorbent assay

µA Microampere

µg Microgram

7

µl Microliter

mA Miliampere

mg Miligram

ml Mililiter

mM Milimolar

MPTS (3-mercaptopropyl)trimethoxysilane

NADH Reduced nicotinamide adenine dinucleotide

NASBA Nucleic acid sequence-based amplification

NRC National Reference Center

NS Nonstructural

OD Optical density

OFIS Optical fiber immunosensor Ω Ohm

Pm Permeability

PBS Phosphate buffered saline

PBST Phosphate buffered saline tween

PMT Photomultiplier tube

PCR Polymerase chain reaction

PMMA Polymethyl metacrylate

Py Pyrrole py-NH2 Pyrrole amine

PPy Polypyrrole

PpyBz Polypyrrole-benzophenone

PRNT Plaque reduction neutralization test

UV Ultraviolet

Urs Urease

RLU Relative light unit

RT-PCR Reverse transcriptase PCR

RNA Ribonucleic acid

SLEV Saint Louis encephalitis virus

SEM Scanning electron microscopy

S/N Signal to noise ratio

SPAD Single photon avalanche diode

SM Skim milk powder

V Volt

v/v Volume/volume

w/v Weight/volume

w/w Weight to weight

WNV West Nile virus

WHO World Health Organization

YFV Yellow fever virus

8

Abstract

Infection with dengue virus (DENV) has emerged as the most important vector-borne viral

disease in tropical areas and it continues to expand geographically. The four serotypes of DENV

that cause human disease are transmitted by Aedes mosquitoes. A reliable diagnosis remains a

crucial step towards the control of DENV disease in human populations. The main goal of this

research was to develop a luminescence based biosensor for the detection of anti DENV

antibodies in human sera samples. The immunoassay was based on a colorimetric IgM capture,

enzyme-linked immunosorbent assay (MAC-ELISA), routinely used by the National Reference

Center for Arboviruses based in French Guiana. The detection of human anti-DENV IgM using

colorimetric MAC-ELISA, chemiluminescent MAC-ELISA and chemiluminescent silica based

optical fiber immunosensor (OFIS) was compared. An internal panel of reference sera was used

and 86 sera samples were screened. Compared to standard colorimetric MAC-ELISA, the

chemiluminescent OFIS had a lower detection limit, 10 times lower than the chemiluminescent

MAC-ELISA and 100 times lower than the colorimetric MAC-ELISA. Therefore the

colorimetric and chemiluminescent MAC-ELISA’s are more suitable for high and intermediate

levels of anti-DENV IgM present in sera samples, whereas the chemiluminescent OFIS is also

useful at low analyte concentration, with sensitivity and specificity of 98.1% and 87.0%,

respectively. Taking into account the lower limit of detection and the high correlation with the

established methods using the known panel, the OFIS technology reported here is reliable,

simple to perform, fast, cost effective, and a field operable analytical tool.

Furthermore an alternative new optic-conductive poly methyl methacrylate (PMMA) fiber

configuration was employed for the construction of biosensing platforms and thoroughly

characterized. Various parameters like time deposition, process temperature, activator plus

pyrrole monomer concentrations were examined in the study. The morphology and permeability

of the PMMA optic-fibers were investigated to examine mass transfer ability. Cyclic

voltammetry and amperometry techniques were applied to characterize the electrical features of

the surface and charge transfer. Then the platform potential was demonstrated by the

construction of both amperometric and optical biosensors - characterization and optimization of

the PMMA platform demonstrating conductive, stable, thin, controllable and light-transmissible

film features. Thus this approach can be useful for improving the efficiency and reproducibility

9

for mass production of optic fiber probes. Finally, optic-conductive biosensors will open the

door to a broad number of applications based on a platform that will include both conductivity

and light transmission.

One of the limitations of current diagnostic tools for DENV is the lack of specificity and

safety of the antigen employed as part of the available diagnostic tools. Thus there is a constant

serological effort to develop alternative antigens that will help to improve the detection of anti-

dengue antibodies and allow working with non hazardous materials. Thus in the last part of this

work an alternative antigen based on hybrid protein between ED3 of the gpE of each DENV

serotype and a mutant alkaline phosphatase (PhoA) from E.coli was adapted to

chemiluminescent based ELISA. The chemiluminescent based ELISA was compared to the

colorimatric one and to the routinely used colorimetric MAC-ELISA.

10

1. Introduction

1.1. Dengue viruses

1.1.1. Emerging Flavivirus diseases

Dengue fever (DF) is an emerging arborviral disease caused by infection with DENV a

member of the family Flaviviridae, genus Flavivirus [1-3]. Emerging diseases are defined as

diseases that have newly appeared in a population or have existed previously but are rapidly

increasing in incidence or geographic range [4-5]. Mosquito-borne members of the genus

Flavivirus in the family Flaviviridae (Fig. 1) provide some of the most important examples of

emerging diseases, as well as one of the earliest documented diseases that has spread into a new

geographic area such as Yellow fever virus (YFV) from West Africa into the Americas in the

17th and 18th centuries. More recently, the enormous resurgence of DENV in the tropical and

subtropical areas of the world, the emergence of West Nile virus (WNV) in North America, and

the spread of Japanese encephalitis virus (JEV) through much of Asia and into Oceania, have

been recorded [3, 5].

The Flavivirus genus contains over 70 viruses, most of them arthropod-borne (arboviruses)

through mosquitoes or ticks, while some still have no known vector [5-6]. The type species of

the genus is YFV, through which the genus and family derive its name [3, 5]. Although all

flaviviruses are serologically related, they can still be distinct, the most important of which are

DENV and JEV and a less serologically cohesive YFV group [5-6].

11

Fig. 1. Flavivirus classification. The relationships between selected flaviviruses are shown in the

dendrogram on the left. The serological (serocomplex) and phylogenetical (clade and cluster)

classifications of these flaviviruses are shown on the right [3, 6].

Flaviviruses are zoonoses that depend on animal species other than humans for their

maintenance in nature, with the exception of the dengue viruses. Humans are usually incidental

and dead-end hosts that do not contribute to the natural transmission cycles DENV however,

have adapted completely to humans and are maintained in large urban areas in the tropics in

human-mosquito-human transmission cycles that no longer depend on animal reservoirs,

although such reservoirs are still maintained in the jungles of western Africa and southeast Asian

in a sylvatic cycle, monkey-mosquito-monkey (Fig. 2) [5, 7]. Unlike the impact that widespread

sylvatic transmission of YFV has on human disease, the contribution of the observed sylvatic

cycle of dengue transmission to human infection is unknown, but appears to be minimal [8].

DENV is transmitted to humans through the bite of an infected Aedes mosquito. Aedes aegypti,

a highly domesticated mosquito, is by far the predominant vector of DENV, but transmission can

also be sustained by Aedes albopictus [8-9].

12

Fig. 2. Transmission of dengue viruses. Because of the high level of viraemia resulting from DENV

infection of humans, the viruses are efficiently transmitted between mosquitoes and humans without the

need for an enzootic amplification host. DENV is spread principally by the Aedes aegypti mosquito,

which breeds in domestic and peridomestic water containers, increasing the frequency of contact between

mosquitoes and humans. In addition, a sylvatic cycle for dengue transmission has been documented in

western Africa [10] and southeast Asia [8, 11].

1.1.2. Epidemiology and public health concerns

DENV is endemic in most urban centers of the tropics and subtropic areas since a dramatic

increase in urbanization created ideal conditions for increased transmission of mosquito borne

dengue disease [5]. The World Health Organization (WHO) estimates that there is a 30-fold

increase in DENV incidence in the last 50 years (Fig. 3a.) with up to 100 million cases, and over

500,000 cases of dengue hemorrhagic fever (DHF) / dengue shock syndrome (DSS) occurring

each year, including nearly 25,000 fatal cases, primarily in children under the age of 15 [3, 12-

13]. Prior to the 1970s, only five countries located in Southeast Asia had reported DHF.

However, DHF has now been documented in >60 countries, and DENV is endemic in >100

13

countries (Fig. 3b.), including most of Southeast Asia, southern and central America, the

Caribbean and South Pacific regions. DENV is present in Africa, but intense disease outbreaks

are rarely reported, which may partly be due to limited surveillance [2, 8, 14-15]. For the time

being, the global distribution of the mosquito vectors, (genus Aedes) is comparable to that of the

malaria vector, and an estimated 2.5 billion people live in areas at risk for epidemic transmission

(Fig. 4). Many of the factors responsible for this dramatic reappearance of epidemic DF are still

not well understood. However, it is clear that demographic and societal changes such as

population growth, urbanization and modern transportation contribute greatly to the increased

incidence and geographical spread of dengue activity. Thus, the combination of expansion in

geographic distribution of both viruses and mosquito vectors, has led to a current global dengue

pandemic [5, 7-8].

Fig. 3. The global resurgence of DF and DHF over the past half century, by incidence (a.) and by

country (b.)[5].

14

Fig. 4. Dengue: its current distribution, and countries with Aedes aegypti and at risk introduction [5].

1.1.3. Dengue virus strains and 'immune protection'

There are four distinct serotypes of DENV, designated DENV-1, DENV-2, DENV-3 and

DENV-4, each causing human disease [1-3, 12]. Dengue disease has a wide spectrum of

expression: from asymptomatic infection or influenza like syndrome to severe disease including

DHF/DSS. Moreover, in a small percentage of cases, severe dengue occurs with unusual

manifestations such as hepatitis, encephalopathy or rhabdomyolysis. In general, the majority of

DENV infections are either asymptomatic or only mildly symptomatic. Most symptomatic

infections present as classic DF, which includes high fever, myalgia, retro-orbital headache, joint

pain, pain behind the eyes, vomiting, abdominal pain and nausea, loss of appetite and usually a

macular rash. A minority of people infected with DENV develop DHF/ DSS. The acute phase of

DHF/ DSS begins as with DF, then suddenly patients develop a severe vascular permeability

syndrome that may lead to shock and death (Fig. 5.) [5, 8]. DF is usually not fatal (case fatality

rate 1- 5%) when supportive therapy is available. Nevertheless, a significant economic burden is

carried by communities in poor health resource settings. Despite an increase in health and

economic impact, there is currently no available anti-dengue vaccine or specific therapy for the

treatment of DENV infection [8, 16].

15

Fig. 5. The range of dengue disease. The case definitions of DF, DHF and DSS are indicated as

provided by the WHO [14]. For a diagnosis of DHF Grade I, each of the four criteria listed in part b of

the figure must be met. There is a contention from some clinicians that this requirement results in an

under-reporting of severe dengue disease as a patient with only two or three severe conditions would be

classified as having DF [17]. A generalized time course of the events associated with DF, DHF and DSS

is indicated in part d of the figure. The incubation period before the development of signs of infection

generally ranges from 4 to 7 days. Hypovolemic shock can develop during the late stage of disease and

usually lasts 1 to 2 days [8].

16

Symptoms of illness generally appear 4–7 days after a mosquito bite [5]. The viremic phase of

infection occurs shortly before the onset of clinical symptoms, and generally lasts around 5 days

[18-19]. Typically, in a non-immune individual, anti-DENV IgM antibodies develop 5–6 days

after the primary infection (that is the first infection by one of the four DENV serotypes) and

anti-DENV IgG antibodies appear and increase after 7–10 days. During the secondary infection,

defined by an infection occurring from one of the three other DENV serotypes after a primary

infection, high levels of IgG antibodies are detectable 3–4 days after fever onset while IgM

antibodies are lower and in some cases can be absent [12, 20-21].

Dengue infection induces a life-long protective immunity to the homologous serotype but

confers only partial and transient protection against subsequent infections by the other three

serotypes. Both retrospective and prospective studies have demonstrated that secondary infection

by a different dengue serotype or multiple infections with various DV serotypes are the most

significant individual risk factors for DHF/DSS [22-25]. The pathogenesis of DHF/DSS is not

very well understood, nor the immune host conditions that enable the severe disease. The

observed alterations in coagulation and vascular permeability in DHF/DSS are believed to arise

from a combination of increased virus replication; increased death of cells from infection or

cytotoxic immune cells or antibodies; complement activation; and increased release of

inflammatory mediators by infected cells or immune cells. The role of the immune response in

DHF/DSS is performed as the hypothesis of the presence of circulating non-neutralizing, cross-

reactive antibodies in a previously infected individual allows for enhancement of infection,

through increased uptake of virus into target cells via Fc receptors (Fig. 6.) [8, 26] - this

phenomenon being termed antibody dependent enhancement (ADE) [8]. This leads to a

complement activation and enhanced infection of monocytic cells, resulting in the release of

cytokines, lysis of cells, and the release of intracellular enzyme and activators, with a subsequent

plasma leakage and shock [27-28].

17

Fig. 6. Model for antibody-dependent enhancement of DE3V replication. ADE of virus replication

occurs when heterotypic, non neutralizing Ab (antibody) present in the host from a primary DENV

infection binds to an infecting DENV particle during a subsequent heterotypic infection but cannot

neutralize the virus. Instead, the Ab–virus complex attaches to the Fcγ receptors (FcγR) on circulating

monocytes, thereby facilitating the infection of FcγR cell types in the body not readily infected in the

absence of antibody. The overall outcome is an increase in the overall replication of virus, leading to the

potential for more severe disease [8, 29].

1.1.4. Virus structure

DENV is a single-stranded positive-sense RNA virus; the genomic RNA is approximately 11

kb in length. The DENV virion is a spherical, enveloped virus that has a diameter of

approximately 50 nm. The genomic RNA is translated to give a rise to a large polyprotein

precursor, which is cotranslationally processed by host cell and virus-specified proteases to yield

the individual viral proteins. Like other Flaviviruses the DENV is composed of three structural

proteins, designated C (capsid protein), M (membrane) and E (envelope protein), while prM is

the intracellular precursor of the M protein (Fig.7) [20]. The membrane precursor, prM, is

believed to aid in the folding of the E glycoprotein and both are integrated in the lipid bilayer of

the virion by two transmembrane regions that surround a nucleocapsid of unknown structure [3].

18

At a late stage of virion assembly in the trans-Golgi network, prM is cleaved by furin, which

results in a rearrangement of the M and E proteins on the virion surface yielding mature

infectious virions. The surface of the mature DENV virion is smooth with the envelope proteins

aligned in pairs parallel to the virion surface [20]. The E glycoprotein mediates cell attachment

and fusion and is also the major target of protective antibodies. The E glycoprotein can be

divided into three structural or functional domains: the central domain; the dimerization domain

which presents a fusion peptide; and the receptor-binding domain. Virions enter cells by

receptor-mediated endocytosis, which is followed by fusion of the viral and cellular membranes

mediated by the E protein under acidic conditions within the endosome [30]. The nonstructural

(NS) proteins are NS1, NS2a, NS2b, NS3, NS4a, NS4b and NS5. There is ~ 40% amino acid

identity among the flaviviruses E proteins. The numbers and positions of potentially

glycosylated residues are not conserved among different strains of the same virus and it has been

suggested that carbohydrate moieties on the virus surface might modulate specificity of receptor

binding [3]. Due to this high similarity between flavivirus members, it is difficult to develop

diagnostic tools without cross reactivity.

19

Fig.7. DE3V structure. a. DENV structural and non-structural proteins. b. Mature DENV virion and

the E proteins domains [8].

1.2. Diagnostic tools for dengue

1.2.1. Laboratory diagnosis

Diagnoses of DENV infection on the basis of clinical syndromes are not reliable, and should

be confirmed by laboratory studies. Since dengue disease has a large spectrum of conditions -

from asymptomatic infection to influenza-like syndrome to severe disease including DHF/DSS -

the laboratory tests need to fit all of the aforementioned stages. Furthermore, as mentioned

20

above, secondary infection by a different dengue serotype or multiple infections with various

DENV serotypes are the most significant individual risk factors for DHF/DSS. Therefore, the

need to identify the various DENV serotypes during the course of DENV disease is imperative.

Knowing the serotype according to the virus or corresponding antibody (Ab) detection can assist

in monitoring people with high risk to develop DHF/DSS. Early diagnosis, followed by

supportive care and symptomatic treatment through fluid replacement are the keys to survival in

cases of severe dengue infection [31]. Moreover rapid and accurate dengue diagnosis is

important for effective control of dengue outbreaks [32]. Accurate and efficient diagnosis of

dengue is also important for epidemiological studies and for the clinical management and

evaluation of individual patients, particularly if new and specific therapeutic agents develop.

Accuracy in diagnosis is also important for pathogenesis studies and vaccine/ therapeutic

research [33]. The diagnosis is even more complex because of the co-circulation of other

arboviruses in most countries where DENV is endemic. For instance, the other arboviruses co-

circulating with the DENV are: the JEV in South-East Asia, the Saint-Louis encephalitis virus

(SLEV) and the YFV in Latin America, or the WNV in the Caribbean [5, 34]. Thus there is a

growing demand for trustworthy, reliable, cost effective, fast and field operable applications for

detection and differentiation of DENV infection in the acute and convalescent phase of illness.

Currently, dengue diagnosis, during the acute phase of the disease, is based on virus isolation

using cell culture [35], genome detection using reverse transcription polymerase chain reaction

(RT-PCR) [36-37] and NS1 antigen detection that is secreted in the serum of the patient during

the viremic period [38-41]. Serological tests for detection of anti-DENV antibodies primarily

rely upon specific ELISA for IgM and IgG detection. In addition, haemagglutination inhibition

(HI) [42-44] and plaque reduction neutralization tests (PRNT) are used for detection of total

anti-DENV antibodies [32, 34]. Table 1 summarizes advantages and disadvantages of the main

laboratories’ use of serological and virological dengue diagnostic assays and Table 2

summarizes some commercial kits for DENV detection.

21

Virological Diagnosis

The main problem with early diagnosis of DENV is the narrow window of time (about 5

days) available for successful detection of the virus [18-19]. Moreover, some DENV-infected

patients do not seek immediate medical care, as the initial manifestations are usually

asymptomatic or a typical fever of undefined cause. Thus, in a majority of cases, the diagnostic

test needs to be based on detection of anti-DENV antibodies [31]. Virological diagnosis usually

involves cell culture (generally mosquito cell line) with fluorescence-labeled antibodies of

various specificities. This technique is relatively sensitive but it takes several days and may not

always be successful due to the small amount of viable virus in the sample. Moreover it requires

a P3 lab, which usually exists only in a reference laboratory [12, 32, 34, 45-46]. Despite these

disadvantages, this application remains the “gold standard” technique for DENV detection [12,

14, 47]. Viral RNA can be detected, according to the serotype, with a high degree of sensitivity

by using RT-PCR. RT-PCR is definitely the most sensitive test, since it is able to detect dengue

viruses up to the 7th-10th day after the onset of the symptoms [12, 32, 34, 45-46, 48]. The RT-

PCR disadvantages are the cost and sophisticated equipment required [31, 49].

An increasing number of publications has revealed the use of real-time RT-PCR assays for

the detection of DENV in acute-phase sera samples [50-55]. Principle advantages of this method

over regular RT-PCR application are rapidity, high sensitivity and specificity. Currently, there

are several chemistries used for the detection of PCR product during real-time PCR: e.g., DNA

binding fluorophores, the 5′ endonuclease, adjacent linear and hairpin oligoprobes and the self-

fluorescing amplicons [56]. The most widely used is the 5′ endonuclease chemistry (TaqMan).

This is promising technology with potential to develop multiplex PCR protocol with up to four

fluorophores in a single tube (i.e. detection of four different DENV serotypes). In an attempt to

develop a simple, reliable, and universal RT-PCR protocol with systemic detection and

differentiation of the various DENV, a real-time quantitative RT-PCR system based on SYBR

Green-I DNA dye binding fluorophores was developed [55]. The rationale is to develop a

diagnostic system with an automated platform using real-time PCR equipment and a universal

RT-PCR protocol that allows multiple primer sets designed and tested without the need to

change the RT-PCR conditions [55]. The main advantage of the SYBR Green real-time RT-PCR

method on the TaqMan assay is simplicity in primer design and the capability to use universal

22

RT-PCR protocols suitable for the detection of multiple target sequences [55, 57]. Moreover an

isothermal nucleic acid sequence-based amplification (NASBA) assay was optimized to amplify

viral RNA of all four DENV serotypes by a set of universal primers and to type the amplified

products by serotype-specific capture probes. The NASBA assay involved the use of silica to

extract viral nucleic acid, which was amplified without thermocycling [58]. The main advantage

of this assay over a PCR technique is that it is entirely isothermal and is conducted at 41°C.

Thus, it would be suitable for epidemiological studies in the field [58]. Nevertheless this

technique is in development and not yet being used for routine diagnostics. Viral antigen

detection is also possible using ELISA [46]. Currently, a kit based on sandwich ELISA to test

secreted NS1 has been developed (Bio-Rad) ( Table 2) [13].

1.2.1.1. Serological diagnoses

Since there is cross-reactivity between flaviviruses serological diagnoses have some

limitations in areas where other arboviruses are circulating or in individuals previously

vaccinated with the yellow fever vaccine or Japanese encephalitis vaccine. Traditionally, the HI

test was used to detect and differentiate between primary and secondary DENV infections due to

its simplicity and sensitivity. However many investigators have questioned this general

applicability of using the HI test where two or more flaviviruses are co-circulating because IgG

antibodies measured are typically broadly flavivirus reactive. This test also requires paired sera

samples and does not recognize the virus serotype [32, 34]. PRNT is the most sensitive and

specific serological test for DENV diagnosis. Due to its high specificity, PRNT can be used to

identify the infecting serotype in primary dengue infections, since a relatively monotypic

response is observed in the patients’ serum during the convalescent phase. In secondary and

tertiary infections, the determination of the infecting serotype by PRNT is not always reliable.

The main disadvantages of this method are its high cost, time consumption and the expertise

necessary to perform it, with associated technical difficulties [32, 34]. Moreover it requires a P3

lab, which usually exists only in a reference laboratory. Both HI and PRNT are not suitable for

high throughput screening of large collections of sera.

23

Capture ELISAs for IgM (MAC-ELISA) and IgG (GAC-ELISA) antibodies or IgG detection

using indirect ELISA are routinely used for the serological analyses of DENV infections, as they

are simple and allow large numbers of samples to be tested. A limitation of these tests is that

specificity is generally lacking, particularly in the case of secondary infections. The envelope

and membrane (E/M)-specific IgM capture ELISA has become the most powerful assay for the

serodiagnosis of DENV infection [12, 59]. Commercial IgG and IgM kits for the detection of

DEN antibodies are available and are based mainly on MAC/ GAC-ELISA, Indirect IgM/IgG

ELISA, IgM/IgG DipStick and IgM/IgG blot [59-60]. However these kits are relatively

expensive due to the high costs associated with antigen production, and thus unaffordable for use

in the economically depressed countries where dengue is mostly prevalent. Furthermore most of

these tests rely on the use of whole virus antigens (produced in tissue culture or suckling mice

brain) for the detection of anti-dengue antibodies in patient sera, and are consequently associated

with an inherent biohazard risk. One kit, which has replaced the whole virus antigen with insect

cell-expressed dengue envelope protein, eliminates this risk but these kits present expenses due

to the high costs associated with antigen production. Apart from this, a major shortcoming of the

commercial kits is that they do not differentiate between infections due to dengue and other

flaviviruses, and between dengue serotypes [31]. IgA-specific capture ELISA has also been

developed for blood sample [61] and for saliva [42]. Several tests are trying to discriminate

between primary and secondary DENV infection. For this purpose HI is the reference test

recommended by the WHO [34] but there is also an ELISA test based on IgM/IgG ratio [34, 43-

44].

Today, new techniques for detecting the virus or subviral components and antibodies are in

development [32, 34, 45], but still there is a great need to standardize and improve current

dengue diagnostics. Development of more sensitive, specific, rapid and cost effective diagnostic

tools, along with field operability for underdeveloped regions of the world, is of increasing

importance. As a result, several groups have focused on developing biosensors for detection of

DENV using chip and quartz crystal microbalance or anti-DENV IgG antibodies with vertical

cavity surface emitting lasers [62-66]. Moreover since working with a virus potentially

infectious to humans requires extra safety precautions; there is constant need to develop

alternative antigens to replace the whole virus antigen in diagnostic tests [31].

24

Table 1: advantages and disadvantages of the main laboratories’ use of serological and virological

dengue diagnostic assays.

Advantages Disadvantages

Virus detection using

Cell culture

• Relatively sensitive

• Specific to serotype

• Consider to be "Gold standard" for viral

detection

• Time consuming (~ 10-21

days)

• Required P3 lab

• Expensive

Genome detection

using RT-PCR

• High specificity also according to the

serotype

• High sensitivity

• Relatively fast (~ 1-2 days)

• Expensive (specially in

undeveloped countries)

• Sophisticated equipment Virological

Genome detection

using Real time RT-

PCR

• High sensitivity

• High specificity also according to the

serotype

• Relatively fast

• Quantifies measurements

• Expensive equipment and

reagents

• Sophisticated equipment

• Not easy to standardize

IgM detection using

MAC-ELISA

• Simple

• High throughput method

• Fast with in house protocol (1-2 days)

• Existence of commercial tests

• Reliable

• Easy to perform

• Considered to be the routine diagnostic test

for serological detection

• Lack of specificity.

IgG detection using

indirect ELISA

• Simple

• High throughput method

• Fast with in house protocol (1-2 days)

• Reliable

• Easy to perform

• Lack of specificity

Serological

HI

• Chip

• Sensitive

• Simple, but requires skilled technician

• Non specific to species

• Lack of specificity

• The need for paired sample

• Time consuming

25

• Considered to be "Gold standard" for

serological detection

PRNT

• Most sensitive and specific serological test

• Detection according to the serotype, but

only in primary dengue infection

• Time consuming

• Requires skilled technician

• Requires P3 lab

26

Table 2: Commercial kits for anti DENV detection.

Ref. % specificity % sensitivity Detection time Method type Kit

]59 ,67[ 100 98 4h ELISA - IgG MRL diagnostics

]60[ 84.4 99 4h ELISA – IgM capture PanBio

]59 ,67[ 100 100 2.5h ELISA – IgG PanBio

]60[ 90.6 77.8 15min IgM and IgG -rapid test PanBio – Dengue

Duo Cassette

]59[ 89 71 2h Fluorescent (IgM) Progene

]59[ 86 96 1.5h dipstick Integrated

diagnostics

]59[ 92 100 8h IgM blot Genelabs

]60[ 79.9 98.6 6h IgM capture Focuse Diagnostics

]60[ 84.6 61.5 4h Indirect IgM detection Omega diagnostics

]60[ 97.8 62.3 4h IgM capture Omega diagnostics

]60[ 90 60.9 15-20min IgM and IgG rapid

tests

Standard

diagnostics

]60[ 76.6 97.7 1.5h IgM rapid tests Pentax

]60[ 86.7 20.5 15min IgM and IgG rapid

tests

Zephyr

biomedicals

]13[ 97.9 60.4 3h≈ NS1 antigen detection PanBio

]13[ 100 81.5-82.4 15-30min NS1 antigen detection Bio-Rad

27

1.3. The biosensor technology

A biosensor is defined as a self-contained integrated device, which is capable of providing

specific quantitative or semi-quantitative analytical information using a biological recognition

element (biochemical receptor), which is retained in direct spatial contact with a transduction

element. The term biosensor generally refers to a small, portable, analytical device that is

capable of detecting chemical or biological materials selectively and with a high sensitivity [68-

70]. Biosensors include four components (Fig. 8): a recognition module (biochemical receptor),

specific binding chemistry (immobilization), a transduction module and a data evaluation

module [71]. Biosensors are usually classified either by their bioreceptor or their transducer

type. For molecular recognition, the module consists of a biological or biomimetic system that

utilizes a biochemical mechanism. The most common forms of bioreceptors used in biosensors

are based on the following interactions: antibody/antigen (immunosensors) [72], nucleic acid

(genosensor) [73] and enzymatic [74]. Progressions in genetic engineering, protein engineering

and polymer chemistry have greatly expanded the availability and quality of recognition

elements. The immobilization layer enables binding of the biological element to the sensor and

helps reduce a non-specific signal. This layer should guarantee reproducibility, accessibility to

the binding site of the target, stabilize the recognition element as well as its chemical and/ or

physical properties and reduce non-specific binding. The transducer converts a biochemical

signal, resulting from the interaction of a recognition biological component, into a measurable

electronic signal. A wide variety of physical transducers are available: e.g., optic,

electrochemical, acoustic or thermal [71, 75]. Optical transducers may use luminescence,

absorption or surface plasmon resonance to convey specific signal properties. Suitable

transducing systems can be adapted in a biosensor assembly depending on the nature of the

biochemical interaction with the species of interest [76].

28

Fig. 8. Biosensor elements. Biosensors include four components: recognition module (biochemical

receptor), specific binding chemistry (immobilization), transduction module and data evaluation module.

The recognition element interacts with the analyte to create a signal which is detected by the transducer

with output recorded (adapted from http://www.jaist.ac.jp/~yokoyama/biosensor.html).

1.3.1. Chemiluminescence based biosensors

Fluorescence is used to describe the light emission that occurs when a molecule in an excited

state relaxes to its ground state. The various types of luminescence differ from the source of

energy to obtain the excited state. In chemiluminescence, the energy is produced by a chemical

reaction. Since excitation is not required for sample radiation, problems frequently encountered

in photoluminescence as light scattering or source instability are absent in chemiluminescence.

Chemiluminescent measurements are becoming increasingly popular due to their high

sensitivity, low background, wide dynamic range and comparatively inexpensive

instrumentation [77]. Compared to fluorescence, luminometry needs neither an excitation source

nor interference filters, nor does it suffer from photobleaching, thus it is the most promising

technology to be adapted on a chip. Luminometry is up to 100,000 times more sensitive than

absorption spectroscopy and over 1000 times more sensitive than fluorometry [78]. A well

designed luminometer can detect as little as 0.6 picograms of adenosine triphosphate (ATP) or

0.1 femtograms of luciferase, two common luminescent analytes.

29

1.3.1.1. The biochemistry of chemiluminescence

Numerous substrates have already been used in the field of chemiluminescence. Most of the

chemiluminescent bioassays are currently based on luminol (3-Aminophthalhydrazide) and its

derivatives, alone or coupled to light enhancers (Fig. 9). Luminol is a chemical that exhibits blue

chemiluminescence (λ=430nm), when mixed with an appropriate oxidizing agent. To exhibit its

luminescence, the luminol must first be activated with an oxidant. Usually, a solution of

hydrogen peroxide (H2O2) and a hydroxide salt in water is used as the activator. In the presence

of a catalyst such as an iron compound, the H2O2 is decomposed to form oxygen and water.

Enzymes in a variety of biological systems may also catalyze the decomposition of H2O2. When

luminol reacts with the hydroxide salt, a dianion is formed. The oxygen produced from the H2O2

then reacts with the luminol dianion. The product of this reaction, organic peroxide, is very

unstable and immediately decomposes with the loss of nitrogen to produce 5-aminophthalic acid

with electrons in an excited state. As the excited state relaxes to the ground state, the excess

energy is liberated as a photon, visible as blue light.

Fig. 9. Chemiluminescent reaction produced by the catalysis of H2O2 by horseradish peroxidase (HRP)

and the charge transfer to luminal (C8H7N3O2). Light is detected during the relaxation of a molecule from

an excited state to a ground state [79].

30

1.3.1.2. Enzymes used in chemiluminescence reactions

In aprotic media (dimethylsulphoxide or dimethylformamide), only oxygen and a strong

base are required for chemiluminescence [80]. In protic solvents (water, water solvent mixtures

or lower alcohols), various oxygen derivatives (molecular oxygen, peroxides, superoxide anion)

can oxidize luminol derivatives but catalysis either by enzymes or by mineral catalysts is

required [81]. Since the beginning, many catalysts have been proposed [80, 82-83]: enzymes

such as microperoxidase, myeloperoxidase, HRP, catalase, xanthine oxidase [84-86],

haemoglobin especially haptoglobin [87], deuterohemin or mineral catalysts such as molecular

ozone and halogens or persulphate anion or Fe(III), Co(II) and Cu(II) cations as well as their

complexes. More recently, the bacterial peroxidase from Arthromyces ramosus characterized by

a very high turn-over has been proposed and a hundred times increase in sensitivity was claimed

[88-89]. Moreover, many enzymes or enzyme mixtures that produce oxygen derivatives as by-

products have been involved in chemiluminescent detection. Alkaline phosphatase (AP), b-D-

galactosidase and b-glucosidase in the presence of indoxyl conjugates as substrates [90], lactate

oxidase [91], acylCoA synthetase and acylCoA oxidase [92] or diamine oxidase [93] produce

H2O2; 3-a hydroxysteroid deshydrogenase [94] or glucose-6-phosphate deshydrogenase release

NADH which reduces, in the presence of 1-methoxy-5-methylphenazinium methylsulphate,

molecular oxygen to H2O2 which generates light in the luminol microperoxidase system.

Chemiluminescent has been extensively used for the ultrasensitive detection of labels in

immunoassays. Chemiluminescence based assays relying on AP and HRP labels have been the

most popular types [95-96].

1.3.1.3. Chemiluminescence based optical fiber immunosensors

Optical fiber sensors based on chemiluminescence are ideal transducers governed by Snell’s

law. Fiber-optic biosensors use optical fibers as the transduction element, and rely exclusively

on optical transduction mechanisms for detecting target biomolecules. In this method the photon

reading instrument is based on a photomultiplier tube (PMT) (Fig.10). They have the following

advantages [97]: (1) geometric convenience and flexibility; (2) low cost production; (3) inert and

therefore non-hazardous; (4) free of electrical interference; (5) dielectric, therefore protected

against atmospheric disturbances; (6) small volume economizes reagents thus enabling

31

portability and easier access to difficult areas; (7) robust with high tensile strength; (8) silica

composition enables macromolecular conjugation via silanization; (9) allows solid-phase

characterization of the analyte; (10) potentially long interaction lengths permitting remote signal

transmission; (11) light transmission with minimal loss; (12) high efficiency coupling occurs in

the blue region which is ideal for chemiluminescence; (13) optical multiplicity; (14)

polyvalence, as an optrode system, so can be adapted easily from one antigen–Ab system to

another; (15) amenable to mass production; (16) permits multiple antigen detection via fiber

bundles. Additionally, it has been demonstrated that chemiluminescence based silica OFIS are

more sensitive than their analogous colorimetric and chemiluminescent ELISA counterparts [98-

100]. The combined advantages of chemiluminescence based OFIS led to several studies in the

field of diagnostic tools for whole organisms, nucleic acids, IgM and IgG antibodies with high

specificity and sensitivity [98, 100-103], in addition to bioluminescence from genotoxicants

[104-105]. Optical fibers however, have some disadvantage when designing the biosensor: (1)

background caused by ambient light interference. (2) immobilization chemistry is not always

easy to accomplish. (3) fiber to fiber differences can cause great difficulty in normalizing

signals, one to another [71, 106].

32

Fig.10. Descriptive scheme of fiber optic based biosensor with the setups for IgM anti DE3V

detection A. Photon counting unit. A1. Hamamatsu HC135-01 PMT Sensor Module. A2. PMT fixation

ring A3. Manual shutter (71430, Oriel). A4. Fiber holder that prevents the movement of the fiber inside

the photon counting unit. A5. Fiber optic. A6. Connection wire of PMT to computer. A7. Electricity

cable. B. The outside handle of manual shutter that enables light access to the PMT. C. Immobilization

unit C1. fiber optic, C2. 100µl pipette tip, C3. Conical tube cup, C4. Point of fixation of fiber, C5.

Optical fiber core C6. Biorecognition elements according to MAC-ELISA chemiluminescent OFIS [107]

C7. Test samples. E. Connection to computer [108].

33

1.3.1.4. Detection of chemiluminescence: photon-counting system

The detector is an essential component of the biosensor which influence on the overall

performances of the biosensor, both sensitivity and specificity. Its function is to convert the

received optical signals into electrical, which are then amplified before further processing.

Nowadays, there are many kinds of photodetectors with availability of integrated commercial

products incorporating analysis software; the efficiency previously reviewed [109-111]. A PMT

is efficient, sensitive and accurate light detection that is commonly used in analytical

luminescence methods [112]. A PMT is constructed from a glass envelope with a high vacuum

inside, which houses a photocathode, several dynodes, and an anode. Detected photons strike the

photocathode material with electrons produced as a consequence of the photoelectric effect.

These electrons are multiplied by the process of secondary emission. The electron multiplier

consists of a number of electrodes called dynodes. Each dynode is held at a more positive

voltage than the previous one. As the electrons move toward the first dynode, they are

accelerated by the electric field and arrive with much greater energy. Upon striking the first

dynode, more low energy electrons are emitted, and these electrons in turn are accelerated

toward the second dynode and so on through the dynode chain. Finally, the electrons reach the

anode, where the accumulation of charge results in a sharp current pulse indicating the arrival of

a photon at the photocathode. PMTs have very low noise and presently, photon counting using a

PMT still seems to be the most cost effective way to detect very low light levels [111].

Following the development of avalanche photodiodes (APD) single photon avalanche diodes

(SPAD) have also been developed for photon counting purposes. SPAD are semiconductor

devices based on a reverse biased p–i–n junction and operate in a non-proportional

multiplication mode analogous to a Geiger Müller tube [110, 112]. The reverse bias is held

slightly above rather than below the breakdown voltage for the junction. The electric field is

sufficiently high to sustain an avalanche of carrier multiplication via secondary ionization once a

primary electron–hole pair has been photo-induced by absorption in the depletion layer. The

diode current is turned off passively either by limiting the current flowing with a suitable

resistor, or actively by lowering the bias voltage after the onset of the avalanche [112]. In the

PMT the primary photoelectron is emitted from the photo cathode into a vacuum and then

multiplied by secondary electron emission. The shower of secondary electrons is collected by the

anode and produces a current impulse at the output. In the SPAD a conduction electron is

34

excited internally, which triggers an avalanche breakdown. A characteristic feature of a SPAD is

the extremely small variance of the amplitude of the output pulses even if more than one primary

photo-electron was to be excited by the light pulse. The PMT produces, on the other hand,

output pulses with high variance (depending on the type) and the mean amplitude is proportional

to the number of primary photo electrons [112]. Presently, photon counting using PMTs still

seems to be the most cost effective way to detect very low light levels and they have quite large

photo cathode areas, if such are needed by the application. SPAD-detectors will surely become

more and more attractive as time goes by, their development proceeds further and the prices

decrease.

1.3.2. Electrochemical biosensors

1.3.2.1. Introduction to electrochemistry

Electrochemistry is a branch of science, which encompasses chemical and physical processes

involving the transfer of charge. Electrochemistry is used to study the loss of electrons

(oxidation) or gain of electrons (reduction) that a material undergoes during a reaction. These

reactions can provide information about the concentration, kinetics, reaction mechanisms,

chemical status, and other behavior of a chemical species in solution. Electrochemical detection

is based on monitoring changes of an electrical signal due to an electrochemical reaction at an

electrode surface, usually as a result of an imposed potential or current. Electrochemical

processes can be studied and can be understood using, e.g., voltammetry, polarography,

chronopotentiometry, cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS),

linear sweep techniques, chronoamperometry, pulsed techniques.

1.3.2.1.1. Cyclic voltammetry

CV is an electrochemical analysis method which consists of scanning linearly the potential

of a stationary working electrode (in an unstirred solution). During the potential sweep, the

potentiostat measures the current resulting from the applied potential. The resulting current–

potential plot is termed a CV. CV is the most widely used technique for acquiring qualitative

information about electrochemical reactions. In particular, it offers a rapid location of redox

35

potentials of the electro-active species, and convenient evaluation of the effect of the media on

the redox process [113].

1.3.2.1.2. Electrochemical impedance spectroscopy

EIS is an effective technique for probing the features of chemically-modified electrodes and

for understanding electrochemical reaction rates. Impedance is the totally complex resistance

encountered when a current flows through a circuit made of combinations of resistors,

capacitors, or inductors. Electrochemical transformations occurring at the electrode–solution

interface can be modeled using components of the electronic equivalent circuitry that correspond

to the experimental impedance spectra. The impedance of the interface, derived by application of

Ohm’s law, consists of two parts, a real number Z′ and an imaginary one, Z″ [113].

1.3.2.1.3. Electropolymerization

Conducting polymers can be prepared via chemical or electrochemical polymerization, using

an electric potential for monomer polymerization. The advantages of the electropolymerization

process are the simple and reproducible procedures in which the thickness and adherence of the

electrode coating are fairly easily controlled by the duration and intensity of the applied current,

the monomer composition and concentration, the solvent, and the reaction conditions [114-115].

The properties of the polymer can also be controlled by grafting substituents to a functionalized

polymer, or by entrapping biomolecules [116].

1.3.2.2. Electrochemical biosensor

An electrochemical biosensor uses an electrochemical transducer. It is considered to be a

chemically modified electrode as electronic, semi or ionic conducting material coated with a

biochemical film [68]. Electrochemicals are known to play important roles in the research and

development of biosensors. Electrochemical biosensors are being used increasingly for

monitoring synthetic and biological processes. The electrochemical techniques are known to

help in the speedy development of biosensors for continuous, real-time, inexpensive, and in vivo

36

monitoring of many components in clinical laboratories and industries. Compared to optical,

mass, and thermal sensors, electrochemical sensors are especially attractive because of their

remarkable detection limits, experimental simplicity, and low cost. They hold a leading position

among the presently available sensors that have reached the commercial stage and which have

found a vast range of important applications in the fields of clinical, industrial, environmental,

and agricultural analyses.

1.3.2.3. Amperometric biosensors

Amperometric biosensors measure the current produced at a working electrode during the

oxidation or reduction of a product or reactant, usually at a constant applied potential with

respect to a reference electrode. The most important factor affecting the operation of

amperometric biosensors is the electron transfer between catalytic molecules, usually oxidases or

dehydrogenases, and the electrode surface, most often involving a mediator and/or a conducting

polymer [117]. When constructing an amperometric immunosensor, mediators are widely used.

Mediators are artificial electron transferring agents that can readily participate in the redox

reaction with the biological component and thus help in rapid electron transfer. A mediator is a

low molecular weight redox couple, which shuttles electrons from the redox center of the

enzyme to the surface of the indicator electrode. During the catalytic reaction, the mediator first

reacts with the reduced enzyme and then diffuses to the electrode surface to undergo rapid

electron transfer [118]. The effective combination of immunochemistry and electrochemistry in

an analytical device could provide the basis of direct electrical detection of a wide range of

analytes with great sensitivity and specificity [119].

1.4. Alternative fiber for optic biosensors

Silica-based fiber optic biosensors are a well investigated platform being used to immobilize

various biomaterials (enzymes, antibodies, antigen or whole cells) [98-103]. However, silica

fiber chemical modifications are still unreliable fiber-to-fiber, accordingly an alternative

configuration scenario would be useful to improve the efficiency and reproducibility for putative

mass production of optic fiber probes. PMMA optical fibers are alternative fiber optics

37

displaying very good optical properties. While also proving to be inexpensive and easy to

prepare, they are also easier to cleave in comparison to a more commonly used silica based

fibers. The PMMA fiber surface shows a hydrophobic and favorable environment for protein

adsorption. However, in order to achieve more stable bio-conjugate linking, chemical

modification procedures would need to be implemented to conjugate the surfaces.

1.5. Alternative antigen

As detailed above, one of the limitations of current diagnostic tools is the lack of specificity

and safety of the antigen employed as capture moietie. A constant exertion of effort in

developing alternative antigens that will help to improve the detection of anti-dengue antibodies

is made.

1.5.1. Recombinant Protein and Epitopes

The envelope protein (E) plays a key role in numerous aspects of the viral life cycle and viral

pathogenesis including virion assembly, membrane fusion, receptor binding, blood cell

hemagglutination, and induction of a protective immune response [20, 120]. The E ectodomain

consists of three domains: Domain I, Domain II, and Domain III (E3). The E3 has been proposed

to function as the putative receptor-binding domain of DENV [3]. The E protein is the most

immunogenic of all the dengue viral proteins eliciting the first and longest lasting antibodies.

Consequently, much research has been spent on trying to find immunodominant epitopes based

on the E protein [31, 121-122]. The non structural protein NS3 [31, 123] and especially NS1 [31,

123-125] are reported to elicit significant Ab responses, particularly in secondary infections; so

they can also be used for this purpose.

38

2. Aims and expected significance of the research

Dengue diagnoses have come a long way and today, new techniques for detecting the virus

and anti-DENV Ab are available. Problems still remain with those methods requiring the

development of a more sensitive, specific, rapid and cost effective diagnostic tool. The main

problems are the cross reactivity between the dengue serotype and Flavivirus members, narrow

time window to detect the virus and the need for a large spectrum of tests that will fit into the

broad range of dengue diseases. The main goal of the proposed research was to develop a

diagnostic tool for the detection of anti DENV antibodies in human sera samples. The ultimate

biosensor provided a simple, reliable, fast, and field implement table analytical tool. This goal

was achieved in a few stages:

1. Development of a diagnostic tool based on a luminescence biosensing platform to detect

anti dengue antibodies in sera samples in order to increase the sensitivity of the bioassay.

2. Application to clinical issues: implement the diagnostic tool on samples from an endemic

area.

3. Increase the specificity of the bioassay technique and avoid cross-reactivity by using new

antigen and bio-molecules.

4. Technical improvement of silica optical fiber biosensors in order to improve the

sensitivity of the biosensors.

39

3. The logical structure and order of published articles within the

thesis

This thesis is based on a collection of these articles (the full articles is attached in the next

chapter and on CD):

• Danit Atias, Yael Liebes, Vered Chalifa-Caspi, Laetitia Bremand, Leslie Lobel, Robert S. Marks & Phillipe Dussart. "Chemiluminescent optical fiber immunosensor for the detection of IgM antibodies to dengue virus in humans", Sensors and Actuators B: Chemical. 140 (2009) 206–215.

• Danit Atias*, Khalil Abu-Rabeah*, Sebastien Herrmann, Julia Frenkel, Dorith Tavor,

Serge Cosnier, Robert S. Marks, "Poly(methyl metacrylate) conductive fiber optic transducers as dual biosensor platforms" Biosensors and Bioelectronics 24 (2009) 3683–3687.

• Khalil Abu-Rabeah*, Danit Atias* , Sebastien Herrmann, Julia Frenkel, Dorith Tavor,

Serge Cosnier and Robert S. Marks. "Characterization of electrogenerated polypyrrole-benzophenone films coated on poly (pyrrole-methyl metacrylate) optic-conductive fibers", Langmuir 2009, 25(17), 10384–10389. * Both authors contributed equally to this work but the work will be published only in this thesis.

In the first article, a new diagnostic tool was reported that was developed based on a

chemiluminescent OFIS, for the detection of anti-DENV immunoglobulin M (IgM) in human

sera samples. The immunoassay was based on a colorimetric MAC-ELISA, routinely used by

the National Reference Center for Arboviruses based in French Guiana. The detection of human

anti-DENV IgM, using colorimetric MAC ELISA, chemiluminescent MAC-ELISA and

chemiluminescent OFIS were compared. An internal panel of reference sera was used and 86

sera samples were screened. In this work was shown that the OFIS technology reported in the

article is reliable, simple to perform, fast, cost effective, and a field operable analytical tool.

40

In the second article was developed an alternative optic-conductive fiber configuration applied in

the construction of biosensing platforms. This new approach is based on applying the chemical

polymerization of pyrrole onto the surface of PMMA fibers to create a polymer – a conductive

surface, onto which an additional photo-active polypyrrole-benzophenone (PpyBz) film is

electrochemically generated upon the fiber surface. Irradiation of the benzophenone groups

embedded in the Ppy films with UV radiation (350 nm) formed active radicals that allowed the

covalent attachment of the desired bioreceptors. Characterization of the amperometric

biosensing matrix was accomplished by using a model Urease (Urs) through EIS and

amperometry. Both techniques have shown a low charge transfer resistance and a high

sensitivity. Thereafter, the construction of an optical biosensing matrix based on HRP production

of photons was carried out. The high signal to noise (S/N) ratio indicated clearly that this

approach can serve as a new platform to replace glass optical fibers based on biosensors.

In the third article it was characterized and optimized a new platform that was developed and

reported in the second article. The platform was shown as conductive, stable, thin, controllable

and with light-transmissible film features. Various parameters like time deposition, process

temperature, activator plus pyrrole monomer concentrations were examined in the study. The

morphology and permeability of the optic-fiber PMMA fibers were investigated to examine

mass transfer ability. Cyclic voltammetry and amperometry techniques were applied to

characterize the electrical features of the surface and charge transfer. Then the platform potential

was demonstrated by the construction of both amperometric and optical biosensors.

41

4. Original publications

4.1. Article 3o. 1 (p. 42 – 51)

Danit Atias, Yael Liebes, Vered Chalifa-Caspi, Laetitia Bremand, Leslie Lobel,

Robert S. Marks & Phillipe Dussart. "Chemiluminescent optical fiber

immunosensor for the detection of IgM antibodies to dengue virus in humans",

Sensors and Actuators B: Chemical. 140 (2009) 206–215.

4.2. Article 3o. 2 (p. 52 – 56)

Danit Atias, Khalil Abu-Rabeah, Sebastien Herrmann, Julia Frenkel, Dorith

Tavor, Serge Cosnier, Robert S. Marks, "Poly(methyl metacrylate) conductive

fiber optic transducers as dual biosensor platforms" Biosensors and Bioelectronics

24 (2009) 3683–3687.

4.3. Article 3o. 3 (p. 57 – 62)

Khalil Abu-Rabeah, Danit Atias , Sebastien Herrmann, Julia Frenkel, Dorith

Tavor, Serge Cosnier and Robert S. Marks. "Characterization of electrogenerated

polypyrrole-benzophenone films coated on poly (pyrrole-methyl metacrylate)

optic-conductive fibers", Langmuir 2009, 25(17), 10384–10389.

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Sensors and Actuators B 140 (2009) 206–215

Contents lists available at ScienceDirect

Sensors and Actuators B: Chemical

journa l homepage: www.e lsev ier .com/ locate /snb

hemiluminescent optical fiber immunosensor for the detection of IgM antibodyo dengue virus in humans

anit Atiasa,b, Yael Liebesc, Vered Chalifa-Caspid, Laetitia Bremande, Leslie Lobela,obert S. Marksb,d,f,∗, Phillipe Dussarte

Department of Virology, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva 84105, IsraelDepartment of Biotechnology Engineering, Ben-Gurion University of the Negev, Beer-Sheva 84105, IsraelDepartment of Environmental Engineering, Ben-Gurion University of the Negev, Beer-Sheva 84105, IsraelThe National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Beer-Sheva 84105, IsraelLaboratory of Virology, Centre National de Reference des Arbovirus et virus influenza, région Antilles Guyane, Instituet Pasteur de la Guyane, 97300 Cayenne, French GuianaThe Ilse Katz, Center for Meso and Nanoscale Science and Technology, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel

r t i c l e i n f o

rticle history:eceived 15 January 2009eceived in revised form 22 March 2009ccepted 23 March 2009vailable online 31 March 2009

eywords:ptical fiber immunosensor (OFIS)hemiluminescent

gMengue virus

a b s t r a c t

Infection with dengue virus has emerged as the most important vector-borne viral disease in tropicalareas and it continues to expand geographically. The four serotypes of dengue virus (DENV) cause humandisease and are transmitted by Aedes mosquitoes. A reliable diagnosis remains a crucial step towards thecontrol of dengue disease in human populations. In this work, a new diagnostic tool was developed basedon chemiluminescent optical fiber immunosensor (OFIS), for the detection of anti-DENV immunoglob-ulin M (IgM) in human serum samples. The immunoassay was based on a colorimetric IgM capture,enzyme-linked immunosorbent assay (MAC-ELISA), routinely used by the National Reference Center forarboviruses based in French Guiana. The detection of human anti-DENV IgM using colorimetric MAC-ELISA, chemiluminescent MAC-ELISA and chemiluminescent OFIS was compared. An internal panel ofreference sera was used and 86 sera samples were screened. Compared to standard colorimetric MAC-

iagnostics ELISA, the chemiluminescent OFIS had a lower detection limit, 10 times lower than the chemiluminescentMAC-ELISA and 100 times lower than the colorimetric MAC-ELISA. Therefore the colorimetric and chemi-luminescent MAC-ELISA’s are more suitable for high and intermediate levels of anti-DENV IgM presentin serum samples, whereas the chemiluminescent OFIS is also useful at low analyte concentration, withsensitivity and specificity of 98.1% and 87.0%, respectively. Taking into account the lower limit of detectionand the high correlation between the established methods with the known panel, the OFIS technologyreported here is reliable, simple to perform, fast, cost effective, and a field operable analytical tool.

. Introduction

Dengue fever (DF) is caused by infection with the dengue virusDENV). DENV is a, mosquito-borne virus, and a member of the fam-ly Flaviviridae, genus Flavivirus. There are four distinct serotypesf DENV, designated DENV-1, DENV-2, DENV-3 and DENV-4 [1–3].ENV is endemic in most urban centers of tropical and subtropical

reas of the world [4]. To date, DF is the most important arthro-od borne disease in humans with an estimated 100 million casesnd over 500,000 cases of dengue hemorrhagic fever (DHF)/denguehock syndrome (DSS) occurring each year, including nearly 25,000

∗ Corresponding author at: Department of Biotechnology Engineering, Ben-urion University of the Negev, P.O. Box 653, Beer-Sheva 84105, Israel.el.: +972 8 647 7182; fax: +972 8 647 2857.

E-mail address: rsmarks@bgu.ac.il (R.S. Marks).

925-4005/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.snb.2009.03.044

© 2009 Elsevier B.V. All rights reserved.

fatal cases, primarily in children [5]. The World Health Organiza-tion (WHO) estimates that there has been a 30-fold increase in DFincidence in the last 50 years [6]. Expanding in geographic distri-bution both viruses and mosquito vectors, genus Aedes, led to acurrent global dengue pandemic [4,7]. Dengue disease has a widespectrum of expression: from asymptomatic infection or influenzalike syndrome to severe disease including DHF/DSS. Moreover, ina small percentage of cases, severe dengue occurs with unusualmanifestations such as hepatitis, encephalopathy or rhabdomyoly-sis [4,8–11]. Currently there is no available anti-dengue vaccine orspecific therapy for the treatment of DENV infection [12]. Symptomsof illness generally appear 4–7 days after a mosquito bite [4]. The

viremic phase of infection occurs shortly before the onset of clinicalsymptoms, and generally lasts around 5 days [13,14]. Typically, ina non-immune individual, anti-DENV IgM antibodies develop 5–6days after the primary infection (that is the first infection by one ofthe four DENV serotypes) and anti-DENV IgG antibodies appear and

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ncrease after 7–10 days. During the secondary infection, defined byn infection occurring from one of the three other DENV serotypesfter a primary infection, high levels of IgG antibodies are detectable–4 days after fever onset while IgM antibodies are lower and inome cases can be absent [15–17].

Sensitive and reliable diagnostics, followed by supportive carend symptomatic treatment through fluid replacement are theeys to survival in cases of severe dengue infection [18]. More-ver, rapid and accurate dengue diagnosis is important for effectiveontrol of dengue outbreaks and for epidemiological studies [19].urrently, dengue diagnosis, during the acute phase of the dis-ase, is based on virus isolation using cell culture [20], genomeetection using reverse transcription polymerase chain reactionRT-PCR) [21,22] and NS1 antigen detection that is secreted in theerum of the patient during the viremic period [23–26]. Serologi-al tests for detection of anti-DENV antibodies primarily rely uponpecific ELISA for IgM and IgG detection. In addition, haemaggluti-ation inhibition (HI) [27–29] and plaque reduction neutralizationests (PRNT) are used for detection of total anti-DENV antibodies30]. Traditionally, the HI test was used to detect and differentiateetween primary and secondary DENV infections due to its simplic-

ty and sensitivity. However many investigators have questionedhis general applicability of using the HI test where two or moreaviviruses are co-circulating because IgG antibodies measured areypically broadly flavivirus reactive. This test also requires pairederum samples. On the other hand, PRNT is time consuming and islso not suitable for high throughput screening of large collectionsf serum specimens.

The main problem with early diagnosis of DENV is the narrowindow of time (about 5 days) available for successful detection

f the virus [13,14]. Moreover, some DENV-infected patients do noteek immediate medical care, as the initial manifestations are usu-lly asymptomatic or a typical fever of undefined cause. Thus, in aajority of cases, the diagnostic test needs to be based on detec-

ion of anti-DENV antibodies [18]. As such, commercial MAC-ELISAs the standard method for serological analysis of DENV infectionsecause it is simple and allows for testing of large numbers of sam-les in parallel [30]. The envelope and membrane (E/M)-specific

gM capture ELISA has become the most powerful assay for theerodiagnosis of DENV infection [16,31]. Commercial MAC-ELISAits are available [32]; however these kits are relatively expensiveue to the high costs associated with antigen production, and thusnaffordable for use in the economically weaker countries whereengue is mostly prevalent. Finally, serological tests, in general,ave limitations in areas where other arboviruses are circulating,r in individuals previously vaccinated against yellow fever (YF) orapanese encephalitis, because of high cross-reactivity [33–39].

Although dengue diagnosis has made much progress, since HInd mouse inoculation were the only options available, with devel-pment of ELISA using recombinant proteins [31,40–42], there istill a great need to standardize and improve current dengue diag-ostics. Development of more sensitive, specific, rapid and costffective diagnostic tools, along with field operability for relativelynderdeveloped regions of the world, is of increasing importance.s a result, several groups have focused on developing biosensors

or detection of DENV using chip and quartz crystal microbalancer anti-DENV IgG antibodies using vertical cavity surface emittingasers [43–47].

Chemiluminescent measurements are becoming increasinglyopular due to their high sensitivity, low background, wideynamic range and relatively inexpensive instrumentation [48].

ompared to fluorescence, luminometry needs neither an exci-ation source or interference filters, nor does it suffer fromhotobleaching, thus it is the most promising technology to bedapted on a chip. Optical fiber sensors based on chemilumines-ence are ideal transducers governed by Snell’s law. They have many

tors B 140 (2009) 206–215 207

advantages that have been previously described [49]. Furthermoreit has been demonstrated that chemiluminescence based opticalfiber immunosensors are more sensitive than their analogous col-orimetric and chemiluminescent ELISA counterparts [50–52]. Thecombined advantages of chemiluminescence based OFIS led toseveral studies in the field of diagnostic tools for whole organ-isms, nucleic acids, IgM and IgG antibodies with high specificityand sensitivity [50,52–55], in addition to bioluminescence fromgenotoxicants [56,57]. We describe below the development of adiagnostic tool, based on chemiluminescent OFIS, for the detectionof anti-DENV IgM antibodies in human serum samples. This biosen-sor can form the basis of a simple, reliable, fast, cost effective, andfield operable analytical tool. Furthermore we demonstrated for thefirst time serum screening for IgM using chemiluminescent OFIS.

2. Materials and methods

2.1. Chemicals and biochemicals

Viral RNA extraction was carried out using the QIAamp®

Viral RNA Mini kit of Qiagen (Qiagen S.A., Courtaboeuf, France)(52906). Phosphate buffer saline (PBS) (P4471), skim milkpowder (SM) (70166), monoethanolamine (MEA) (E9508), 6-maleimidohexanoic acid n-hydroxysuccinimide ester (EMCS)(M9794), (3-mercaptopropyl)trimethoxysilane (MPTS) (M1521),polyethylene glycol sorbitan monolaurate (Tween® 20 (T))(P1379), dimethyl sulfoxide (DMSO) (D2650) were purchasedfrom Sigma–Aldrich (L’Isle d’Abeau Chesnes, France). Lumines-cence measurements were carried out using the Immuno-starHRP Chemiluminescent kit (170-5040) from Bio-Rad Laboratories(Marnes la Coquette, France). Colorimetric assays were carried outusing 3,3′,5,5′-tetramethylbenzidine (TMB) (T8665), anti-humanIgM (-chain specific) (I2386) and anti-mouse IgG (-chain spe-cific) peroxidase conjugate (A3673), which were purchased fromSigma. DENV-2 antigen (Ag) was obtained from the National Ref-erence Center (NRC) of Arboviruses, Institut Pasteur de la Guyane,French Guiana. The DENV-2 Ag was prepared by extraction frombrains of suckling mice with the sucrose–acetone method and wasshown to have cross-reactivity with the three other DENV serotypes[58]. Homologous mouse ascitic fluid containing anti-DENV IgG wasalso provided by the NRC [59]. The experimentation authorizationnumber for the production of these reagents from mice is 973-3/0702306, delivered by the Direction of the Veterinary Servicesfrom French Guiana to the Institut Pasteur de la Guyane.

2.2. Detection of dengue viruses by RT-PCR

Viral RNA was extracted from acute-phase serum usingQIAamp® Viral RNA kit as recommended by the manufacturer.Then, 5 l of RNA (≈150–250 ng) was mixed with 200 ng of randomhexamer primers, and first-strand cDNA synthesis was performedwith the SuperScriptTM First-Strand Synthesis System for RT-PCR(Invitrogen Life Sciences Technologies, UK) according to the man-ufacturer’s recommendations. The first run of RT-PCR analysis andsubsequent semi-nested PCR analysis were performed following apreviously described procedure [21].

2.3. Human sera

2.3.1. Internal panel of reference seraThe NRC, using its serum collection, has constituted a pool of

serum positive to anti-DENV-2 IgM. Bio-Rad Laboratories, using thispool from the NRC, kindly provided a panel of six serum samplescontaining different concentrations of anti-DENV IgM. The sampleswere referred to as panel point #1 to point #6, where panel point #1contained the lowest titer of anti-DENV-2 IgM. Another serum was

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eferred to as panel point #0, containing no anti-DENV-2 IgM anti-odies, which was obtained from a pool of serum from the Nationallood Bank of France. Each point from the panel has been calibrated

or anti-DENV IgM detection using the dengue IgM Capture ELISAest (E-DEN01M) from Panbio (Brisbane, Australia).

.3.2. Human field seraA total number of 86 sera samples from the collection of the NRC

ere tested in this study. The database provided information abouthe age and sex of the patients, the date of serum collection, clini-al symptoms and information regarding yellow fever vaccinationtatus, which is mandatory in French Guiana, and the day on whichlinical symptoms first appeared (onset of fever taken as day 0, i.e.rst 24 h).

Samples were collected during the acute phase of the diseaseday 0 to day 4) and/or the convalescent phase, i.e. 7 days orater after fever onset. All sera were first tested for anti-DENVgM antibodies by a routinely used colorimetric MAC-ELISA [59]nd then further divided into three groups. The first group con-isted of convalescent phase sera (n = 39) from patients positiveor anti-DENV IgM antibodies. The DENV strain of the samplesas defined by RT-PCR that was performed on acute phase sera

amples previously taken from the respective patients [21]. Theamples were found positive to DENV-1 (n = 5), DENV-2 (n = 25),ENV-3 (n = 5) or DENV-4 (n = 4). For a few additional patient sam-les, lacking respective acute phase samples, the DENV serotypeas defined according to epidemiological information relating to

he dengue serotype isolated in French Guiana at the time of infec-ion. These samples included those containing anti-DENV-2 IgMntibodies (n = 6) and anti-DENV-3 IgM antibodies (n = 8). The sec-nd group of samples consisted of acute phase sera samples (n = 23)rom patients presenting dengue-like syndrome (temperature of38.5 C, arthralgia, headache, and/or myalgia) for whom recentENV infection was ruled out by a negative RT-PCR and nega-

ive anti-DENV IgM assay, and confirmed with a second bloodample negative for anti-DENV IgM antibodies. The third groupf convalescence sera (n = 10) are samples that were found to bequivocal in colorimetric MAC-ELISA for anti-DENV IgM detec-ion.

.4. Detection of anti-DENV IgM antibodies by ELISA

MAC-ELISA was performed as described by Kuno et al. [60] withinor adjustments. A volume of 100 l of 4 g/ml concentration

f anti-human IgM antibodies was inserted into each well of a pre-ashed 96-well microtiter plate (MaxiSorp, Nunc) with PBS. Thelate was covered and was incubated for 2 h at 37 C. Following

ncubation, the plate was washed thrice to remove unbound anti-odies with PBS containing 0.1% Tween 20 (PBS-T) (pH 7.4). Thenera samples, consisting of the panel and controls, were diluted:100 in PBS containing 0.5% Tween 20 and 5% skim milk (pH 7.4)PBS-T-SM), and a volume of 100 l of each sample was added intohe plate in duplicates and incubated for 1 h at 37 C. After wash-ng the plate with PBS-T to reduce background and increase theensitivity of the assay, 100 l of DENV-2 Ag diluted 1:100 in PBS--SM was added to each well. After incubation at 4 C overnight,he plates were washed with PBS-T. Then, 100 l of mouse asciticuid containing murine anti-DENV IgG diluted 1:1000 was added

nto each well. Finally, 100 l of solution containing the secondaryntibodies, anti-mouse IgG peroxidase labeled, diluted 1:2000 wasdded to each well. Each of the last two steps was subjected to

ncubation for 1 h at 37 C followed by washing. For the colorimet-ic measurements, 100 l of the TMB substrate was added into eachell and incubated at room temperature for 20 min. Optical den-

ity was measured at 620 nm using 960 colorimeter (Metertech,aiwan) and reported using the instrument software in OD (opti-

tors B 140 (2009) 206–215

cal density). Positive results were defined as higher than threetimes the mean value of the negative control, and negative resultsas lower than twice that the mean value of the negative control.Values obtained between these ranges were defined as equivo-cal.

Chemiluminescence measurements were performed by addingoxidizing reagent and luminol into the wells in a 1:1 ratio.Measurements were carried out using a standard luminometer(Thermolabsystems-Luminoskan Ascent) and data was collectedusing Luminoskan Ascent software and reported in RLU (relativelight units). A total of 20 measurements were performed at a rate of1 s between each reading. A cut-off value was set according to thenegative control (panel point #0) and confidence range was ±10%.For both methods each experimental point was repeated in tripli-cate and for each triplicate, average and standard deviation werecalculated.

2.5. Preparation of optical fibers modified with anti-human IgM

Preparation of the optical fibers was performed according tothe method described by Herrmann et al. [50] and Salama et al.[51] with some modifications. Silica fibers SFS400/440 SuperguideG UV–vis (Fiberguide Industries, Stirling, USA) with an originalnumerical aperture (NA) of 0.22, a core of 400 m in diameter(refractive index of 1.457 at 633 nm) and a surrounding 40 m silicacladding (refractive index of 1.44 at 633 nm), followed by a 150 mthick silicon buffer and finally a 210 m thick transparent Tefzel®

jacket were used throughout all experiments. The length of any sin-gle fiber used in these experiments was approximately 20 cm. TheTefzel® jacket and silicon buffer were stripped away mechanicallyusing a fiber stripping tool (Micro-Strip®, from Micro-ElectronicsInc., USA), leaving a 0.5 cm nude optical fiber core tip.

Preparation of the fibers for the covalent immobilization of theanti-human IgM antibodies consists of the following major steps:removal of major micro-contaminants from the newly exposedfiber by soaking them in a 1:1 methanol 97% (v/v) HCl solutionfor 45 min heated to 64 C; drying under air/CO2 flow; enhance-ment of hydroxyl-group exposure on the silica surface of theoptical fiber tips by contact with 96% (v/v) sulfuric acid for 45 min;quick rinsing in ddH2O followed by acetone and then dryingunder air/CO2 flow to successively clear sulfuric acid residuals.The silanization procedure was then performed on the fibers in areactor chamber under constant argon flow of 2.5 l/min using (3-mercaptopropyl)trimethoxysilane (MPTS). A total volume of 1 mlMPTS was manually injected into an argon saturated chamber andthe silanization reaction occurred during 40 min at room temper-ature and ambient pressure. As a result, MPTS molecules werecovalently attached to the hydroxylated glass optical fiber sur-face by a gas phase deposition resulting in a functional thiolgroup (Fig. 1A). Following the silanization step, the fibers wererinsed in ethanol for 5 min and then in ddH2O in order to removeunattached MPTS molecules. Thereafter the modified fibers weredipped in a 1:9 (v/v) DMSO/PBS (pH 6.8) solution containing 4 mMof 6-maleimidohexanoic acid N-hydroxysuccinimide ester (EMCS)for 60 min at room temperature (Fig. 1B). The EMCS serves as ahetero-bi-functional cross-linker molecule: the maleimide groupof the EMCS covalently binds the free thiol group on the silanizedfiber, and the ester group of the EMCS creates an amide bondwith an amino group located on the anti-human IgM surface. Fol-lowing EMCS incubation, the fibers were first rinsed in 1:9 (v/v)DMSO/PBS (pH 7.4), then with PBS (pH 7.4) solution to remove

the non-bound cross-linker molecules and finally dipped overnightin PBS (pH 7.4) containing 4 g/ml of anti-human IgM antibod-ies solution at 4 C. These steps allow the covalent attachmentof the anti-human IgM antibodies to the optical fiber surface(Fig. 2.1).

D. Atias et al. / Sensors and Actuators B 140 (2009) 206–215 209

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ig. 1. Chemical modification to the chemiluminescent OFIS via silanization. (A) Bifilica fiber core, followed by (B) reaction of their thiol groups to maleimide group o

.6. Detection of human anti-DENV IgM antibodies using theptical immunosensor

Anti-DENV IgM detection, by the optical immunosensor, waserformed on the same rational basis as the above describedLISA with modifications and described in Fig. 2. In brief, thenti-human IgM modified fibers were rinsed twice with PBS-TpH 7.4) and dipped in blocking solution (1 M MEA: 0.5% PBS-T:% SM) (Fig. 2.2). The fibers were then introduced to the serumamples diluted in PBS-T-SM (pH 7.4) (Fig. 2.3). A set of 5–10bers were prepared for each experimental point. Another groupf fibers was used as a negative control (panel point #0). Recog-ition of bound anti-DENV IgM molecules was done by dippinghe fibers with DENV-2 antigen diluted 1:100 (v/v) in PBS-T-SMpH 7.4) (Fig. 2.4). Subsequently, the fibers were dipped in mousenti-DENV IgG antibodies diluted 1:1000 (v/v) in PBS-T-SM (pH.6) (Fig. 2.5) and then in goat anti-mouse IgG peroxidase labelediluted 1:2000 (v/v) in PBS-T-SM (pH 7.4) (Fig. 2.6). All incubationsccurred for 30 min at room temperature with 50 l solutions forach fiber. After each step the fibers were washed from unboundolecules by dipping twice each fiber for 5 min, in 100 l 0.1%

BS-T.

.7. Signal measurement for the chemiluminescent optical fibermmunoassay

The instrument set-up for the optical fiber chemilumines-

ent measurements has been previously described [50,51,53,56].Hamamatsu HC135-01 photo multiplier tube (PMT) sensor mod-

le was used for chemiluminescent measurements. The far end ofhe fiber was held by a fiber holder (FPH-DJ, Newport) and placednto an adjustable single-fiber mount (77837, Oriel). Monitoring of

ig. 2. Biosensor assembly on the modified OFIS. (1) Ester moiety of the cross-linker allolocking reagent to minimize non-specific binding. (3) Serum sample is tested for anti-DEouse anti-DENV antibodies. (6) Chemiluminescent detection of goat anti-mouse IgG. (7

nal silane molecule (MPTS) is covalently bound to exposed hydroxyl groups on theo-bifunctional cross-linker (EMCS).

the chemiluminescent signal and data handling in real time wasperformed with LabView (version 3.1, National Instruments Cor-poration). The last step of the procedure (Fig. 2.7), reading of thechemiluminescent reaction, is achieved by placing the immunosen-sor optical fibers in a 400 l sample tube containing the combinedoxidizing reagent and enhanced luminol reagent solutions in a1:1 (v/v) ratio. Chemiluminescent readings were integrated for 1 sand each measurement was obtained by taking a mean value ofphoton counts during 10 s. The mean of each experimental pointand the standard deviation were calculated as RLU (relative lightunits).

2.8. Data analysis and normalization for the chemiluminescentoptical fiber immunoassay

Since the above detailed chemiluminescent OFIS is a novel tech-nique, it needs to be further optimized, as differences in results mayoccur while performing the same measurements at different times.Variation in the results could probably be improved by automatingwashing and fiber cleaving. Nevertheless, all experiments demon-strated the same behavior and tendency. To obviate the problem ofvariation between experiments, data was normalized to eliminatethe effect of assay variation on the results and final conclusions.For each experiment, therefore, panel point #0 was tested as anegative control. The average of this point was compared to thevalue in the other experiments and all of the results were normal-ized according to this ratio. This normalization technique enables

not only deduction of the inter-experimental differences, but alsofacile determination of a cut-off, therefore deciding if the sampleis positive or negative. The cut-off value was set according to thenegative control (panel point #0) and the empiric confidence rangewas adjusted according to the methods ±10%.

ws covalent linking of the capture antibody through the amino group. (2) AddingNV-IgM. (4) Detecting specific anti-DENV IgM via DENV-2 antigen. (5) Introducing

) Luminous signal is developed and reflected into the optical fiber.

2 Actuators B 140 (2009) 206–215

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Fig. 3. Calibration curves. The curves were obtained by using serum dilutions rang-ing from 1:101 to 1:107. () Positive serum (panel point 6), () negative serum(panel point 0). (A) Colorimetric MAC-ELISA, (B) chemiluminescent MAC-ELISA and(C) chemiluminescent OFIS. The regression curves and the linear range were ana-lyzed by ORIGIN Lab version 6. The regression curves were calculated by applying the

10 D. Atias et al. / Sensors and

.9. Comparing the diagnostic tools using statistical analysis

The following statistical approaches were used to estimatehe diagnostic sensitivity and specificity: sensitivity = [Tp/(Tp + Fn)]

100, specificity = [Tn/(Tn + Fp)] × 100; where Tp represents therue positive sera, Fn the false negative sera, Tn the true negativeera, and Fp the false positive sera [61].

. Results and discussion

Three immunoassays were employed and compared for theetection of IgM antibodies against DENV in human sera samples:olorimetric MAC-ELISA, chemiluminescent MAC-ELISA and chemi-uminescent OFIS. The use of internal panel reference sera andamples from endemic areas enable a reliable comparison betweenifferent diagnostic methods.

.1. Calibration curves

The calibration curve describes the general behavior of theetecting system of anti-DENV IgM according to the MAC-ELISArotocol (Fig. 3). The curves were plotted using a set of dilutions ofhe analyte solution ranging from 1:101 to 1:107. For each method,ositive serum (panel point #6) and negative serum (panel point0) were tested. The curves were further used to determine the

mmunosensors essential parameters such as the dynamic range,ensitivity and the lower detection limit of the different procedures.

The regression model demonstrated a good correlation betweenhe serum dilutions and the reported signal, as shown by the2 values: r2 for the sigmoid graph was 0.99 for colorimetricnd chemiluminescent ELISA and 0.98 for OFIS. Evaluation of theetection limit describes the highest analyte dilution effectivelyetectable by the system. This limit is determined by the high-st dilution before the dilution where the control sample (panel0) average signal value with three times its standard deviation,xceeds the analyte sample average signal value. The detection limitor the colorimetric MAC-ELISA, chemiluminescent MAC-ELISA andhe OFIS was approximately 1:104, 1:105 and 1:106, respectively.he OFIS has a detection limit 10 times lower than the chemi-uminescent MAC-ELISA and 100 times lower than colorimetric

AC-ELISA. In each procedure, experimental points based on con-rol serum demonstrated a low, homogenous and linear basedignal.

The linear range parameter, also known as the working ranger dynamic range gives the titer range where the detection sys-em shows a proportional relation between the serum titer andhe measured signal. Clinical diagnostics usually require a serumilution that belongs to this range in order to guarantee the puta-ive detection of comparable amounts of target antibodies. Theynamic ranges are visualized in Fig. 3 in the top right corner ofheir corresponding curve. In this study, the dynamic ranges were:178–1:3160 for the colorimetric MAC-ELISA and 1:562–1:5620or the chemiluminescent MAC-ELISA and 1:102–1:106 for thehemiluminescent OFIS. The linear range parameter was betweenand 2 orders of magnitude for the colorimetric and chemilumi-

escent MAC-ELISA and 4 orders of magnitude for the OFIS. The r2

f the linear range was with good relation between the serum dilu-ions and the reported signal for all three methodologies and was.99 for colorimetric ELISA, 0.97 for chemiluminescent ELISA and.98 for chemiluminescent OFIS.

Analytical sensitivity of the tool is defined as the slope of theinear range. By extrapolation, it also represents the power to dis-riminate between two different concentrations A and B because its based on the ratio (YA − YB)/(XA − XB), where Y represents the sig-al measured and X is the serum dilution. The chemiluminescent

logistic sigmoid four-parameter curve regression model described by the followingequation: y = A2 + [A1 − A2/(1 + (X/X0)p)]. The linear range which are visualized in thetop right corner of each curve were calculated by applying a two parameter linearregression equation: y = b log(X) + a.

OFIS slope was higher (1255) relative to chemiluminescent ELISA

(473) and to colorimetric ELISA (1.18).

An additional advantage of the OFIS is the duration of the proce-dure, thus time to obtain results is shorter compared to MAC-ELISAwhen taking the analyte binding incubation as the first step of the

D. Atias et al. / Sensors and Actuators B 140 (2009) 206–215 211

Fig. 4. Screening of panel reference sera. A comparison between 4 diagnosticsmethod to reveal anti-DENV IgM antibodies from known panel using: (A) PanbiocMcp

ahtawpcM

tOtcTmis

3

asMOdt#t

Fig. 5. Signal-to-noise ratio of panel reference sera. Signal values (anti-DENV IgMantibodies) are divided by the noise values (control serum) in order to follow the

signal. The Panbio commercial MAC-ELISA kit showed a higherS/N ratio than the colorimetric MAC-ELISA and the chemilumines-cent methodologies, pointing out that the background signal has agreater influence in this procedure. It is understandable that the cal-ibration and development of a commercial kit with high S/N ratios

ommercial MAC-ELISA kit, (B) colorimetric MAC-ELISA kit, (C) chemiluminescentAC-ELISA, (D) chemiluminescent OFIS. *Significantly higher compared to negative

ontrol (p-value <0.05), using analysis of variance (ANOVA). Note that in all methods,anel 4 onwards had a p value <0.001 compared to the negative control.

ssay. The incubation time in chemiluminescent OFIS is shorter byalf compared to MAC-ELISA. Moreover when the MAC-ELISA pro-ocol is used, there is an overnight incubation after adding the DENVntigen, therefore the duration of the procedure is around 20 hhile in OFIS it is around 3 h. Even if, with in-house MAC-ELISArotocols, we replace the overnight incubation by 2 h in humidhambers at 37 C, OFIS remains shorter than the conventionalAC-ELISA protocols.From these results it can be claimed that the OFIS is more sensi-

ive for detection of anti-DENV IgM. Moreover, chemiluminescentFIS has the advantage of shorter duration of the procedure. The

wo established methodologies, colorimetric and chemilumines-ent MAC-ELISA, provided good reproducibility of measurements.he reproducibility of chemiluminescent OFIS, which is a newethod, is not as good as the MAC-ELISA. Factors such as the bind-

ng chemistry and improvements in consistency of the cleaved fiberurface may provide better consistency.

.2. Screening of panel reference sera

Another way to compare between different methods is to testpanel of reference sera. A panel of reference sera was therefore

creened using the Panbio commercial MAC-ELISA kit, colorimetricAC-ELISA, chemiluminescent MAC-ELISA and chemiluminescent

FIS, with 3–10 replicates per panel (Fig. 4). Since the panel sera wasiluted and stabilized by Bio-Rad Laboratories it is not known whathe exact dilution for each point was. It is known that panel point1 contains the lowest titer of anti-DENV-2 IgM, panel point #6

he highest and panel point #0 does not contain anti-DENV-2 IgM.

evolution of the background signal. We use ANOVA analysis to compute S/N ratio.The graph comparison between four diagnostics method to reveal anti-DENV IgMantibodies from known panel using: () Panbio commercial MAC-ELISA kit, () col-orimetric MAC-ELISA, (*) chemiluminescent MAC-ELISA and () chemiluminescentOFIS.

For each method, the results of panel points #1 to #6 were checkedto see if they were significantly positive compared to the negativecontrol (panel point #0), using analysis of variance (ANOVA), whichyielded a p-value (Fig. 4) and signal-to-noise ratio (S/N) for eachpanel point (Fig. 5).

Colorimetric MAC-ELISA, chemiluminescent MAC-ELISA andOFIS were significantly positive (p-value <0.05) from panel point #2and above, only the Panbio commercial MAC-ELISA kit was foundsignificant from panel point #1. S/N ratio was received by dividingthe signal values (anti-DENV IgM antibodies) by the noise values(control serum) in order to follow the evolution of the background

Fig. 6. Standardization of panel reference sera. The results from Fig. 3 were standard-ized using Partek genomics suiteTM. The original results for each method were shiftedto give a mean 0, and further scaled to have a standard deviation of 1. The graphcomparison between four diagnostics method to reveal anti-DENV IgM antibodiesfrom known panel using: () Panbio commercial MAC-ELISA kit (), colorimetricMAC-ELISA, (*) chemiluminescent MAC-ELISA and () chemiluminescent OFIS.

212 D. Atias et al. / Sensors and Actua

Table 1Summary of panel screening according to cutoff of each method. Cut-off value wasset according to the negative control (panel 0) and confidence range was adjusted tothe methods. For chemiluminescent OFIS and chemiluminescent ELISA: ±10%. Forcolorimetric ELISA, positive results were defined as higher than three times the valueof the negative control, and negative results as lower than twice that value.

Panel no. 0 1 2 3 4 5 6

Panbio Kit − − −/+ + + + +CCC

ifr

wm

fsporei

3

uettdM#p

pic#

TSwtO

(

C

P

N

E

(

SS

olorimetric ELISA − − − + + + +hemiluminescent ELISA − + + + + + +hemiluminescent OFIS − + + + + + +

s achieved with large investment by companies. Further researchocusing on the OFIS detection method may also yield improved S/Natios.

To compare the different methods on the same scale, the resultsere standardized such that the values from each method had aean of 0 and a standard deviation of 1 (Fig. 6).This was computed by subtracting the original method mean

rom each value, and by further dividing each value by the originaltandard deviation. Subsequently, Pearson’s correlation was com-uted between the standardized data of each method to that of eachf the other methods. All method pairs were shown to be highly cor-elated, with r > 0.97. The similarity between the methods is alsovident from the curves in Fig. 6, albeit some differences did existn panel points #3 and #4.

.3. Selection of cut off values and confidence range

The cut-off values of the colorimetric MAC-ELISA, commonlysed for the detection of anti-DENV IgM antibodies, are alreadystablished. The positive results were defined as higher than threeimes the value of the negative control, and negative results as lowerhan twice that value. Values obtained between these ranges wereefined as equivocal. The cut-off values of the Panbio commercialAC-ELISA kit are not published but they considered panel point0 as pure negative, panel point #1 as negative (very high dilution),anel point #2 as equivocal and panel points #3 to #6 as positive.

Since OFIS employs a new detection methodology and has notreviously been used for detection of anti-DENV IgM antibodies

t was necessary to set cut-off values and a confidence range. Theut-off value was set according to the negative control (panel point0) and empiric confidence range was defined as to ±10%. This cal-

able 2creening of human sera (A) summary of the screening results to reveal anti-DENV IgM anas set up according to the negative (panel 0) and confidence range was ±10%. The result w

hat found positive in chemiluminescent OFIS was similar to the one in chemiluminesceFIS and chemiluminescent ELISA. (B) The specificity and sensitivity of the methods.

A)

olorimetric ELISA DENV serotypes

ositive DENV-1DENV-2DENV-3

DENV-4

egative Negative

quivocal DENV-3

B)

Chemiluminescent ELISA

pecificity 95.6%ensitivity 100%

tors B 140 (2009) 206–215

culation was implemented for both chemiluminescent MAC-ELISAand chemiluminescent OFIS. Table 1 summarizes the result definingwhich panels are considered to be positive or negative accordingto cut-off and confidence range values. From that table it is clearthat chemiluminescent based methods are more sensitive. More-over, we have to take into account that for each method, there wasa different confidence range and that panel point #1 did not pro-duce a signal significant enough to be considered positive by thechemiluminescent methods. The reliability of this different confi-dence range for the chemiluminescent methods came from the factthat these methods react with the same pattern of high correlationto the colorimetric MAC-ELISA and Panbio commercial MAC-ELISAkit.

3.4. Screening of human sera

A total of 86 serum samples from the collection of the NRC havebeen tested for anti-DENV IgM antibodies (Table 2). The sera sam-ples were divided into three groups: positives to anti-DENV IgM,negatives, and equivocal samples, which were classified accordingto the colorimetric MAC-ELISA, the routine diagnostic tool in theNRC. The positive samples were from all four distinct serotypes(DENV-1, DENV-2, DENV-3 and DENV-4). Two new methods foranti-DENV IgM detection were used: chemiluminescent OFIS andchemiluminescent MAC-ELISA. The biological recognition entitywas based on colorimetric MAC-ELISA with the required modifi-cations for OFIS.

The group that was defined as positive (by colorimetricMAC-ELISA) was also found to be positive by chemiluminescentMAC-ELISA (Table 2A and B). Thus the diagnostic sensitivity of thismethod was 100%. In chemiluminescent OFIS, one sample from 53samples was found to be “False negative” which reduced the diag-nostic sensitivity to 98.1%. In the group that was defined as negative(by colorimetric MAC-ELISA), one sample out of 23 samples wasfound to be “False positive” in chemiluminescent MAC-ELISA whichreduced the diagnostic specificity to 95.6%. In chemiluminescentOFIS, three out of 23 samples were found to be “False positive”which reduced the diagnostic specificity to 87.0%. However one of

those three samples was also found to be positive in chemilumi-nescent MAC-ELISA. In the group that was defined as equivocal (bycolorimetric MAC-ELISA), all of the samples except for one, werepositive. This sample was found to be equivocal in chemilumines-cent MAC-ELISA and chemiluminescent OFIS.

tibodies using chemiluminescent OFIS and chemiluminescent ELISA. Cut-off valueas divided to 3: (−) negatives (+) positives and (−/+) equivocal. *One of the samples

nt ELISA. **The same sample has been found to be equivocal in chemiluminescent

Chemiluminescent ELISA Chemiluminescent OFIS

5/5+ 5/5+29/29+ 31/31+13/13+ 12/13+

1/13−No samples were checked 4/4+

22/23− 19/23−*1/23+ *3/23+

1/23 (−/+)

8/9+ 9/10+**1/9 (−/+) **1/10 (−/+)

Chemiluminescent OFIS

87.0%98.1%

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D. Atias et al. / Sensors and

Although colorimetric MAC-ELISA is the routine way to detectnti-DENV IgM antibodies, it is not considered a gold standard. Thushere is the possibility that “false positives” are “true positives”sing a more sensitive method. To support this claim it can be seenhat one of the “false positives” was found to be positive by bothhemiluminescent ELISA and chemiluminescent OFIS.

When affected with a dengue-like syndrome the patient willome to the outpatient clinic 3–4 days after the onset of fever. Athis point, the patient will have a low titer for anti-DENV IgM pri-

arily in secondary DV-infection, and a test could show negativeENV genome detection by RT-PCR and/or negative anti-DENV IgMetection by colorimetric MAC-ELISA. Using more sensitive meth-ds may detect such cases. Since it has been shown earlier that theetection limit of chemiluminescent OFIS and chemiluminescentAC-ELISA are lower than colorimetric MAC-ELISA, they may reveal

hat these “false positives” are indeed “true positives” with perhapslow titer of anti-DENV IgM. However, to increase the specificityf the antibody detection, technology based on chemiluminescentFIS might be improved in the future with recombinant proteinserive from the glycoprotein E.

. Conclusions

The results presented in this study demonstrate that a sen-itive and rapid semi-quantitative chemiluminescent OFIS cane used for the detection of anti-DENV IgM in human serumamples. In comparison to standard colorimetric MAC-ELISA pro-edures, it has been revealed that: (1) the chemiluminescent OFISas a lower detection limit than ELISA methodologies; (2) col-rimetric and chemiluminescent MAC-ELISA are reliable systemsor high and intermediate levels of serum containing anti-DENVgM, whereas chemiluminescent MAC-ELISA has a lower detec-ion limit; (3) the chemiluminescent OFIS has a reliable workingange at very low IgM concentrations with an analytical sensitivitynabling a semi-quantitative test, (4) chemiluminescent proce-ures are at least 10–100 times more sensitive than the respectiveolorimetric test enabling detection of very low analyte concentra-ions, (5) chemiluminescent OFIS is advantageous over colorimetricnd chemiluminescent MAC-ELISA, since it is much more rapid.6) However, the two established methodologies – colorimetricnd chemiluminescent MAC-ELISA – have better reproducibili-ies. These results support previous studies that demonstrated thedvantages of using chemiluminescent methods, especially OFIS,s a useful diagnostic tool for viral detection [50,52]. Although theiagnostic specificity of the chemiluminescent OFIS was 87.0%, itid have a high diagnostic sensitivity (98.1%), lower detection limitnd a very high correlation between the established methods withknown panel. Moreover OFIS is rapid, cost effective, simple to

erform and has the potential to work under field conditions pro-iding a reliable option for anti-DENV IgM detection in human seraamples.

In this study we employed DENV Ags that were extractedrom suckling mouse brains using a sucrose–acetone technique. Tomprove the specificity of chemiluminescent OFIS for anti-DENVgM detection, it could be very useful to develop a new proto-ol using recombinant protein (from envelop, membrane or NS1)hat was previously tested with colorimetric MAC-ELISA for dengueiagnosis or even for seroepidemiological studies [31,40,41]. Finally,onsidering the existence of pauci- or asymptomatic forms of theengue disease, technologies such chemiluminescent OFIS, withigh sensitivity, may be very useful for diagnosis.

cknowledgments

This research was funded by grant from the European Commis-ion (DENFRAME contract no. FP6-2003-INCO-DEV2-517711). We

[

tors B 140 (2009) 206–215 213

would like to thank Khalil Abu-Rabeah and Sebastien Herrmannfrom the Department of Biotechnology Engineering, Ben-GurionUniversity, Israel for their help in this work, and Marc Tabouret fromBio-Rad Laboratories, for the stabilization of the internal panel ofreference sera used in this study. We would also like to thank GinaHuges for her expert editorial help.

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44] C.F.R. Mateus, M.C.Y. Huang, C.J. Chang-Hasnain, J.E. Foley, R. Beatty, P. Li,B.T. Cunningham, Ultra-sensitive immunoassay using VCSEL detection system,Electron. Lett. 40 (11) (2004).

45] M.C.Y. Huang, Mateus, F.R. Carlos, Foley, E. Jonathan, Beatty, Robert, Cun-

ningham, T. Brian, Chang-Hasnain, J. Connie, VCSEL optoelectronic biosensorfor detection of infectious diseases, IEEE Photon. Technol. Lett. 20 (6)(2008).

46] A.J. Baeumner, Schlesinger, A. Nicole, Slutzki, S. Naomi, J. Romano, E. Mi Lee,Richard A. Montagna, Biosensor for dengue virus detection: sensitive, rapid,and serotype specific, Anal. Chem. 74 (2002) 142–1448.

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48] C. Dodeigne, L. Thunus, R. Lejeune, Chemiluminescence as diagnostic tool. Areview. Talanta 51 (3) (2000), 415–439.

49] R.S. Marks, E. Bassis, A. Bychenko, M.M. Levine, Chemiluminescent opticalfiber immunosensor for detecting cholera antitoxin, Opt. Eng. 36 (12) (1997)3258–3264.

50] S. Herrmann, B. Leshem, S. Landes, B. Rager-Zisman, R.S. Marks, Chemilumines-cent optical fiber immunosensor for the detection of anti-West Nile virus IgG,Talanta 66 (1) (2005) 6–14.

51] O. Salama, S. Herrmann, A. Tziknovsky, B. Piura, M. Meirovich, I. Trakht, B.Reed, L.I. Lobel, R.S. Marks, Chemiluminescent optical fiber immunosensor fordetection of autoantibodies to ovarian and breast cancer-associated antigens,Biosens. Bioelectron. 22 (7) (2007) 1508–1516.

52] A. Sobarzo, J.T. Paweska, S. Herrmann, T. Amir, R.S. Marks, L. Lobel, Optical fiberimmunosensor for the detection of IgG antibody to Rift Valley fever virus inhumans, J. Virol. Methods 146 (1–2) (2007) 327–334.

53] B. Leshem, G. Sarfati, A. Novoa, I. Breslav, R.S. Marks, Photochemical attach-ment of biomolecules onto fibre-optics for construction of a chemiluminescentimmunosensor, Luminescence 19 (2) (2004) 69–77.

54] T. Konry, A. Novoa, Y. Shemer-Avni, N. Hanuka, S. Cosnier, A. Lepellec, R.S.Marks, Optical fiber immunosensor based on a poly(pyrrole-benzophenone)film for the detection of antibodies to viral antigen, Anal. Chem. 77 (6) (2005)1771–1779.

55] A. Petrosova, T. Konry, S. Cosnier, I. Trakht, J. Lutwama, E. Rwaguma,A. Chepurnov, E. Muhlberger, L. Lobel, R.S. Marks, Development of ahighly sensitive, field operable biosensor for serological studies of Ebolavirus in central Africa, Sensors Actuat. B: Chem. 122 (2) (2007) 578–586.

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Biographies

Atias Danit received her B.Med.L.Sc. degree (2002) fromthe School of Medical Laboratory Science, Faculty of HealthSciences Ben Gurion University of the Negev, Israel. Shereceived her M.Med.Sc. degree (2005), with academicdistinction, from the Department of Microbiology andImmunology, Faculty of Health Sciences Ben Gurion Uni-versity of the Negev, Israel. Now she is studying her PhDin the Department of Virology, Faculty of Health Sciencesand the Department of Biotechnology Engineering, Fac-ulty of Engineering Ben Gurion University of the Negev,Israel. Her research work is concerned with lumines-cence based biosensors for dengue diagnostics. Her work

includes molecular biology, optical immuno-biosensors, phage display, immuno-virology techniques.

Yael Liebes received her B.Sc. in Biotechnology Engineer-ing (2006) and M.Sc. in Environmental Engineering (2008)

versity of Negev and her research is concentrating on solidstate nanopores for stochastic sensing of proteins.

Actua

I

the Institut Pasteur in Paris, France. He has worked as avirologist at the laboratory from the Institut Pasteur de laGuyane since 2002, where he is now in charge of the CentreNational de Réference des Arbovirus et Virus Influenza régionAntilles Guyane. His research interests include diagnosisand epidemiology of dengue virus and other arboviruses.

D. Atias et al. / Sensors and

Vered Chalifa-Caspi received her B.Sc. degree from theFaculty of Life Sciences at the Hebrew University ofJerusalem, Israel. She then continued for M.Sc. and PhDstudies at the Department of Biological Regulation at theWeizmann Institute of Science, Rehovot, Israel, where shegraduated on 1996. During her post-doc and subsequentlyas a research associate at the Department of MolecularGenetics and the Bioinformatics Unit of the WeizmannInstitute of Science she was one of the founders and devel-opers of the GeneCards® database. In 2003 she moved toBen-Gurion University to establish and head the Bioin-formatics Core Facility, which is now part of the National

nstitute of Biotechnology in the Negev.

Laëtitia Bremand has worked as a laboratory technicianat the laboratory of virology since 2006. She is in chargeof the molecular diagnosis of dengue and is also involvedin the serological diagnosis of arboviruses.

Leslie Lobel is a Senior Lecturer in the Department ofVirology & Developmental Biology at Ben Gurion Univer-sity. He earned his B.A., Summa Cum Laude from ColumbiaCollege of Columbia University in Chemistry and enteredthe Medical Scientist Training Program at the College ofPhysicians and Surgeons of Columbia University earning

the M.D.-Ph.D. degrees in 1988. His doctoral work was inRetrovirology under Stephen Goff at Columbia University.After postdoctoral work in developmental biology on C.elegans in the laboratory of H. Robert Horvitz at M.I.T.,he returned to the Department of Medicine at ColumbiaUniversity before moving to the Department of Virology

tors B 140 (2009) 206–215 215

at Ben Gurion University. He set up a laboratory of immunovirology and cancerimmunology at BGU in 2003. His work focuses in part on the isolation of totallyhuman monoclonal antibodies to a variety of viral diseases that currently lack effec-tive treatment, such as Hepatitis C and avian influenza as well as RSV, ebola and RiftValley Fever. In addition, the laboratory is studying the humoral immune responseto cancer and to this end is isolating totally human monoclonal antibodies that arecancer specific from cancer patients and healthy adults.

Robert S. Marks received his B.Sc. in biological sciencesand his M.Sc. in cell biology and physiology from Univer-sity of California, Santa Barbara, USA. He then completedhis PhD in chemical immunology at the Weizmann Insti-tute of Science, Rehovot, Israel, in 1992 on an enhancedmucosal vaccination method using silica microparticlescoated with synthetic peptides. From 1992 to 1994, he wasa research associate at the University of Cambridge, UK,designing a fluorescent adiabatically tapered optical fiberimmunosensor. In 1995 he moved to the Ben-Gurion Uni-versity of the Negev, where he is now a tenured AssociateProfessor and head of a Biosensor Group with projects on

bioluminescence whole-cell bioreporter biosensors, chemiluminescence fiber opticbiosensors, amperometric biosensors, biomaterials, enzyme nanolithography andother projects.

Philippe Dussart received his Pharm D from University ofChâtenay-Malabry – Paris XI – France, in 2001. He gradu-ated in 2002 from the course of Systematic Virology from

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Biosensors and Bioelectronics 24 (2009) 3683–3687

Contents lists available at ScienceDirect

Biosensors and Bioelectronics

journa l homepage: www.e lsev ier .com/ locate /b ios

hort communication

oly(methyl metacrylate) conductive fiber optic transducers as dualiosensor platforms

anit Atias a,b,1, Khalil Abu-Rabeah b,1, Sebastien Herrmann b, Julia Frenkel c,orith Tavor c, Serge Cosnier d, Robert S. Marks b,e,f,∗

Department of Virology, Ben-Gurion University of the Negev, P.O. Box 653, Beer-Sheva 84105, IsraelDepartment of Biotechnology Engineering, Ben-Gurion University of the Negev, P.O. Box 653, Beer-Sheva 84105, IsraelDepartment of Chemical Engineering, Sami Shamoon College of Engineering Bialk/Basel Sts., Beer Sheva 84100 IsraelDépartement de chimie moléculaire, UMR CNRS 5250, Institut de Chimie Moléculaire de Grenoble, FR CNRS 2607, Université Joseph Fourier Grenoble I,P 53, 38041 Grenoble, Cedex 9, Grenoble, FranceNational Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, P.O. Box 653, Beer-Sheva 84105, IsraelILSE Katz Center for Meso and Nanoscale Science and Technology, P.O. Box 653, Beer Sheva 84105, Israel

r t i c l e i n f o

rticle history:eceived 22 January 2009eceived in revised form 5 April 2009ccepted 23 April 2009vailable online 3 May 2009

a b s t r a c t

Herein the development of an alternative optic-conductive fiber configuration applied for the construc-tion of biosensing platforms. This new approach is based on applying the chemical polymerization ofpyrrole onto the surface of polymethyl metacrylate (PMMA) fibers to create a polymer—a conductive sur-face, onto which an additional photoactive polypyrrole-benzophenone (PpyBz) film is electrochemicallygenerated upon the fiber surface. Irradiation of the benzophenone groups embedded in the Ppy films with

eywords:oly(methyl metacrylate)iosensoryrrole-benzophenoneptic fiberyrrole

UV radiation (350 nm) formed active radicals that allowed the covalent attachment of the desired biore-ceptors. Characterization of the amperometric biosensing matrix was accomplished by using a modelUrease (Urs) through electrochemical impedance spectroscopy (EIS) and amperometry. Both techniqueshave shown a low charge transfer resistance (340 k) and a high sensitivity (12.3 A mM−1 cm−2). There-after, the construction of an optical biosensing matrix based on horseradish peroxidase (HRP) productionof photons was carried out. The high signal to noise (S/N) ratio (1600) indicated clearly that this approach

rm to

can serve as a new platfo

. Introduction

Fiber optic biosensors are diagnostic tools with specialized fea-ures (Marks et al., 1997) that may be utilized in a variety ofelds (medical, pharmaceutical, environmental, defense, biopro-essing or food). The fiber optic device serves as the transductionlement and the transmitted signal is often proportional to theoncentration of a chemical or biochemical to which the biologicallement reacts (Bosch et al., 2007; Leung et al., 2007; Marazuela andoreno-Bondi, 2002; Wolfbeis, 2004). Therefore it may be used in

variety of spectroscopic techniques, such as chemiluminescence,

bsorption, fluorescence, phosphorescence or surface plasmon res-nance (SPR) (Bosch et al., 2007). Silica-based fiber optic biosensorsave been published as serving a platform for the immobiliza-

∗ Corresponding author at: Department of Biotechnology Engineering,en-Gurion University of the Negev, P.O. Box 653, Beer-Sheva 84105, Israel.el.: +972 8 6477182; fax: +972 8 6472857.

E-mail address: rsmarks@bgu.ac.il (R.S. Marks).1 Both authors contributed equally to this work.

956-5663/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.bios.2009.04.035

replace glass optical fibers based on biosensors.© 2009 Elsevier B.V. All rights reserved.

tion of various biomaterials (enzymes, antibodies, antigen or wholecells) (Herrmann et al., 2005; Konry et al., 2005; Leshem et al.,2004; Petrosova et al., 2006; Salama et al., 2007; Sobarzo etal., 2007). We have demonstrated that chemiluminescence-basedoptical fiber immunosensors (OFIS) are more sensitive than theiranalogous colorimetric and chemiluminescent-based ELISA coun-terparts (Herrmann et al., 2005; Konry et al., 2005; Leshem et al.,2004; Petrosova et al., 2006; Salama et al., 2007; Sobarzo et al.,2007). However, silica optic fiber chemical modifications are stillunreliable fiber-to-fiber, accordingly alternative configuration sce-narios would be useful to improve the efficiency and reproducibilityfor putative mass production of optic fiber probes. Furthermore,optic-conductive biosensors would open the door to a numberof wide applications based on a platform that will include bothconductivity and light transmission. The alternative fiber optic plat-form which we tested, was Ppy-coated. PMMA optical fibers display

very good optical properties while also proving to be inexpensiveand easy to prepare and also easier to cleave in comparison to morecommonly used silica-based fibers. The PMMA fiber surface showsa hydrophobic and favorable environment for protein adsorption.However, in order to achieve a more stable bio-conjugate linking,

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684 D. Atias et al. / Biosensors and

hemical modification procedures would need to be implementedo conjugate the surfaces; which in turn would necessitate organicolvents that could damage the fiber surface. PMMA particles werehown to be coated by pyrrole polymerization in an aqueous dis-ersion medium (Ferenets and Harlin, 2007; Omastova et al., 1998;mastova and Simon, 2000). This deposition process results in a

hin, transparent, conductive and stable Ppy film (Ferenets andarlin, 2007; Fredj et al., 2008; Wang et al., 2001).

This procedure was adapted to coat PMMA fibers with pyrroleolymerized chemically at low temperature, which then display

ncreased hydrophilicity when compared to naked PMMA fibers.he additional step to attach the biomaterial components wasccomplished through electropolymerization of an intermediateunctionalized pyrrole (biotin, amine or benzophenone) and ofhese three tested, the finest results were obtained using benzophe-one as it was best suited to the required parameters (conductive,table, thin, controllable and light-transmissible films). The elec-ropolymerization of the PyBz monomer was successfully achievedn an aqueous phase and the observed films showed that the colorf the Ppy films varied from light grey to black indicating a sta-le and typical Ppy film. Irradiation of the benzophenone groupsmbedded in the Ppy films using UV radiation (350 nm) activatedhe benzophenone groups and allowed the attachment of proteina-eous enzymes (Urs or HRP) to the fiber surface. The subsequenteasurements showed high sensitivity (12.3 A mM−1 cm−2) and

igh S/N ratio (1600) when compared to unmodified PMMA fiber,espectively.

. Materials and methods

.1. Reagents and materials

Tris–HCl (77-861); pyrrole (13170-9); lithium perchlorate431567); Urs (9002-13-5); urea (57-13-6); ammonium persulfate8% (APS) (248614); p-toluene sulphonic acid (PTSA) (402885);,1′-ferrocenedicarboxylic acid, 96% (FeCN) (215-068-9); isopropyllcohol (IPA) (67-63-0); HRP (P8375) were purchased fromigma–Aldrich (St. Louis, USA) and used as received. PMMA fibers3 mm diameter unjacketed, NT53-833), mirror and a professionalber cutter (N54-013) were purchased from Edmund optics (San

ose, USA). Luminescence measurements were carried out usinghe Immuno-star HRP Chemiluminescent Kit (170-5040) purchasedrom Bio-Rad Laboratories. PyBz was synthesized as described else-

here (Cosnier and Senillou, 2003).

.2. Preparation of the PMMA optic-conductive fiber biosensor

.2.1. Chemical polymerization of pyrrole onto the PMMAber-optic endface

PMMA optical fibers (3 mm core diameter) were cleaved man-ally using a professional fiber cutter with a length of 2.6 cm. Thebers were first soaked in isopropyl alcohol for 5 min, followed byacuum desiccation for 5 min and finally an additional 5 min ofipping in double distilled water (ddH2O). The fibers were driedith airflow through a 45 m filter, after which they were fur-

her dried in an oven at 47 C for 20 min and underwent vacuumesiccation for 5 min. The clean fibers were dipped into an 1.5 mlube, containing a mixture 1:1:1 (v:v:v) of the following cold solu-ions: 25 mmol APS, 25 mmol PTSA and 7.5 mmol pyrrole; to allow

he chemical polymerization of pyrrole monomers. The fibers wereipped into the eppendorf immediately after introducing the pyr-ole. The 1.5 ml tube was mixed by vortex and transferred to ance bath (5 C) (Ferenets and Harlin, 2007) for 4.5 min, after whichhe fibers were moved into ddH2O and gently vortexed, rinsed indH2O and dried by airflow.

ctronics 24 (2009) 3683–3687

2.2.2. Electrochemical polymerization of PyBz onto thePpy-coated PMMA fiber-optics

PMMA fibers coated with Ppy films were used as working elec-trodes to enable the electrogeneration of an additional conductiveand functional layer consisting of PyBz monomers so as to elec-tropolymerize PyBz onto fiber-optic tips. This new layer wouldthen enable the subsequent immobilization of the desired recep-tor protein. The electropolymerization was processed by applying aconstant voltage of 0.85 V lasting 10 min. The electropolymerizationsolution used was prepared by mixing 10 mM PyBz free monomerwithin a 0.1 M of lithium perchlorate aqueous solution.

2.2.3. Immobilization of bioreceptors onto the transducer surfaceThe enzymes Urs or HRP (concentration 0.5 mg/ml) were linked

to the PpyBz film through UV −350 nm irradiation (Fig. 1). In orderto produce the desired activation radiation we used a 1000 W Xelamp (Oriel 6271) mount connected to a light condenser (Oriel66021). The light was reflected through a dichroic mirror (Oriel66226). The spectrum radiation was then condensed into themonochromator (Oriel 77250) using the appropriate lenses (Oriel,plano convex lenses). After this, a 350 nm wavelength light output,with a light intensity of 100 mW cm−2 (as measured by an OphirOptronics power-meter Nova reader, PD300-UV) was projected for7 min (which was the established optimal time in our lab) into thefar end of the optical fiber that was coated at its near end with PpyBzand soaked in 0.5 mg/ml of HRP solution during irradiation (Konryet al., 2008; Leshem et al., 2004; Petrosova et al., 2006). The irradi-ation of the PyBz film created radicals enabling the attachment ofthe enzymes covalently. The fibers were then washed with ddH2Oto remove unattached proteins.

2.3. X-ray photoelectron spectroscopy (XPS) analysis and contactangle measurements

An ESCALAB system with parallel X-ray photoelectron imaging(XPI) analysis of small features at a resolution approaching 1 mmwas used. The ESCALAB 250 combines the auger electron spec-troscopy (AES) and scanning auger electron mapping (SAM). Thevacuum in the operation chamber was 2 × 10−9 mbar. XPS analy-sis was performed using a monochromatic Alk- X-ray source witha 500 m surface of analysis and X-ray gun set at 15 kV, 150 W.High resolution spectra of the elements were taken with a passenergy at 15 eV, and a peak position normalized according to C1s(284.6 eV). Static contact angle measurements were taken at 20 Cwith a sessile drop method using an OCA 40 system (Dataphysics,German).

2.4. Electrochemical instrumentation

The amperometric and impedance measurements, as well as theelectropolymerization, were carried out using a PGSTAT30 poten-tiostat and a conventional electrochemical cell (Metrohm). Themodified fibers were used as the working electrodes; a saturatedAg–AgCl–KCl electrode (Ag/AgCl) was used as a reference electrode.A platinum (Pt) wire, placed in a separate compartment containingan aqueous solution of 0.1 M Tris–HCl buffer electrolyte (pH 7), wasused as a counter electrode for the amperometric measurements;while in the electropolymerization procedure, a Pt wire was used asa counter electrode and immersed directly in a 0.1 M LiClO4 aqueoussolution. EIS faradic measurements were conducted in a solutioncontaining 2 mM FeCN, in which the electrode set up was the same

as that used in the electrochemical deposition. An alternating cur-rent sinusoidal signal of (10 mV) amplitude was used and the directpotential was set to 0.7 V. The value of the impedance was deter-mined over a range 10–105 Hz, and the impedance measurementswere performed with a PGSTAT30 electrochemical workstation. The

D. Atias et al. / Biosensors and Bioelectronics 24 (2009) 3683–3687 3685

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ig. 1. Amperometric and optical biosensor constructions, (1) Modification of PMMAf PyBz, (3) irradiation of benzophenone moieties by UV and attachment of the enzf urea concentration in the amperometric biosensor case (A) and light recording inhe wells in a 1:1 ratio.

apacitance values and resulting data modelled were calculated andatched with the fit and stimulation software integrated in the FRA

oftware (Eco Chemie B.V. Utrecht Netherlands).

.5. HRP chemiluminescence measurement

Chemiluminescence measurements were performed by apply-ng hydrogen peroxide and luminol into fiber holding wells in a:1 ratio. Measurements were carried out using a single photonvalanche diode (SPAD) photodetector based on a SPAD mod-le purchased from SensL (PCMplus module, 10 m sensor). Dataas collected using SensL Integrated Environment software and

ecorded in RLU (relative light units).

. Results and discussion

Steric hindrance forces formed by benzophenone functionalroups limited the chemical polymerization of PyBz onto the nakedMMA fiber surfaces. To overcome this obstacle a chemical poly-erization of pyrrole was carried out to form a conductive surface,hich enabled the subsequent electrochemical polymerization of

yBz with the ultimate target of an efficient immobilization ofeceptor proteins. Polymerization time was set to 4.5 min, whileonger polymerization periods resulted in rather unstable quality

ith non-uniform films. These samples required additional clean-ng in order to remove the loosely fixed film layers.

.1. X-ray photoelectron spectroscopy (XPS)

The shift on binding energy for samples in the O1s region fromMMA (532 eV) to PMMA-Ppy (531 eV) was attributed to the inten-ity increase of the O1s featuring the polymerization of pyrrole

s surface by chemical polymerization of pyrrole, (2) electrochemical polymerizationrs or HRP to the benzophenone groups, (4) monitoring of the current as a function

hemiluminescence’s biosensor (B) when adding oxidizing reagent and luminol into

molecules on the PMMA surface. This was further supported bythe observation that the O1s intensity increased substantially as aresult of PyBz electropolymerization on the PMMA-Ppy which cor-related directly to the binding energy shift to higher values as theelectropolymerization tightened the thin polymeric film structure(C/O increases relative to PMMA-Ppy).

3.2. Surface hydrophilicity

To gain a better understanding of the modification, the surfacehydrophilicity was characterized by measuring its contact anglewhich for PMMA was 78.4, very similar to the values recordedin the literature (83) (Bull et al., 2006). The PMMA-Ppy fiber hasdisplayed a better contact angle (48) due to a very thin film ofPpy coating and the existence of amine groups on the fiber surface.The contact angle was further decreased in the PMMA-Ppy-PpyBzcase showing a better hydrophlicity (18), when compared to nakedPMMA, which directly correlates to the large amount of hydroxylgroups on the PpyBz film. The resulting hydrophilic surface pro-vided an excellent microenvironment for enzyme immobilization.

The morphology of the surface was investigated by scanningelectron microscopy (SEM). The SEM study of Ppy films demon-strated that the surface was covered with grains (diameter ofapproximately 10–100 nm). The body of the film was approximatelyhundred nanometers thick. The PpyBz films created a smooth layerupon the Ppy films.

3.3. Electrochemical impedance analysis

Fig. 2 presents the imaginary part versus the real part of themodified fiber impedance which shows typical characteristics ofconducting polymers. The high frequency semicircular regions in

3686 D. Atias et al. / Biosensors and Bioele

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ig. 2. Equivalent electrical circuit of EIS data and Nyquist plot for (A) PMMA-PpyB) PMMA-Ppy-PpyBz (C) PMMA-Ppy-PpyBz-Urs in 0.1 M Tris–HCl (pH 7).

he PMMA-Ppy case showed good conductivity (when compared tohe non conductive PMMA), with a radius of curvature equivalento 500 k, indicating a moderate electrical resistance of the chargeransfer, while the value of the radius in the PMMA-Ppy-PpyBzas approximately 3 times lower (160 k), due to the additional

ayer of PpyBz generated on the PMMA-Ppy fiber surface whichurther decreased the electrical charge transfer resistance. In theMMA-Ppy-PyBz-Urs case the charge transfer resistance increasedo higher values as the protein on the surface contributed to theecreasing values of the analyte diffusion, hence increasing theharge resistance accordingly to 400 k. In order to characterizehe response of the monolayer Urs-modified sensor, the apparent

ichaelis–Menten constant Kappm can be calculated for the immobi-

ized enzyme by the amperometric method (Shu and Wilson, 1976),here J is the steady state current density, Jmax is the maximum

urrent density under conditions of enzyme saturation, and C ishe urea concentration. Kapp

m calculated from the Lineweaver–Burk/J vs. 1/C plot is as the relation with a slope of Kapp

m /Jmax and anntercept of 1/Jmax.

/J = Kappm /(JmaxC−1) + 1/Jmax (1)

ypical Kappm and Jmax values were calculated as 1.1 mM and

0 A cm−2, respectively. These values were comparable with thosereviously reported for the immobilization of Urs in electrochemi-ally prepared Ppy films (Gambhir et al., 2001) and indicate a stericindrance toward the diffusion of the products of the enzyme reac-ion toward the fiber surface as shown in Fig. 3 (due to the existencef the bulky enzyme on the surface). The capacitance values ofMMA-Ppy-PpyBz and PMMA-Ppy fibers modelled and fitted byandles circuit were 2 × 10−7 F and 1 × 10−6 F, respectively. Thesealues indicate that the PMMA-Ppy-PyBz set up provides a betteronductivity than PMMA-Ppy alone.

.4. Amperometric analysis

In the optical PMMA fiber surface modification process pre-ented in Fig. 1, pyrrole was polymerized chemically on the fiberurface to create a conductive surface, and then PpyBz films werelectropolymerized by applying a voltage at 0.85 V. Benzophe-one and most of its derivatives absorb photons at approximately50 nm, resulting in the promotion of one electron from a non-onding sp2-like n-orbital of oxygen to an antibonding *-orbital

f the carbonyl group. The actual electron deficient oxygen n-orbitalecomes electrophilic and therefore interacts with weak C–H -onds, resulting in hydrogen abstraction to complete the half-filled-orbital. When amines or similar heteroatoms are in the vicinity ofhe exited carbonyl, an electron transfer step may occur, followed

ctronics 24 (2009) 3683–3687

by proton abstraction from an adjacent group. In biological sys-tems the most effective H-donors include backbone C–H bonds inamino acids; thus, methylene groups of amino acid side chains aregood candidates providing abstractable hydrogens (Leshem et al.,2004). This enables the enzyme attachment and the catalytic reac-tion of the analyte that could be detected by either electrochemicalor optical means. The analytical performance of such a platform wasdemonstrated by constructing an amperometric biosensor for ureamonitoring. The detection principle of urea is based on the carbamicacid oxidation. As shown in Fig. 1, Urs catalyzed the hydrolysis ofurea to form intermediate products (carbamic acid and ammonia).The electrolysis was conducted by applying a 1.1 V to oxidize thecarbamic acid (three electron oxidation) (Wang et al., 2007) whichcreated a current that correlated to the urea concentration.

The PMMA-Ppy-PpyBz-Urs fiber in Fig. 3A exhibits a better per-formance than those of the controls PMMA-Ppy-PpyBz-Urs withoutUV irradiation (Fig. 3B), and PMMA-Ppy-Urs without PyBz linker(Fig. 3C). The biosensor PMMA-Ppy-PyBz-Urs performance indi-cates that the irradiation of the PyBz did activate the benzophenoneand did attach the enzyme Urs specifically. Furthermore, the sen-sitivity of the PMMA-Ppy-PyBz-Urs (12.3 A mM−1 cm−2) probewas 4 times higher than the set up without having UV radia-tion treatment (2.3 A mM−1 cm−2), indicating that the enzymewas attached covalently to the benzophenone group althoughsome may bind non-specifically. The biosensor limit of detec-tion, dynamic range, response time and sensitivity were 0.5 mM,0–10 mM, 2 s and 12.3 A mM−1 cm−2, respectively. These valuesindicate significant improvement compared to the values reportedin the literature (limit of detection 0.2 mM, dynamic range 0–5 mM,response time 40 s and 0.47 A mM−1 cm−2 sensitivity) (Rajesh etal., 2005). Hence this biosensor presents rapid, sensitive and effi-cient analytical performance.

3.5. Light measurements and calibration curves

During the chemiluminescent reaction, the light is emitted in alldirections (360 C) from the solution just above the fiber interface.Thus a certain amount of photons are not directed into the fiberand are lost into the measuring chamber. In order to decrease thelight loss and improve the sensitivity of our system, an elliptic mir-ror was placed above the well containing the chemiluminescentreaction and was calibrated. For this purpose, 10 l of a 1 mg/mlHRP solution diluted by 1:100 was mixed with 30 l of the chemi-luminescent substrate into the reaction well. The light level wasfirst measured without the reflecting system (control) and thenthe mirror was placed above the fiber at a distance ranging from4 to 10 cm. The mirror was finally situated 9 cm above the fiberbased on the measurements displayed in Fig. 3H. Consequently, aserial dilution of HRP was used to examine the PMMA modifiedfiber surface as a platform for light transmission. A chemilumines-cence mix was added to the well and the signal recorded as relativelight units (RLU). Eight HRP dilutions were tested in triplicate inthe biosensor (102–106). The results are displayed in Fig. 3G. A lin-ear distribution can be observed in the optical system down to adilution of 1:1,000,000 of the HRP stock solution (1 mg/ml) with astrong correlation factor (r2 = 0.99).

The ultimate target of this research was to demonstrate theapplicability of a platform that would enable the attachment ofbiomolecules to a fiber surface so as to transmit light efficiently. Thiswas demonstrated by the attachment of the enzyme HRP (Fig. 1)to the fiber surface, introducing hydrogen peroxide and luminol

reagents into the measurement cell in a 1:1 ratio and collecting thelight by a single photon avalanche diode photo-detector (Ashkenaziet al., 2009). The observed values for the sample (Fig. 3D) PMMA-Ppy-PyBz-HRP have displayed very sensitive results (1600 S/N)obtained on a small integrated optical system, when compared with

D. Atias et al. / Biosensors and Bioelectronics 24 (2009) 3683–3687 3687

Fig. 3. Calibration curves amperometric measurements corresponding to (A) PMMA-Ppy-PyBz-Urs/UV, (B) PMMA-Ppy-PpyBz-Urs, (C) PMMA-Ppy-Urs/UV modified PMMAfiber as a function of urea concentration ranging from 0 to 60 mM. E = 1.1 V vs. Ag/AgCl. Light measurements corresponding to (D) PMMA-Ppy-PyBz-HRP/UV, (E) PMMA-Ppy-PyBz-HRP, (F) PMMA-Ppy-HRP/UV modified PMMA fiber. Chemiluminescence measurements were performed by adding oxidizing reagent and luminol into the wells in a1 ilutiono a good . Thep

tisu

4

nboossPe

A

UsF

R

AB

:1 ratio, (G) HRP calibration in the PMMA/SPAD reader configuration. Eight HRP dbserved in the optical system until a dilution of 1:1,000,000 of the stock HRP withistance ranging from 4 to 14 cm in order to improve the light collection efficiencylaced in the mirror point (9 cm).

he same set up without UV radiation treatment (11 S/N), therebyndicating that the enzyme was linked specifically to the transducerurface which was further supported by the value of the same setp but without PyBz (16 S/N).

. Conclusions

In this study we have demonstrated the development of an alter-ative fiber-optic configuration designated for the construction ofiosensing platforms. This new approach was developed and basedn the chemical polymerization of pyrrole applied onto the surfacef PMMA fibers to create a conductive surface, which enabled theubsequent electrogeneration of photoactive PpyBz upon the fiberurface. Irradiation of the benzophenone groups embedded on thepy films using UV radiation (350 nm) enabled the attachment ofnzymes based sensing matrices.

cknowledgement

The authors gratefully acknowledge Dr. Boris Polyak from Drexelniversity, College of Medicine, USA, for his valuable assistance andcientific discussions of the results in the frame of this work androumin Natalya for the XPS measurements.

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10384 DOI: 10.1021/la901174p Langmuir 2009, 25(17), 10384–10389Published on Web 06/11/2009

pubs.acs.org/Langmuir

© 2009 American Chemical Society

Characterization of Electrogenerated Polypyrrole-Benzophenone Films

Coated on Poly(pyrrole-methyl metacrylate) Optic-Conductive Fibers

Khalil Abu-Rabeah,†,3 Danit Atias,†,‡,3 Sebastien Herrmann,† Julia Frenkel, ) Dorith Tavor, )

Serge Cosnier,^ and Robert S. Marks*,†,§,#

†Department of Biotechnology Engineering and ‡Department of Virology and §National Institute forBiotechnology in the Negev, Ben-Gurion University of the Negev, P.O. Box 653, Beer-Sheva 84105, Israel,

)Department of Chemical Engineering, Sami Shamoon College of Engineering, Bialk/Basel Sts., Beer Sheva84100 Israel, ^Departement de Chimie Moleculaire, UMR CNRS 5250, Institut de Chimie Moleculaire de

Grenoble, FR CNRS 2607, Universite Joseph Fourier Grenoble I, BP 53, 38041 Grenoble, Cedex 9, Grenoble,France, and #ILSE Katz Center for Meso and Nanoscale Science and Technology, P.O. Box 653, Beer Sheva

84105, Israel. 3Both authors contributed equally to this work

Received April 3, 2009. Revised Manuscript Received May 28, 2009

A conductive surface was created for the development of a biosensing platform via chemical polymerization ofpyrrole onto the surface of poly(methyl methacrylate) (PMMA) fibers, with a subsequent electrogeneration of aphotoactive linker pyrrole-benzophenone (PyBz) monomer on the fiber surface. Irradiation of the benzophenonegroups embedded in the polypyrrole (Ppy) films byUV (350 nm) formed active radicals, allowing covalent attachment ofthe desired biomaterials. Characterization and optimization of this platform were carried out, with the platformshowing conductive, stable, thin, controllable, and light-transmissible film features. Various parameters such as timedeposition, process temperature, and activator plus pyrrole monomer concentrations were examined in the study. Themorphology and permeability of the optic-fiber PMMA fibers were investigated to examine mass transfer ability. Cyclicvoltammetry and amperometry techniques were applied to characterize the electrical features of the surface and chargetransfer. The platform potential was then demonstrated by the construction of both amperometric and opticalbiosensors.

1. Introduction

Fiber optic biosensors are diagnostic tools with special fea-tures1 that may find uses in a variety of fields, for example,medical, pharmaceutical, environmental, defense, bioprocessing,or food. The fiber optic device serves as the transduction element,and the transmitted signal is proportional to the concentration ofa chemical or biochemical to which the biological element reacts.Therefore, it has been used in a variety of spectroscopic techni-ques, for example, chemiluminescence, absorption, fluorescence,phosphorescence, or surface plasmon resonance (SPR).2-5 Silica-based fiber optic biosensors are a well investigated platform beingused to immobilize various biomaterials (enzymes, antibodies,antigens, or whole cells). Furthermore, it has been shown pre-viously that chemiluminescence-based silica optical fiber immu-nosensors (OFISs) are more sensitive than their analogouscolorimetric and chemiluminescent-based enzyme linked immu-

noadsorbent assay (ELISA) counterparts.6-11However, silica fiberchemical modifications are still unreliable fiber-to-fiber, so alter-native configuration scenarios would be useful to improve theefficiency and reproducibility for putativemass production of opticfiber probes. To this end, previously, a new and alternativeapproach was developed and reported.12 This approach was basedon poly(methyl methacrylate) (PMMA) optical fibers owing totheir excellent optical properties, low cost, and elasticity as com-pared to themore commonly used silica-based fibers. Although thePMMA fiber surface showed a hydrophobic and favorable envir-onment for protein adsorption and could be used without furthermodification for immobilization, we explored an alternative con-jugation of the surface, as polypyrrole-based coatings were seen toreduce nonspecific binding of proteinaceous macromolecules.

PMMA particles have been modified by pyrrole chemicalpolymerization in an aqueous dispersion medium,13-15 resultingin a thin, transparent, and conductive polypyrrole (Ppy) film thatis environmentally stable.14,16,17 Consequently, this procedure

*Corresponding author. Telephone: +972 8 6477182. Fax: +972 86472857. E-mail: rsmarks@bgu.ac.il.(1) Marks, R. S.; Bassis, E.; Bychenko, A.; Levine, M. M. Opt. Eng. 1997, 36,

3258–3264.(2) Bosch, M. E.; Sanchez, A. J. R.; Rojas, F. S.; Ojeda, C. B. Sensors 2007, 7,

797–859.(3) Marazuela, D.; Moreno-Bondi, M. C. Anal. Bioanal. Chem. 2002, 372, 664–

682.(4) Wolfbeis, O. S. Anal. Chem. 2004, 76, 3269–3283.(5) Leung, A.; Shankar, P. M.; Mutharasan, R. Sens. Actuators, B 2007, 125,

688–703.(6) Herrmann, S.; Leshem, B.; Landes, S.; Rager-Zisman, B.; Marks, R. S.

Talanta 2005, 66, 6–14.(7) Salama, O.; Herrmann, S.; Tziknovsky, A.; Piura, B.; Meirovich, M.;

Trakht, I.; Reed, B.; Lobel, L. I.; Marks, R. S. Biosens. Bioelectron. 2007, 22,1508–1516.(8) Sobarzo, A.; Paweska, J. T.; Herrmann, S.; Amir, T.;Marks, R. S.; Lobel, L.

J .Virol. Methods 2007, 146, 327–334.

(9) Leshem, B.; Sarfati, G.; Novoa, A.; Breslav, I.; Marks, R. S. Luminescence2004, 19, 69–77.

(10) Konry, T.; Novoa, A.; Shemer-Avni, Y.; Hanuka, N.; Cosnier, S.; Lepellec,A.; Marks, R. S. Anal. Chem. 2005, 77, 1771–1779.

(11) Petrosova, A.; Konry, T.; Cosnier, S.; Trakht, I.; Lutwama, J.; Rwaguma,E.; Chepurnov, A.; Muhlberger, E.; Lobel, L.; Marks, R. S. Sens. Actuators, B2006, 122, 578–586.

(12) Atias, D.; Abu-Rabeah, K.; Herrmann, S.; Frenkel, J.; Tavor, D.; Cosnier,S.; Marks, R. S. Biosens. Bioelectron., in press; doi:10.1016/j.bios.2009.04.2.

(13) Omastova, M.; Simon, F. J. Mater. Sci. 2000, 35, 1743–1749.(14) Ferenets, M.; Harlin, A. Thin Solid Films 2007, 515, 5324–5328.(15) Omastova, M.; Pavlinec, J.; Pionteck, J.; Simon, F.; Kosina, S. Polymer

1998, 39, 6559–6566.(16) Fredj, H. B.; Helali, S.; Esseghaier, C.; Vonna, L.; Vidal, L.; Abdelghani, A.

Talanta 2008, 75, 740–747.(17) Wang, L. X.; Li, X. G.; Yang, Y. L.React. Funct. Polym. 2001, 47, 125–139.

DOI: 10.1021/la901174p 10385Langmuir 2009, 25(17), 10384–10389

Abu-Rabeah et al. Article

was used by us previously to modify and coat PMMA fibers withpyrrole, thus creating a conductive surface used later to electro-polymerize functional pyrrole. The best reactive material forimmobilization was shown with pyrrole-benzophenone (PyBz)electropolymerization presenting conductive, stable, thin, con-trollable, and light-transmissible film features. The modifiedsurface was irradiated by UV (350 nm) to activate the benzophe-none groups embedded in the polypyrrole-benzophenone(PpyBz) films, which resulted in covalent coupling of biomole-cules.

Results showed characterization and optimization of the optic-conductive PMMA fiber platform. The main target optimizationprocess was tomatch conductivity and light transmission betweenPMMA fibers. The Ppy coats imparted the surface with con-ductivity essential for the later electrochemical treatment, but theultimate use as an optic platform required high light transmissionefficiency. Arriving at the above-mentioned features, this studypresents various parameters such as time deposition, processtemperature, and activator plus pyrrole concentrations. Themorphology and permeability of the optic-fiber PMMA fiberswere investigated to study themass transfer ability of the setup. Inaddition, the electrical features were examined and characterizedby cyclic voltammetry and amperometry techniques. Finally, theoptic-conductive PMMA fiber platform was used to constructamperometric and optical biosensors.

2. Experimental Section

2.1. Reagents. Tris-HCl (77-861), pyrrole (13170-9), lithiumperchlorate (431567), urease (Urs) (9002-13-5), urea (57-13-6),ammonium persulfate 98% (APS, 248614), p-toluene sulfonicacid (PTSA, 402885), isopropyl alcohol (IPA, 67-63-0), 1,10-ferrocenedicarboxylic acid, 96%Fe(CN) (215-068-9), horseradishperoxidase (HRP) (P8375), polyethylene glycol sorbitan mono-laurate (Tween20 (T), P7949), skim milk powder (SM, 70166),andN-ethyl-N0-(3-dimethylaminopropyl)carbodiimidehydrochlor-ide (EDAC, E1769) were purchased from Sigma-Aldrich (St.Louis, MO) and used as received; N-hydroxysulfosuccinimide(NHSS, 21335) was purchased from Pierce; PMMA fibers (3 mmdiameter unjacketed, NT53-833), mirror, and a professional fibercutter (N54-013) were purchased from Edmund optics (San Jose,CA); PyBz, Biotin-Py,18,19 and PyNH2

20-22 were synthesized asdescribed elsewhere; luminescencemeasurementswere carried outusing the Immuno-star HRP Chemiluminescent Kit (170-5040)from Bio-Rad Laboratories. Chemiluminescence measurementswere conducted by applying hydrogen peroxide and luminol intofiber holding wells in a 1:1 ratio.

2.2. Preparation of PMMAOptic-Conductive Fiber Bio-

sensors. 2.2.1. Chemical Polymerization ofPyrrole onto thePMMA Fiber-Optic Endface. PMMA optical fibers (3 mmcore diameter) were cleaved manually using a professional fibercutter. First, the fibers were soaked in isopropyl alcohol, soni-cated, and dipped in ddH2O, each procedure for 5min. The fiberswere driedwith an air flow through a 45μmfilter, dried in anovenat 47 C for 20min, and vacuumed for 5min.The clean fibersweredipped into a 1.5 mL tube, containing a mixture 1:1:1 (v:v:v) ofsequentially added cold solutions of 25 mmol APS, 25 mmolPTSA, and 7.5 mmol pyrrole to allow the chemical polymeriza-tion of pyrrole monomers, while the fibers were dipped intothe eppendorfs immediately after introducing the pyrrole. The

mixture was vortexed and placed in an ice bath (5 C)14,23 for 4.5min. The fibers were then placed in ddH2O, gently vortexed,rinsed in ddH2O, and dried by airflow.

2.2.2. Electrochemical Polymerization of PyBz onto thePpy-Coated PMMA Fiber-Optics. PMMA fibers coated by aPpy film were used as working electrodes to enable the electro-generation of an additional conductive and functional layer usingPyBz or PyNH2 monomers in order to electropolymerize themonto fiber optic tips. The desired receptor protein would subse-quently be immobilized onto these new layers. The electropoly-merization process was achieved by the application of a constantvoltage of 0.85V (for PyBz) or 0.93V (for PyNH2) during 10min;the solutionusedwaspreparedbymixingPyBz orPyNH2 (10mMboth) free monomers in 0.1 M of a lithium perchlorate aqueoussolution.

2.2.3. Immobilization of Bioreceptors onto the TransducerSurface. The enzymes, either Urs or HRP (0.5 mg mL-1), werelinked to thePpyBz film (Figure 1) through irradiationusing aUVlamp (1000W, 350 nm) for 7 min, thereby creating radicals thatenabled the covalent attachment of the enzymes. Further washingof the fiber with ddH2O removed unattached proteins. HRP wasalso linked to PyNH2 films using the cross-linkers (NHSS andEDAC) in 2-(N-morpholino)ethanesulfonic acid (MES) buffersolution (0.1MpH6), dipped into anErlenmeyer flask containing3 mMNHSS and 6 mM EDAC and HRP (0.5 mg mL-1),24 andconstantly stirred for 3 h at RT. The fibers were washed twice toremove nonspecific bonding with phosphate buffer saline (PBS)containing 0.5% Tween 20 (PBS-T) (pH 7), then dipped in ablocking solution (0.5% PBS-T/5% SM) for 30 min, and washedagain three times, for 30min inddH2O (pH5), ddH2O (pH8), andPBST (pH 7).

2.3. Electrochemical Instrumentation. The amperometricmeasurements and cyclic voltammograms (CVs) as well as theelectropolymerization were conducted using a PGSTAT30potentiostat and a conventional electrochemical cell (Metrohm).The modified fibers were used as the working electrodes, asaturated Ag-AgCl-KCl electrode (Ag/AgCl) was used as a refe-rence electrode, and a Pt wire as a counter electrode. For theelectropolymerizationprocedure, a 0.1MLiClO4aqueous solutionwas used,whereas for the amperometricmeasurements an aqueoussolution of 0.1 M Tris-HCl buffer electrolyte (pH=7) was used.

The CVs were recorded and swept between -0.3 and 0.8 Vversus Ag/AgCl. These limits were wide enough to include areversible redox reaction and narrow enough to avoid overoxida-tion remaining within the water window. A scan rate of 100 mV/swas used. Before each cyclic voltammogram, several conditioningcycles were swept to ensure stability in the film. A solution of0.1 M Tris-HCl buffers (pH 7) was used as the electrolyte in athree-electrode cell.

2.4. Amperometric Detection of Urs Activity. Theenzyme catalyzed the hydrolysis of urea, and the products weredetected at the electrode surface through their oxidation. Theamperometric detection was carried out in 1.5 mL of aqueoussolution of 0.1 M Tris-HCl buffer electrolyte (pH= 7). Themodified electrode was potentiostated at 1.1 V vs the referenceelectrode until reaching equilibration.25 Urea (0.1 M) was intro-duced into the monitoring system within the concentration rangeof 0-60mM.The current detectionwasmonitored andmeasuredby using the PGSTAT30 potentiostat equipped with GPES4software.

2.5. Permeability Studies of the Optic Fiber. The perme-ability ofPMMAfibers coatedwithPpy andwith Ppy-PpyBz-Urswas examined by using a rotating-fiber electrode (RFE) equippedwith a rotation controller. The experiments were conducted atdifferent rotation speeds using Fe(CN) (2mM) in 0.1MTris-HCl

(18) Cosnier, S.; Senillou, A. Chem. Commun. 2003, 414–415.(19) Cosnier, S.; Lepellec, A. Electrochim. Acta 1999, 44, 1833–1836.(20) Rajesh; Bisht, V.; Takashima, W.; Kaneto, K. Biomaterials 2005, 26, 3683–

3690.(21) Naji, A.; Marzin, C.; Tarrago, G.; Cretin, M.; Innocent, C.; Persin, M.;

Sarrazin, J. J. Appl. Electrochem. 2001, 31, 547–557.(22) Katritzky, A. R.; He, H. Y.; Jiang, R. Tetrahedron Lett. 2002, 43, 2831–

2833.

(23) Machida, S.;Miyata, S.; Techagumpuch, A. Synth.Met. 1989, 31, 311–318.(24) Polyak, B.; Geresh, S.; Marks, R. S. Biomacromolecules 2004, 5, 389–396.(25) Wang, X.; Watanabe, H.; Sekioka, N.; Hamana, H.; Uchiyama, S.

Electroanalysis 2007, 19, 1300–1306.

10386 DOI: 10.1021/la901174p Langmuir 2009, 25(17), 10384–10389

Article Abu-Rabeah et al.

(pH=7) as a redox probe. The permeability (Pm) of the coatingswas estimated using RFE experiments at different rotation rates.The results were analyzed and examined by applying eqs 1-4 thatwere developedbyGough andLeypoldt26 describing the variationof steady state limiting current Ilim with the mass transport for acoated RFE.

1=I lim ¼ 1=I s þ 1=Im ð1Þ

I s ¼ 0:62nFAC0Ds2=3ν-1=6ω1=2 ð2Þ

Im ¼ nFAKC0Dm=δ ¼ NFC0Pm ð3Þ

Pm ¼ KDm=δ ð4ÞTermsDs andDm are the diffusion coefficients of the substrate inthe bulk solution and in the membrane, respectively; ν is thekinematic viscosity of the solution; ω is the rotation rate of theRFE; δ is the thickness of the membrane; K is the partitionequilibrium constant of the substrate between solution andmembrane; A is the electrode surface; n is the number of ex-changed electrons, and C0 is the substrate concentration. Equa-tion 1 is composed of two terms, where the first represents thecurrent flow under the same conditions, PMMAcoatedwith Ppy,and is therefore characteristic of the diffusion of the substrate inthe bulk solution (Levich current, eq 2). The second termaccountsfor the diffusion of the substrate in the coating and depends on theproduct of the partition equilibrium constant K, the diffusionconstant of the substrate in the coating, and the film thickness.

Only the first term of the equation is dependent upon the rotationrate of theRFE. Therefore, a plot of 1/Ilim versus 1/ω1/2 (Figure 5)presents a linear behavior with the same slope as for the firstelectrode with a positive intercept, whose value depends on thepermeability Pm of the membranes (eqs 3 and 4). The relativedeviation of the values was <4%.

2.6. Scanning Electron Microscopy (SEM). SEM micro-graphs were taken by using a JEOK JSM 7400F system with a129 eV resolution for the detector and exposure time of 100 s. Thespecimens were analyzed using energy dispersive X-ray spectro-metry microanalysis analytical equipment installed in the scan-ning electron microscope.

2.7. Spectroscopic Analysis. Infrared measurements wereperformed in transmission mode on a Bruker Equinox 55infrared spectrometer. The Fourier transform infrared (FTIR)spectra were averaged over 128 scans at a resolution of 4 cm-1.Optical densitymeasurementswere carried out using anUltrospec2100 ProUV/Visible spectrophotometer (Biochrom CB4 0FJ,England) at 490 nm.

2.8. HRPChemiluminescenceMeasurement. Chemilumi-nescencemeasurementswere performed by introducing anoxidiz-ing reagent and luminol into fiber holding wells in a 1:1 ratio.Measurements were carried out using a single photon avalanchediode (SPAD) photodetector based on a SPAD module pur-chased from SensL (PCMplus module, 10 μm sensor). Data werecollected using SensL Integrated Environment software andreported in relative light units (RLU).

3. Results and Discussion

3.1. Fiber Optic Preparation and Optimization. CleanPMMAfiberswere dipped into awater-based solution containingAPS, PTSA, and pyrrole 1:1:1 (v:v:v) to permit the chemicalpolymerization of pyrrole monomers. Different polymerizationtimes were used. Thereafter, the fibers were examined usinga spectrophotometer for their light transmission measured at490 nm (Figure 2). The modified PMMA fibers showed goodtransmittance at 3 min of activation (>90%), while a longerprocedure leads to a drastic loss of light transmission capability.However, a conductive film is crucial for the electrochemicaldeposition, and hence, higher polymerization time was definedand set to 4.5 min. This procedure resulted in a very low surfaceresistance (17 KΩ mm-2) but still reasonable light transmission(60%), enabling improved electrochemical deposition of PyBz.

Various experiments were performed to polymerize PyBz andPyNH2 directly on the transducer surface, but the results werevery poor and the procedure with pyrrole was selected as theoptimum in this study.3.2. FTIR Spectroscopy. The FTIR spectrum of PMMA

fibers coated with two layers of Ppy and PpyBz illustrates thedetails of the functional groups present in the PMMA fiber(Figure 3). The sharp intense peak at 1721 cm-1 appeared dueto the presence of the ester carbonyl group stretching vibration.The broad peak ranging from 1260 to 1000 cm-1 can be explainedowing to the C-O (ester bond) stretching vibration. The broadband from 950 to 650 cm-1 is due to the bending of C-H; thebroad peak ranging from 3000 to 2900 cm-1 is due to thestretching vibration. A sharp peak of OH stretch vibration bandappeared at 3570 cm-1, indicating the existence of the benzophe-none group. The medium sharp peak at 1282 cm-1 (amide IIband, interaction between theN-Hbending andC-N stretchingvibration) indicates the existence of the pyrrole. The peak at 3333cm-1 is assigned toN-Hbond,whereas the 2300 cm-1 peak is thestrong C-N band.

Specimens were also analyzed using energy dispersive X-rayspectrometry microanalysis. The shift on binding energy for

Figure 1. Construction of amperometric and optical biosensors.(1) Modification of PMMA fiber surface by chemical polymeriza-tion of pyrrole, (2) electrochemical polymerization of PyBz, (3)irradiationofbenzophenonemoieties byUVandattachmentof theenzyme Urs or HRP to the benzophenone groups, (4) monitoringof the current as a function of urea concentration in the ampero-metric biosensor case (A) and light radiation recording in thechemiluminescence-based biosensor (B) when adding oxidizingreagent and luminol into the wells at a 1:1 ratio.

(26) Gough, D. A.; Leypoldt, J. K. Anal. Chem. 1979, 51, 439–444.

DOI: 10.1021/la901174p 10387Langmuir 2009, 25(17), 10384–10389

Abu-Rabeah et al. Article

samples in the O1s region from PMMA (532 eV) to PMMA-Ppy(531 eV) was attributed to the increase in intensity of the O1sfeaturing the polymerization of pyrrole molecules at the PMMAsurface. This was further supported by the observation that theO1s intensity increased substantially as a result of PyBz electro-polymerization on the PMMA-Ppy, which correlated directly tothe binding energy shift to higher values as the electropolymer-ization tightened the thin polymeric film structure (C/O increasesrelative to PMMA-Ppy).3.3. Electrochemical Characterization of the Modified

Fibers. Characteristic redox responses observed for the PMMA-Ppy, PMMA-Ppy-PpyBz, and PMMA-Ppy-PpyBz-Urs are illu-strated in Figure 4, with the CV of PMMA-Ppy (Figure 4A)

showing higher redox waves, while PMMA-Ppy-PyBz(Figure 4B) and PMMA-Ppy-PyBz-Urs (Figure 4C) revealed ageneral blurring of the redox responses. Such behavior points toan increase in the resistance and sluggish electron transfer, andwas accompanied by a concomitant ion transport; this blurringwas formed by the Bz and Urs existence on the PMMA fibersurface and indicated that the ion diffusionwas slow and reflectedin the loss of well-defined redox behavior. A higher current in thecase ofPMMA-Ppy signified improved response and the ability ofthe polymer to switch more easily between both redox states, as itis accompanied by better counterion transport and charge trans-fer compared to the PMMA-Ppy-PyBz-Urs. The intensity of theoxidation current reflected also the amount of polymerizedmonomeric units, which is directly connected to the enhancedphotografting ability of the polymer deposited on the fiber.3.4. Permeability Studies. Figure 5 shows such a Kou-

tecky-Levich plot of fiber coating with increasing film thickness.The results show that the film coatings behave like a homoge-neous membrane;27 even for more than one layer, the film is stillhomogeneous and no transition frommembrane to pinhole film is

Figure 2. RelationbetweenPpy chemical polymerization timeandfiber light transmittance.

Figure 3. (A) FTIR spectra of PMMA fiber and (B) FTIR spectraof PMMA fiber coated with two layers of Ppy and PpyBz.

Figure 4. Cyclic voltammograms of (A) PMMA-Ppy, (B)PMMA-Ppy-PpyBz, and (C) PMMA-Ppy-PpyBz-Urs in 0.1 MTris-HCl (pH 7). Scan rate at 0.1 V s-1.

Figure 5. Koutecky-Levichplot for (A)PMMAfiber coatedwithPpy and (B) PMMA fiber coated with Ppy-PpyBz. The testsolution was a 2 mM solution of Fe(CN) in water.

(27) Gooding, J. J.; Hall, C. E.; Hall, E. A. H. Anal. Chim. Acta 1997, 349, 131–141.

10388 DOI: 10.1021/la901174p Langmuir 2009, 25(17), 10384–10389

Article Abu-Rabeah et al.

observed, as has often been encountered previously.28,29 A highpermeability was observed from the setup of PMMA-Ppy coatedwith PpyBz (7.9 10-4 cm s-1). This value indicated that theoxidative probes permeated very easily and oxidized on the fibersurface, further supported by the values of the response time andcurrently emphasizing the ability of the coating to behave as ametal-like oxidizing surface.3.5. Scanning ElectronMicroscopy (SEM) Studies. SEM

measurements were performed for PpyBz films electropolymer-ized on PMMA optical fibers coated with Ppy. Figure 6 shows ahomogeneous surface for both PMMA-Ppy and PMMA-Ppy-PpyBz. The PyBz deposition on the PMMA-Ppy created grainsupon the surface, presented in Figure 6B with a different resolu-tion. The film thickness was evaluated to be about 100 nm. Thisformed a thin, transparent conductive film. The PMMA-Ppy-PpyBz morphology showed a smooth and homogeneous surfacedistributed with a few grainy spots attributed to the first layermorphology. Furthermore, the electropolymerization process isknown to be controllable, thus allowing PyBz films to be coated ina smooth and ordered way upon the Ppy layer.3.6. Amperometric Measurements of Urea Based on a

PMMA-Urs Biosensor. In the optical PMMA fiber surfacemodification process presented in Figure 1, pyrrole was polym-erized chemically to create a conductive layer, and then PyBzfilms were electropolymerized there by applying a voltage at0.85 V. The protein attachment was done through irradiationand activation of the benzophenone groups, which createdunstable radicals “attacking” the C-H bond in the proteinskeleton to bind the biological entities to the fiber surface.Enzyme attachment enabled the catalytic reaction of the analytethat could be detected by either electrochemical or optical means.

The analytical performance of such a platform was demonstratedby constructing an amperometric biosensor for urea monitoring.The detection principle of urea is based on carbamic acid oxida-tion. As shown inFigure 1,Urs catalyzed the hydrolysis of urea toform intermediate products (carbamic acid and ammonia). Theelectrolysis was done by applying 1.1 V to oxidize the carbamicacid (three electron oxidation),25 creating a current which corre-lated to the urea concentration.

The PMMA-Ppy-PpyBz-Urs fiber in Figure 7A exhibits abetter performance than those of the controls PMMA-Ppy-PpyBz-Urs without UV irradiation (Figure 7B) and PMMA-Ppy-Urs without a PyBz linker (Figure 7C). The performance ofthe PMMA-Ppy-PyBz-Urs biosensor indicated that the irradia-tion of the PyBz activated the benzophenone and specificallyattached the enzymeUrs. Furthermore, the sensitivity of PMMA-Ppy-PyBz-Urs (12.3 μAmM-1 cm-2) was 4 times higher than thesetup without UV (2.3 μA mM-1 cm-2), indicating that theenzyme was attached covalently to the benzophenone group,although some may bind nonspecifically. The resulting responsetimewas very short (2 s), and the platformwas shown to be highlysensitive, efficient, and rapid for amperometric measurements. At10 C (Figure 7D), the activity almost disappeared, demonstrat-ing that the oxidation current at 1.1 V effectively was due to theenzymatically generated carbamic acid, with the enzyme activitymarkedly decreasing at this temperature.3.7. Luminescent Measurements. The ability of such a

platform enabling the attachment of biomolecules to the fibersurface and to transmit light efficientlywas also investigated in theframework of this research. This was demonstrated by theattachment of the enzyme HRP to the fiber surface, introducinghydrogen peroxide and luminol reagents into the measurementcell and collecting the light by using a single photon avalanchediode (SPAD) photodetector. The observed values for the sample(Figure 8D) PMMA-Ppy-PyBz-HRP have shown very sensitiveresults (1600 signal/noise S/N) when compared with the value

Figure 6. SEM micrographs of PMMA fibers based on the copolymerization of (A) PMMA-Ppy and (B) PMMA-Ppy-PpyBz.

(28) Cosnier, S.; Szunerits, S.; Marks, R. S.; Novoa, A.; Puech, L.; Perez, E.;Rico-Lattes, I. Talanta 2001, 55, 889–897.(29) Situmorang,M.; Gooding, J. J.; Hibbert, D. B.Anal. Chim. Acta 1999, 394,

211–223.

DOI: 10.1021/la901174p 10389Langmuir 2009, 25(17), 10384–10389

Abu-Rabeah et al. Article

(11 S/N) of the same setup without UV (Figure 8E), indicatingthat the enzyme was linked specifically to the transducer surface.This was further supported by the value (16 S/N) of the samesetup but without the photolinker PyBz (Figure 8F). The linkerbiotin-pyrrole and PyNH2 were also used to construct the above-mentioned setup instead of the photolinker PyBz, but the resultsof the PyNH2 sample (Figure 8A) showed very low sensitivity(80 S/N) compared to that of the PyBz. This result is more likelyattributed due to an extremely low efficiency in binding enzyme tothe fiber surface. The pyrrole-biotin and pyrrole-NHSS (notshown) also present poor results when compared to the onesmade with PyBz.

4. Conclusion

In this study we have shown the characterization of a new fiberoptic configuration for the construction of biosensing platforms.This was developed and based on the chemical polymerization of

pyrrole onto the surface of PMMA fibers to create a conductivelayer, enabling the subsequent electrogeneration of photo-active PpyBz upon the fiber surface. Irradiation of the benzophe-none groups embedded in the Ppy films by UV (350 nm) enabledthe attachment of enzyme-based sensingmatrices. The fibers werecharacterized here by various techniques which established theconductive and transparent features that opened a wide range ofputative applications based on the combined use of parallel lightand conductivity measurments.

Acknowledgment. The authors gratefully acknowledge Dr.Boris Polyak fromDrexel University, College ofMedicine, for hisvaluable assistance and scientific discussions of the results in theframe of this work, and Dr. Louisa Meshi for the SEM micro-graphs.

Figure 7. Calibration curves of amperometric measurements cor-responding to (A) PMMA-Ppy-PyBz-Urs/ UV, (B) PMMA-Ppy-PpyBz-Urs, and (C) PMMA-Ppy-Urs/UV (all were tested at RT)and (D) PMMA-Ppy-PyBz-Urs/UV (10 C) as a function of ureaconcentration ranging from 0 to 60mM.E=1.1V versusAg/AgCl.

Figure 8. Luminescentmeasurements corresponding to (A)PMMA-Ppy-PpyNH2-HRP (with NHSS and EDAC), (B) PMMA-Ppy-PpyNH2/HRP (without NHSS and EDAC), (C) PMMA-Ppy-HRP,(D) PMMA-Ppy-PyBz-HRP/UV, (E) PMMA-Ppy-PpyBz-HRP(withoutUV),and(F)PMMA-Ppy-HRP/UVmodifiedPMMAfiber.Chemiluminescence-based measurements were performed by addingoxidizing reagent and luminol into the wells in a 1:1 ratio.

63

5. Summary of materials and methods not found in published

articles.

5.1. Detection of anti-DE3V IgM antibodies using ED3 MAC ELISA

5.1.1. Chemicals and biochemicals

Skim milk powder (SM) (70166), polyethyleneglycol sorbitan monolaurate (Tween® 20 (T))

(P1379) were purchased from Sigma-Aldrich. Phosphate buffer saline (PBSx1) was made of

0.203g NaH2PO4, 1.149g Na2HPO4, 8.5g NaCl, and brought up to 1L with ddH2O. MAC-ELISA

was carried out using goat anti-human IgM (µ-chain specific) (12386), and anti-mouse IgG

peroxidase conjugated (γ-chain specific) (A3673), purchased from Sigma. DENV-2 antigen was

obtained from the National Reference Center (NRC) of Arboviruses, Institut Pasteur de la

Guyane, French Guiana. The DENV-2 Ag was prepared by extraction from brains of suckling

mice with the sucrose-acetone method and was shown to have cross-reactivity with the three

other DENV serotypes [126]. Homologous mouse ascitic fluid containing anti-DENV IgG was

also provided by the NRC [61]. Luminescence MAC-ELISA measurements were carried out

using the Immuno-star HRP Chemiluminescent kit (170-5040) from Bio-Rad Laboratories.

Colorimetric MAC-ELISA assays were carried out using 3,3′,5,5′-Tetramethylbenzidine (TMB)

(T8665), Sigma. Luminescence ED3 MAC-ELISA mmeasurements were carried out using

Immun-Star AP substrate pack (170-5012), purchased from Bio-rad Laboratories. Colorimetric

ED3 MAC-ELISA measurements were carried out in a home made system using p-

nitrophenylphosphate (pNPP) (71768), magnesium sulfate (MgSO4) (M9397), Diethanolamine

(D83303), zinc-chloride (ZnCl2) (208086) all obtained from Sigma-Aldrich or Alkaline

Phosphatase Yellow (pNPP) Liquid Substrate System for ELISA (P7998), purchased from

Sigma-Aldrich.

5.1.2. ED3-PhoA and ED3 MAC-ELISA

ED3-PhoA is a hybrid protein between ectodomain 3 (ED3) of the envelope glycoprotein

(gpE) of each DENV serotype, and a mutant alkaline phosphatase (PhoA) from E.coli. The

hybrid protein was received from Prof. H. Bedouelle, from Institut Pasteur, Paris as part of a

64

DENFRAME 1 collaboration. Colorimetric ELISA based on MAC-ELISA using ED3-PhoA

(refer here as colorimetric ED3 MAC-ELISA) was developed (data not published yet) by Prof.

H. Bedouelle used ED3-PhoA instead of the two last steps of the routinely used colorimetric

MAC-ELISA (Fig.11). Furthermore the ED3 MAC- ELISA has different incubation time

arrangement which make the process shorter.

Fig. 11. Comparison between MAC-ELISA to ED3 MAC ELISA.

5.2. Human sera

5.2.1. Internal panel of reference sera

The NRC used its serum collection to constitute a pool of sera positive to anti-DENV-2 IgM.

Bio-Rad Laboratories, used this pool and kindly provided up with a panel of six seral samples

containing different concentration of anti-DENV IgM. The samples were referred to as panel

point #0 to point #6, where panel point #1 contained the lowest titer of anti-DENV-2 IgM.

Another serum was referred to as panel point #0, containing no anti-DENV-2 IgM antibodies,

which was obtained from a pool of sera from the National Blood Bank of France. Each point

from the panel has been calibrated for anti-DENV IgM detection using the Dengue IgM Capture

1 DENFRAME is a program supervised and financed by the EUROPEA3 COMMISSIO3. The main aim of the DENFRAME project is to improve the management of dengue disease in the human populations of Latin America and Asia.

Goat anti-mouse IgG HRP conj.

Goat anti-human IgM

Human anti-DENV IgM

Dengue antigen

Mouse anti-DENV

Dengue antigen AP conj. (specific to

a serotype)

2

Blocker

1

MAC ELISA ED3 MAC ELISA

Goat anti-mouse IgG HRP conj.

Goat anti-human IgM

Human anti-DENV IgM

Dengue antigen

Mouse anti-DENV

Dengue antigen AP conj. (specific to

a serotype)

2

Blocker

1

MAC ELISA ED3 MAC ELISA

2

Blocker

1

MAC ELISA ED3 MAC ELISA

65

ELISA test (E-DEN01M) from Panbio (Brisbane - Australia). See also article No. 1 paragraph

2.3.1.

5.2.2. Human sera samples

A total number of 36 sera samples from the collection of the NRC were tested in this study.

Samples were collected during the acute phase of the disease (day 0 to day 4) and/or the

convalescent phase i.e. 7 days or later after fever onset. All sera were first tested for anti-DENV

IgM antibodies by a routinely used colorimetric MAC-ELISA [61] and then further divided into

two groups. The first group consisted of convalescent phase sera (n=25) from patients positive

for anti-DENV IgM antibodies. The DENV strain of the samples was defined by RT-PCR that

was performed on acute phase sera samples previously taken from the respective patients [36].

The samples were found positive to DENV1 (n=5), DENV2 (n=10), DENV3 (n=10). The second

group of samples consisted of acute phase sera samples (n=11) from patients presenting dengue-

like syndrome (temperature of ≥ 38.5°C, arthralgia, headache, and/or myalgia) for whom recent

DENV infection was ruled out by a negative RT-PCR and negative anti-DENV IgM assay, and

confirmed with a second blood sample negative for anti-DENV IgM antibodies.

5.2.3. Detection of anti-DE3V IgM antibodies using the routine MAC-ELISA

MAC-ELISA was performed as described by Kuno et al. [127] with minor adjustments. A

volume of 100 µl of 4 µg/ml concentration of anti-human IgM antibodies was inserted into each

well of a pre-washed 96-well microtiter plate (MaxiSorp, Nunc) with PBS. The plate was

covered and was incubated for 2 h at 37°C. Following incubation, the plate was washed thrice to

remove unbound antibodies with PBS containing 0.1% Tween 20 (PBS-T) (pH 7.4). Then sera

samples, consisting of the panel and controls, were diluted 1:100 in PBS containing 0.5% Tween

20 and 5% skim milk (PBS-T-SM), and a volume of 100 µl of each sample was added into the

plate in duplicates and incubated for 1 h at 37°C. After washing the plate with PBS-T to reduce

background and increase the sensitivity of the assay, 100 µl of DENV-2 Ag diluted 1:100 in

PBS-T-SM was added to each well. After incubation at 4°C overnight, the plates were washed

with PBS-T. Then, 100 µl of mouse ascitic fluid containing murine anti-DENV IgG diluted

1:1000 was added into each well. Finally, 100 µl of solution containing the secondary antibodies,

anti-mouse IgG peroxidase labeled, diluted 1:2000 was added to each well. Each of the last two

steps was subjected to incubation for 1 h at 37°C followed by washing. For the colorimetric

66

measurements, 100 µl of the TMB substrate was added into each well and incubated at room

temperature for 20 min. Optical density was measured at 620 nm using Σ960 colorimeter

(Metertech, Taiwan) and reported using the instrument software in OD (optical density). Positive

results were defined as higher than three times the mean value of the negative control, and

negative results as lower than twice that the mean value of the negative control. Values obtained

between these ranges were defined as equivocal.

Chemiluminescence measurements were performed by adding oxidizing reagent and luminol

into the wells in a 1:1 ratio. Measurements were carried out using a standard luminometer

(Thermolabsystems-Luminoskan Ascent) and data was collected using Luminoskan Ascent

software and reported in RLU (relative light units). A total of 20 measurements were performed

at a rate of 1 sec between each reading. A cut-off value was set according to the negative control

(panel point # 0) and confidence range was ±10%. For both methods each experimental point

was repeated in triplicate and for each triplicate, average and standard deviation were calculated.

5.2.4. Detection of anti-DE3V IgM antibodies using ED3 MAC-ELISA

A 96-well plate (MxiSorp, Nunc) was pre-washed with PBSx1 3 times. Every well was

coated with 100µl/well of anti-human IgM diluted 1:500 in PBS x1, and a plate was incubated

overnight in 40°C. Followed the incubation, the plate was washed 3 times with 0.1% PBS-T.

Every well was coated with 100µl/well blocking buffer (0.5% PBS-T- 5% SM), and plate was

incubated at 370C for 1 hour. The plate was washed three times with 0.1% PBS-T, and every

well was coated with 100µl/well sera samples, diluted 1:100 in dilution buffer (0.1% PBS-T- 1%

SM). The plate was incubated at 370C for 1 hour, and washed three times with 0.1% PBS-T.

Every well was coated with 100µl/well working solution of ED3-PhoA [0.2µM ED3-PhoA in

0.5% PBS-T- 5% SM (2:1 respectively)]. The plate was incubated 1 hour at 370C, and washed

three times in 0.1% PBS-T.

5.2.4.1. Colorimetric detection

2mg of pNPP was added to a ml of DEA-Zn buffer DEA-Zn buffer [1 ml MgSO4 1M, 10 ml

Diethanolamine, was adjusted to pH 9.8 with HCl, 0.2 ml ZnCl2, ddW was completed to 100ml

and kept in 4°C]. 100µl/well of pNPP-DEA-Zn solution was added to the plate that was

67

incubated at room temperature usually for 3 to 5 hours in the dark. The plate optical density

(OD) was read at 405nm in room temperature.

5.2.4.2. Luminescent detection

A reaction mixture of 1:20 enhancer in substrate was made usig Immune-Star AP substrate

pack kit. 100µl/well of reaction mixture was added to each well, and the reading was executed

immediately after, using a standard luminometer (ThermoLabSystems-Luminescent Ascent); and

data was collected using Luminoskan Ascent software and reported in RLU (relative light units).

Measurement can be taken also without the enhancer but the signal will be very low.

5.2.4.3. Statistical analysis

The following statistical approaches were used to estimate the diagnostic sensitivity and

specificity: sensitivity = [Tp/(Tp + Fn)]*100, specificity = [Tn/(Tn + Fp)]*100; where Tp

represents the true positive sera, Fn the false negative sera, Tn the true negative sera, and Fp the

false positive sera [128]. The cross reactivity between the expected (by RT-PCR) DENV

serotype to different DENV serotype was calculated as follows: cross reactivity = [Fp/ Tp]* 100.

68

6. Summary of results not found in published articles

As detailed above, one of the limitations of the current diagnostic tools is their lack of

specificity and safety of the antigen. In this chapter an effort was made to adapte alternative

antigens to the routinely used methods in order to improve the detection of anti-dengue

antibodies. This was performed by using recombinant protein (ED3-PhoA) of each DENV

serotype. The ED3-PhoA is a hybrid protein between ED3 of the envelope gpE of each DENV

serotype, and a mutant alkaline phosphatase (PhoA) from E.coli. The E protein is known to be

the most immunogenic of all the dengue viral proteins and has been proposed to function as the

putative receptor-binding domain of DENV [3].

6.1. Detection of anti-DE3V IgM antibodies using ED3 MAC-ELISA

6.1.1. Screening of panel reference sera

In order to astablise the colorimetric ED3 MAC-ELISA protocol in the lab first test were

panel point #6 (contain high lavel of anti-DENV-2 IgM) and panel point #0 (does not contain

anti-DENV-2 IgM) using all four serotypes in order to check the specificity to serotypes (cross

reactivity) (Fig. 12). Furthermore the signal development over time was tested. Panel point #6

was prepared from the NRC pool of sera which is in endemic areas to DENV. The sample was

selected during the 2006 outbreak caused mainly by DENV-2. It seems from the result that the

colorimetric ED3 MAC-ELISA detected the presence of anti-DENV2 IgM antibodies with slight

cross reactivity to DENV1. The signal was developed over time, after adding the substrate,

while the best result was detected after 20 hours but a significant result can be detected also

afther 4-5 hours as suggested in the original protocol. Results were presented as S/N ratio, since

for each serotype, different values were detected for panel point #0.

69

Fig. 12. Colorimetric ED3 MAC-ELISA. Signal values (anti-DENV IgM antibodies- panel reference sera

No. 6) are divided by the noise values (control serum- panel reference sera No. 0). The signal was

measured using colorimetric ED3 MAC-ELISA with all four serotypes: DENV1 (♦), DENV2 (),

DENV3 () and DENV4 (X).

In order to compare between ED3 MAC-ELISA to MAC-ELISA and to test the use of

luminescence instead of colorimetric in ED3 MAC-ELISA system, they were tested using a

panel of reference sera. This panel therefore was screened using the Colorimetric MAC-ELISA

(Fig.13 A) Luminescence MAC-ELISA (Fig.13 B) Colorimetric DENV2 ED3 MAC-ELISA

(Fig.13 C) Luminescence DENV2 ED3 MAC-ELISA (Fig.13 D). For each method, the results of

panel points #0 to #6 were measured and Signal to Noise ratio (S/N) was calculated (Fig.14).

Since the panel of sera was diluted and stabilized by Bio-Rad Laboratories it is not known what

the exact dilution for each point was. It is known that panel point #1 contains the lowest titer of

anti-DENV-2 IgM, panel point #6 the highest and panel point #0 does not contain anti-DENV-2

IgM. All four methods demonstrate the same detection pattern which was also shown using other

diagnostic tools in article No. 1 Fig. 4. S/N ratio was received by dividing the Signal values

(anti-DENV IgM antibodies) by the noise values (control sera) in order to follow the evolution of

the background signal. The Luminescence MAC-ELISA showed a higher S/N ratio than the

other method. Moreover both luminescence methods presented higher S/N than the colorimetric

methodologies, especially in panel point #4-6, which contain higher values of anti-DENV-2 IgM.

0.0

0.5

1.0

1.5

2.0

2.5

0:00 4:00 8:00 12:00 16:00 20:00

Time (hh:mm)

S/N

70

Fig. 13. Screening of panel reference sera. A comparison between 4 diagnostics method to reveal anti-DENV IgM antibodies from known panel using: (A) Colorimetric MAC-ELISA (B) Luminescence MAC-ELISA (C) Colorimetric DENV2 ED3 MAC-ELISA (D) Luminescence DENV2 ED3 MAC-ELISA.

Fig. 14. Signal to noise ratio (S/3) of panel reference sera. Signal values (anti-DENV IgM antibodies) are divided by the noise values (control sera) in order to follow the evolution of the background signal. The graph comparison between four diagnostics method to reveal anti-DENV IgM antibodies from

known panel using: () Colorimetric MAC-ELISA, () Luminescence MAC-ELISA, (∆) Colorimetric DENV2 ED3 MAC-ELISA and () Luminescence DENV2 ED3 MAC ELISA.

0

2

4

6

8

10

0 1 2 3 4 5 6

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RLU

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RLU

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0 1 2 3 4 5 6

OD

A.

B.

C.

D.

71

6.1.2. Screening of human sera

A total number of 36 sera samples from the collection of the NRC were tested in this study

using DENV1-4 ED3 MAC ELISA (Table. 3.). All sera from the collection of NRC were first

tested for anti-DENV IgM antibodies by a routinely used colorimetric MAC-ELISA and then

further divided into two groups. The first group consisted of convalescent phase sera (n=25)

from patients positive for anti-DENV IgM antibodies. The DENV strain of the samples was

defined by RT-PCR that was performed on acute phase sera samples previously taken from the

respective patients. The samples were found positive to DENV1 (n=5), DENV2 (n=10), DENV3

(n=10). The second group of samples consisted of acute phase sera samples (n=11) from patients

presenting dengue-like syndrome for whom recent DENV infection was ruled out by a negative

RT-PCR and negative anti-DENV IgM assay, and confirmed with a second blood sample

negative for anti-DENV IgM antibodies. The result was summarized according to the sensitivity,

specificity and cross reactivity with other serotypes. The specificity was very low (45%) but the

sensitivity off all samples that were defined "positive" to anti-DENV IgM gained high

sensitivity- 90% for DENV2, 3 and 100% to DENV1. High cross reactivity of ED3-DENV with

sample with different serotypes was observed- 60% for DENV1 50% for DENV2 and 90% for

DENV3. The low specificity perhaps be explained by the strong cross-reactivities between the

DENV serotypes and other groups of flaviviruses circulating in French Guinea. Nevertheless

DENV ED3-PhoA hybrids had much less cross-reactivity than with other antigens differentiating

according to serotype. In spite of of that in most cases (82%), the highest signal was observed

with the antigen corresponding to the serotype in charge of the DENV infection. A similar result,

(89%), was also obtained by NRC in Dr. Phillipe Dussart’s laboratory using colorimetric

DENV1-4 ED3 MAC-ELISA (result was kindly provided from Dr. Dussart and Prof. Bedouelle,

full data not shown). An example of three samples from DENV1 infection that was tested by

luminescence DENV1-4 ED3 in our laboratory was also compared to those made by NRC in Dr.

Phillipe Dussart’s laboratory by colorimetric DENV1-4 ED3 MAC-ELISA (Fig. 15). In all three

samples the same pattern was observed, and the highest signal was detected using DENV1 ED3

as expected from the RT PCR that was performed in a parallel sample with the same patient from

the acute phase of the disease. In most of the detection, cross-reactivities were observed, but

since the result of the negative control of the colorimetric DENV1-4 ED3 MAC-ELISA was not

received, the exact rate could not be calculated. Moreover, using luminescence DENV1-4 ED3

72

MAC-ELISA enabled detection after ~20 minutes while using colorimetric DENV1-4 ED3

MAC-ELISA become possible only after 3-5 hours and in these examples the best results were

detected only after 24 hours.

Table. 3. Screening of human sera using ED3 MAC-ELISA, DE3V1-4 ED3. Sera were taken in the

convalescent phase, from patients positive, by routine used colorimetric MAC- ELISA for anti-DENV

IgM antibodies. The DENV strain of the samples was defined by RT-PCR that was performed on acute

phase sera samples previously taken from the respective patients. The second group of samples consisted

of acute phase sera samples where DENV infection was ruled out by a negative RT-PCR and negative

anti-DENV IgM assay, confirmed with a second blood sample negative for anti-DENV IgM antibodies.

The result was summarized according to the sensitivity, specificity and cross reactivity with other

serotypes.

Serotype DE3V1 DE3V2 DE3V3 3egative Total

No. of samples 5 10 10 11 36

Sensitivity 5/5 (100%) 9/10 (90%) 9/10 (90%) - -

Specificity - - - 5/11 (45%) -

Cross reactivity of

ED3-DENV with

sample with different

serotypes

3/5 (60%) 5/10 (50%) 9/10 (90%) - -

73

Fig. 15. Comparison between (A) Colorimetric DENV1-4 ED3 MAC-ELISA that was in performed in

Prof. H. Bedouelle laboratory (B) Luminescence DENV1-4 ED3 MAC-ELISA. Both ELISA was used

the same DENV1 human sera (a-c). OD was measured 24 hours after substrate adding and luminescent

measures after 30 minutes.

Comparison between colorimetric DENV1-4 ED3 MAC-ELISA Luminescence DENV1-4 ED3

MAC-ELISA

A. B.

0

100

200

300

400

Serotypes

RLU

0

100

200

300

400

Serotypes

RLU

0

100

200

300

400

Serotypes

RLU

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

Serotypes

OD405

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

Serotypes

OD405

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

Serotypes

OD405

b.

c.

b.

c.

a. a.

74

7. Overall discussion

7.1. Development of chemiluminescent optical fiber immunosensor for

the detection of IgM antibody to Dengue virus in humans

Three immunoassays were employed and compared for the detection of IgM antibodies

against DENV in human sera samples: colorimetric MAC-ELISA, chemiluminescent MAC-

ELISA, and chemiluminescent silica based OFIS. The two chemiluminescent methods were used

for the first time for the detection of IgM Ab to DENV in humans. The use of internal panel

reference sera and samples from endemic areas enable a reliable comparison between different

diagnostic methods. The results presented in this study demonstrate that a sensitive and rapid

semi-quantitative chemiluminescent OFIS can be used for the detection of anti-DENV IgM in

human sera samples. In comparison to standard colorimetric MAC-ELISA procedures, it has

been revealed that: (1) the chemiluminescent OFIS has a lower detection limit than ELISA

methodologies; (2) colorimetric and chemiluminescent MAC-ELISA are reliable systems for

high and intermediate levels of sera containing anti-DENV IgM, whereas chemiluminescent

MAC-ELISA has a lower detection limit; (3) the chemiluminescent OFIS has a reliable working

range at very low IgM concentrations with an analytical sensitivity enabling a semi-quantitative

test, (4) chemiluminescent procedures are at least 10–100 times more sensitive than the

respective colorimetric test enabling detection of very low analyte concentrations, (5)

chemiluminescent OFIS is advantageous over colorimetric and chemiluminescent MAC-ELISA,

since it is much more rapid. (6) However, the two established methodologies – colorimetric and

chemiluminescent MAC-ELISA – have better reproducibilities. These results support previous

studies that demonstrated the advantages of using chemiluminescent methods, especially OFIS,

as a useful diagnostic tool for viral detection [98, 100]. Although the diagnostic specificity of the

chemiluminescent OFIS was 87.0%, it did have a high diagnostic sensitivity (98.1%), lower

detection limit and a very high correlation between the established methods with a known panel.

Moreover OFIS is rapid, cost effective, simple to perform and has the potential to work under

field conditions providing a reliable option for anti-DENV IgM detection in human sera samples.

In this study, DENV Ags that were extracted from suckling mouse brains using a sucrose-

acetone technique were employed. To improve the specificity of chemiluminescent OFIS for

75

anti-DENV IgM detection, it could be very useful to develop a new protocol using recombinant

protein (from envelope, membrane or NS1). Finally, considering the existence of pauci- or

asymptomatic forms of the dengue disease, technologies such as chemiluminescent OFIS, with

high sensitivity, may be very useful for diagnosis.

7.2. Development and characterization of Poly (methyl metacrylate)

conductive fiber optic transducers as dual biosensor platforms

In this study it was demonstrated the development of an alternative fiber-optic configuration

designed to the construction of biosensing platforms. This new approach was developed and

based on the chemical polymerization of pyrrole applied onto the surface of PMMA fibers to

create a conductive layer, which enabled the subsequent electrogeneration of photo-active PpyBz

upon the fiber surface. Irradiation of the benzophenone groups embedded on the Ppy films using

UV radiation (350 nm) enabled the covalent attachment of the desired biomaterials.

Characterization of the amperometric biosensing matrix was accomplished by using a model Urs

through EIS and amperometry. Both techniques have shown a low charge transfer resistance

and a high sensitivity. Thereafter, the construction of an optical biosensing matrix based on HRP

production of photons was carried out. The high signal to noise (S/N) ratio indicated clearly that

this approach can serve as a new platform to replace glass optical fibers based on biosensors.

Thus thorough characterization and optimization of this platform was carried out. The platform

showed conductive, stable, thin, controllable and light-transmissible film features. Various

parameters like time deposition, process temperature, activator plus pyrrole monomer

concentrations were examined in the study. The morphology and permeability of the PMMA

optic fibers were investigated to examine mass transfer ability. Cyclic voltammetry and

amperometry techniques were applied to characterize the electrical features of the surface and

charge transfer. The thorough characterization of the PMMA fibers established the conductive

and transparent features that opened a wide range of putative applications based on the combined

use of parallel light and conductivity measurements.

76

7.3. Alternative antigen

The production of a new alternative antigen based on hybrid protein between ED3 of the gpE

of each DENV serotype and a mutant alkaline phosphatase (PhoA) from E.coli allows the

development of DENV1-4 ED3 MAC-ELISA. The use of DENV1-4 ED3 MAC-ELISA has two

important advantages compared to MAC-ELISA. Firstly, it saves time in the immunoassay by

skipping two steps, and by performing an overnight incubation after adding anti-human IgM,

allowing faster detection after adding the tested sera. The second one is that using DENV1-4

ED3 gives for the first time the option to perform a serological test for the presence of anti-

DENV IgM antibodies according to infected DENV serotypes. The disadvantage of the system is

that it is based on alkaline phosphatase instead of HRP which in the colorimetric system the

signal development takes a lot of time (3-24 hours). Therefore the main advantage of using

luminescence in this system (based on alkaline phosphatase mutant) is the time saving since the

luminescence signal is rapidly developed compared to colorimetric signals. Moreover

both luminescence methods presented higher S/N than the colorimetric methodologies,

especially in panel point 4-6. Chemiluminescent measurements, especially OFIS and ELISA

based systems are already proven to be more sensitive compared to their colorimetric analogous.

Combining the advantages of DENV1-4 ED3 MAC-ELISA with luminescence can be the basis

of an improved diagnostic tool for the detection of IgM Ab to DENV in humans. Certainly the

specificity of the DENV ED3-PhoA hybrids needs to be improved in order to avoid cross-

reactivity between the DENV serotypes and other groups of flaviviruses. Despite of those

problems the results showed high specificity and in most of the cases the highest signal was

observed with the antigen corresponding to the serotype in charge of the DENV infection. The

validity of the results also came from a similar pattern that was observed by NRC in Dr. Phillipe

Dussart laboratory using colorimetric DENV1-4 ED3 MAC-ELISA. Furthermore the use of

internal panel reference sera from endemic areas enables a reliable comparison between the

different diagnostic methods. High correlation obtained from testing MAC-ELISA and ED3

MAC-ELISA using colorimetric or luminescence detection was observed to verify the potential

of this new method. These satisfactory results can be the basis for wider research to establish the

use of DENV1-4 ED3 MAC-ELISA for detection of IgM Ab to DENV in humans.

77

8. Concluding remarks and future perspectives

Although Dengue diagnosis has made much progress, there is still a great need to standardize

and improve current dengue diagnostics. Development of more sensitive, specific, rapid and cost

effective diagnostic tools, along with field operability for relatively underdeveloped regions of

the world, is of increasing importance. Therefore the main goal of this thesis was to develop a

luminescent based biosensor for the detection of DENV antibodies in human sera samples. In

this work we attempted a modest contribution by developing three luminescent diagnostic tools

for the detection of human anti-DENV IgM: chemiluminescent silica based OFIS,

chemiluminescent MAC-ELISA and chemiluminescent DENV1-4 ED3 MAC- ELISA. All the

techniques were compared to routinely use colorimetric MAC-ELISA. Taking into account the

lower limit of detection and the high correlation with the established methods using the known

panel, the OFIS technology reported here is reliable, simple to perform, fast, cost effective, and a

putative field operable analytical tool.

Moreover, we constructed and characterized a new alternative optic-conductive PMMA fiber

configuration employed for the construction of biosensing platforms. The PMMA platform

demonstrated conductive, stable, thin, controllable and light-transmissible film features. This

approach opened a wide range of putative applications based on the combined use of parallel

light and conductivity measurements. It also can be useful to improve the efficiency and

reproducibility for putative mass production of optic fiber. In the future, further intermediated

functionalized pyrrole can be examined in order to improve the efficiency of the binding and

therefore the technique. Carboxyl, amines and aldehyde groups can be used as intermediate

functionalized pyrrole.

In the last part of this work a new antigen was used in order to increase the specificity of the

bioassay technique and avoid cross-reactivity. The production of a new alternative antigen based

on hybrid protein between ED3 of the gpE of each DENV serotype and a mutant alkaline

phosphatase (PhoA) from E.coli allows the development of DENV1-4 ED3 MAC-ELISA. The

use of DENV1-4 ED3 MAC-ELISA has two important advantages compared to MAC-ELISA:

first, time is saved in the immunoassay, and two, is that using DENV1-4 ED3 gives for the first

time the option to perform a serological test based ELISA for the presence of anti-DENV IgM

antibodies according to infected DENV serotypes. The disadvantage of the system is that it is

78

based on alkaline phosphatase instead of HRP which in the colorimetric system the signal

development takes a lot of time (3-24 hours). This was improved by using luminescence

reaction, since the luminescence signal is rapidly developed compared to colorimetric signals.

Furthermore, a production of recombinant hybrid protein of each DENV serotype conjugated to

HRP can also solve this problem. Special focus is needed to be made on the development of

alternative antigen, and more importantly, according to a serotype, which will improve the

specificity and safety of the current diagnostic methods. Emergent technologies based on

molecular biology such as phage display techniques will benefit diagnostics by producing

molecules that are otherwise unobtainable by traditional approaches. Therefore exploitation of

the advantages of phage display system together with the ongoing development in the biosensor

field will lead to revolutionary diagnostic devices in general and in particular in DENV

detection.

79

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74. Abu-Rabeah, K., B. Polyak, R.E. Ionescu, S. Cosnier, and R.S. Marks, Synthesis and

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immunosensor for detecting cholera antitoxin. Optical Engineering, 1997. 36(12): p. 3258-3264. 98. Herrmann, S., B. Leshem, S. Landes, B. Rager-Zisman, and R.S. Marks, Chemiluminescent

optical fiber immunosensor for the detection of anti-West !ile virus IgG. Talanta, 2005. 66(1): p. 6-14.

99. Salama, O., S. Herrmann, A. Tziknovsky, B. Piura, M. Meirovich, I. Trakht, B. Reed, L.I. Lobel, and R.S. Marks, Chemiluminescent optical fiber immunosensor for detection of autoantibodies to

ovarian and breast cancer-associated antigens. Biosens Bioelectron, 2007. 22(7): p. 1508-16. 100. Sobarzo, A., J.T. Paweska, S. Herrmann, T. Amir, R.S. Marks, and L. Lobel, Optical fiber

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101. Leshem, B., G. Sarfati, A. Novoa, I. Breslav, and R.S. Marks, Photochemical attachment of

biomolecules onto fibre-optics for construction of a chemiluminescent immunosensor. Luminescence, 2004. 19(2): p. 69-77.

102. Konry, T., A. Novoa, Y. Shemer-Avni, N. Hanuka, S. Cosnier, A. Lepellec, and R.S. Marks, Optical fiber immunosensor based on a poly(pyrrole-benzophenone) film for the detection of

antibodies to viral antigen. Anal Chem, 2005. 77(6): p. 1771-9. 103. Petrosova, A., T. Konry, S. Cosnier, I. Trakht, J. Lutwama, E. Rwaguma, A. Chepurnov, E.

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biosensor for serological studies of Ebola virus in central Africa. Sensors and Actuators B-Chemical, 2007. 122(2): p. 578-586.

104. Polyak, B., E. Bassis, A. Novodvorets, S. Belkin, and R.S. Marks, Optical fiber bioluminescent

whole-cell microbial biosensors to genotoxicants. Water Science and Technology, 2000. 42(1-2): p. 305-311.

105. Polyak, B., E. Bassis, A. Novodvorets, S. Belkin, and R.S. Marks, Bioluminescent whole cell

optical fiber sensor to genotoxicants: system optimization. Sensors and Actuators B: Chemical, 2001. 74(1-3): p. 18-26.

106. Ramsden, J.J., Optical biosensors. Journal of Molecular Recognition, 1997. 10(3): p. 109-120. 107. Atias, D., Y. Liebes, V. Chalifa-Caspi, L. Bremand, L. Lobel, R.S. Marks, and P. Dussart,

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in humans. Sensors and Actuators B: Chemical, 2009. 140(1): p. 206-215. 108. Eltzov, E., D. Atias, G. Levi, and R.S. Marks, Dengue virus diagnostics, in Detection Of

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photon avalanche diodes and quenching circuits. journal of modern optics, 2004. 51: p. 1267-1288.

111. Herrmann, S. and R.S. Marks, Chemiluminescnt optical fiber immunosensor. 2007: Wiley. 112. Kulmala, S. and J. Suomi, Current status of modern analytical luminescence methods. Analytica

Chimica Acta, 2003. 500(1-2): p. 21-69. 113. Wang, J., Analytical electrochemistry. 2006: Wiley and sons. 114. Cosnier, S., Recent Advances in Biological Sensors Based on Electrogenerated Polymers: A

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coating of metallic medical devices. Journal of Polymer Science Part A: Polymer Chemistry, 2004. 42(7): p. 1658-1667.

116. Vidal, J.C., E. Garcia, and J.R. Castillo, Electropolymerization of pyrrole and immobilization of

glucose oxidase in a flow system: influence of the operating conditions on analytical

performance. Biosens Bioelectron, 1998. 13(3-4): p. 371-82. 117. Gerard, M., A. Chaubey, and B.D. Malhotra, Application of conducting polymers to biosensors.

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17(6-7): p. 441-456. 119. Porter, R.A., Investigation of electroplated conducting polymers as antibody receptors in

immunosensors. J Immunoassay, 2000. 21(1): p. 51-64. 120. Hurrelbrink, R.J. and P.C. McMinn, Molecular determinants of virulence: the structural and

functional basis for flavivirus attenuation. Adv Virus Res, 2003. 60: p. 1-42. 121. Megret, F., J.P. Hugnot, A. Falconar, M.K. Gentry, D.M. Morens, J.M. Murray, J.J. Schlesinger,

P.J. Wright, P. Young, M.H. Van Regenmortel, and et al., Use of recombinant fusion proteins and

monoclonal antibodies to define linear and discontinuous antigenic sites on the dengue virus

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123. Garcia, G., D.W. Vaughn, and R.M. Del Angel, Recognition of synthetic oligopeptides from

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subcomplex and complex B cell epitopes on the nonstructural-1 glycoprotein. Arch Virol, 1994. 137(3-4): p. 315-26.

125. Wu, H.C., M.Y. Jung, C.Y. Chiu, T.T. Chao, S.C. Lai, J.T. Jan, and M.F. Shaio, Identification of

a dengue virus type 2 (DE!-2) serotype-specific B-cell epitope and detection of DE!-2-

immunized animal serum samples using an epitope-based peptide antigen. J Gen Virol, 2003. 84(Pt 10): p. 2771-9.

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with arthropod-borne viruses. Am J Trop Med Hyg, 1958. 7(5): p. 561-73. 127. Kuno, G., I. Gomez, and D.J. Gubler, Detecting artificial anti-dengue IgM immune complexes

using an enzyme-linked immunosorbent assay. Am J Trop Med Hyg, 1987. 36(1): p. 153-9. 128. Greiner, M. and I.A. Gardner, Application of diagnostic tests in veterinary epidemiologic studies.

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קדחת הדנגילאיבחון ביוסנסור לומינסנטי פיתוח

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תקציר

המועבר לאדם Flaviviridaeהנגרמת על ידי נגיף הדנגי ממשפחת " מתחדשת"קדחת הדנגי הינה מחלה

י פרוקי הרגליים "נגיף הדנגי נחשב כיום כוירוס העיקרי המועבר ע Aedes.-באמצעות נקבת יתוש ממשפחת ה

DENV3 ,DENV2 זנים סרולוגים הגורמים למחלה והנקראים4לנגיף הדנגי ישנם . באזורים טרופים ותת טרופים

,DENV1 וDENV4 . אצל רוב האנשים שנדבקו בנגיף לא מופיעים סימנים קליניים או מופעים סימנים של מחלה

מהמקרים מפתחים מחלה קשה 0.5% -כ. הדומה לשפעת ומתבטאת בעיקר בחום גבוה ונקראת קדחת הדנגי, קלה

העלולה לגרום dengue shock syndrome (DSS) ולעיתים גםdengue hemorrhagic fever (DHF) הנקראת

דשות לאיתור הנגיף השיטות לאבחון קדחת הדנגי התקדמו מאוד בשנים האחרונות וכיום ישנם שיטות ח. למוות

למרות זאת דיאגנוסטיקה אמינה נותרה עדיין שלב מכריע בפיקוח ובקרה על . בעקבות ההדבקהשנוצרווהנוגדנים

. קדחת הדנגי בקרב בני האדם

המטרה העיקרית של מחקר זה הינה פיתוח ביוסנסור לומינסנטי לאבחון נוגדנים כנגד נגיף הדנגי בדגימות סרום

IgM [Colorimetric הושתתה על אליזת צבע לאבחון נוגדנים מסוג(immunoassay) הביולוגית המערכת. של אדם

IgM capture, enzyme-linked immunosorbent assay (MAC-ELISA)] שיטה זו נמצאת . כנגד נגיף הדנגי

ושוו מספר שיטות לאבחון בעבודה זו ה. מדינה אנדמית לנגיף הדנגי, בשימוש רוטיני עי מכון פסטר בגוויאנה הצרפתית

המבוססת על פליטת אור MAC-ELISA, המבוססת על צבעMAC-ELISA: כנגד נגיף הדנגיIgMנוגדנים מסוג

Chemiluminescent silica basedואימיונוסנסור כמילומנסנטי המבוסס על סיבים אופטיים העשויים מזכוכית

optical fiber immunosensor (OFIS)] [.86 כ וניסרקוהשתמשנו ברפרנס פאנלאלו ואה של שיטות לצורך השו

כמילומינסנטי המבוסס על סיבים אופטיים הינו בעל סף בהשוואה בין השיטות נראה כי אימיונוסנסור .סרומים של אדם

-MAC מ 100 המבוססת על פליטת אור ופי MAC-ELISA מאשר 10 נמוך פי lower detection limit)(אבחון

ELISAריכוז בינוני עד גבוה בעוד ב נוגדניםלכן שיטת האליזה יותר מתאימה לאבחון. המבוססת על צבע

אימיונוסנסורסריקת הסרומים באמצעות ה. מתאים גם לאבחון של ריכוזים נמוכים של נוגדנים בסרום אימיונוסנסורה

ואת הקורלציה , האבחון הנמוךכאשר לוקחים בחשבון את סף. 87.0% וספציפיות של 98.1%רגישות של הראתה

אימיונוסנסור כמילומינסנטי המבוסס על סיבים אופטיים ב השימוש ,שיטות המבוססות בבדיקת הרפרנס פאנללהגבוה

.מהיר ובעל אפשרות לשימוש בתנאי שטח, פשוט לביצוע, שהוצג בעבודה זו הינו אמין

poly methylבים אופטיים העשויים מ שימוש בסיהמבוססת על בנוסף בעבודה זו פותחה שיטה חדשה

methacrylate (PMMA)על מנת לאפשר קיבוע כימי יציב של חלבונים . אמפרומטרי- לבניית ביוסנסור אופטי

אליה פולמרו אלקטרוכימית פולמרו כימית על גבי הסיב ויצרו שיכבה יציבה ומוליכהpyrroleמונומרים של , לסיבים

polypyrrole-benzophenone (PpyBz) , הקרנה של קבוצת ה benzophenone באמצעותUV (350 nm)

מערכת זו אופיינה ביסודיות באמצעות פרמטרים רבים כגון ריכוז . אפשרה את הקישור הקוולנטי של החלבון הרצוי

מעבר ה את יכולת לבדוק על מנתאופיינומבנה פני השטח והחדירות של הסיבים . זמן וטמפרטורת התהליך, החומרים

90

יושמו על מנת לאפיין את התכונות האלקטריות של פני השטח ואת amperometryו cyclic voltammetry . חומרב

, דקה, הפוטנציאל של המערכת כביוסנסור אופטי ואמפרומטרי הוכח ואופיין כבעל שיכבה מוליכה. יכולת מעבר הזרם

ההדירות , בית זו יכולה לשמש כבסיס לשיפור היעילותגישה אלטרנטי. יציבה ואמינה בעלת יכולת העברת אור וזרם

מוליך יכול לפתוח את הדלת - כמו כן ביוסנסור אופטי. סנסורים המבוססים על סיבים אופטייםביווהעלות של

.לאפליקציות רבות המשלבות שימוש במעבר אור וזרם

הוא העדר, עיקר בשיטות הסרולוגיות ב ,אחד החסרונות בשיטות הדיאגנוסטיקה הקיימות כיום כנגד נגיף הדנגי

ולרוב הינם מהווים בסיס ליצירת סביבת עבודה בעלת הספציפיות של האנטיגנים הנמצאים בשימוש בשיטות אלה

לכן ישנו מאמץ מתמשך לפיתוח אנטיגנים אלטרנטיביים על מנת לשפר את האבחון של נוגדנים כנגד . סיכון ביולוגי

לכן בחלק האחרון של עבודה זו נעשה שימוש באנטיגן המבוסס על . ודה עם שיטות אלהנגיף הדנגי ואת הבטיחות בעב

פותחו כ . E. coliמ , PhoA, ושל מוטנט של אלקלאין פוספטז, ED3,חלבון איחויי של אפיטופ ויראלי של נגיף הדנגי

יטת אור בהשוואה לשיטה אפיטופים אלו נבדקו באמצעות אליזה המבוססת על פל. חלבוני איחוי אחד לכל סרוטיפ4

שיטה זו אפשרה אבחון סרולוגי על פי זן בזמן קצר יותר מהשיטה הרוטינית וללא צורך .MAC-ELISAהרוטינית

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