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Analytica Chimica Acta 681 (2010) 8–15

Contents lists available at ScienceDirect

Analytica Chimica Acta

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eview

review on viral biosensors to detect human pathogens

ebecca L. Caygill ∗, G. Eric Blair, Paul A. Millnernstitute of Membrane and Systems Biology, University of Leeds, Leeds LS2 9JT, UK

r t i c l e i n f o

rticle history:eceived 3 February 2010eceived in revised form2 September 2010ccepted 23 September 2010

a b s t r a c t

Rapid identification of viruses has important implications for medical healthcare. Current methods foridentification and quantification of particular virus are time consuming and often expensive. Therefore,demand for sensitive and accurate viral biosensors with rapid detection systems is increasing. A handheld biosensing device would give fast, reliable results for identifying and quantitating the number ofvirus particles in a sample. Techniques currently being applied to achieve this aim include electrochemi-

vailable online 1 October 2010

eywords:irus detection

mmunosensorabel-free detection

cal biosensors, based on amperometric, potentiometric and impedance measurement, optical biosensorsusing surface plasmon resonance (SPR), optical fibers and piezoelectric biosensors based on microcan-tilevers. Future research also looks to the use of nanoparticles and novel nanomaterials as alternaterecognition surfaces for use in a variety of sensor formats.

© 2010 Elsevier B.V. All rights reserved.

ticles;zyme

mmunoassayiosensor

R.L. Caygill gained a B.Sc. in Biochemistry with MedicalBiochemistry from the University of Leeds in 2007 andis currently in her final year of study for a Ph.D. withthe Biosensor and Biocatalysis Group at the Universityof Leeds. Her current research is on electrochemicalbased virus sensing, using both bacteriophage (MS2and M13) and human adenoviruses.

Abbreviations: Ab, Antibody; AFM, atomic force microscopy; AuNP, gold nanoparID50, egg infective dose 50%; EIS, electrochemical impedance spectroscopy; ELISA, en

aemagluttinin; HBV, hepatitis B virus; HCV, hepatitis C virus; HIV, human immunodeficielf-assembled monolayer; NASBA, nucleic acid-based sequence amplification; PCR, polyEA, restriction enzyme analysis; RT-PCR, reverse transcription-polymerase chain reactystematic evolution of ligands by exponential enrichment; SPR, surface plasmon resona∗ Corresponding author. Tel.: +44 113 343 3162.

E-mail address: bmb4rlc@leeds.ac.uk (R.L. Caygill).

003-2670/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.aca.2010.09.038

BSA, bovine serum albumin; cDNA, complementary DNA; CdTe, cadmium telluride;-linked immunosorbent assay; EPM, embedded piezoresistive microcantilever; HA,

G.E. Blair gained a B.Sc. in Biochemistry at the Uni-versity of Edinburgh and a Ph.D. in Biological Sciencesat the University of Warwick. He moved to postdoc-toral fellowships in molecular biology and virologyat Aarhus, Denmark; Uppsala, Sweden and NationalInstitute of Medical Research, London, before join-ing the University of Leeds as a lecturer in 1981.His particular research interests are DNA tumourviruses (adenoviruses, papillomaviruses, SV40 andpolyomaviruses), how they enter cells and evade theimmune system. His work aims to understand the bio-chemical mechanisms that underlie immune evasionsince this may reveal new targets to eradicate cancer-

causing viruses.

ency virus; HPV, human papilloma virus; HSV, herpes simplex virus; mSAM, mixedmerase chain reaction; Ppm, parts per million; QCM, quartz crystal microbalance;ion; SAM, self-assembled monolayer; SEM, scanning electron microscopy; SELEX,nce; TB, tuberculosis; TCID50, tissue culture infection dose at 50% endpoint.

a Chimica Acta 681 (2010) 8–15 9

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Table 1Varying sensitivity between types of viral assays in TCID50 (tissue culture infectiondose at 50% endpoint) [25].

Assay Sensitivity (TCID50)

R.L. Caygill et al. / Analytic

P.A. Millner gained a B.Sc. in Biochemistry and aPh.D. in Plant Science at the University of Leeds. Afterpostdoctoral fellowships at Purdue University, USAand Imperial College, he returned to Leeds in 1986as a lecturer. After 15 years as a plant biotechnolo-gist/protein chemist he moved into the area of nano-and bionanotechnology, with particular interests inthe applications of nanoparticles and developmentof biosensors for medical diagnostics, environmentalmonitoring and detection of biological and chemicaltoxins. However, all of his work is united by a deepinterest in bioengineering on the nanoscale by inter-facing biological reagents with surfaces to result in

electrical communication or enhanced activity.

. Introduction

Since Clark and Lyons’ glucose oxidase sensor to detect bloodlucose, in 1962, biosensing technologies have expanded [1].otential biosensor applications are now wide ranging from smallolecule detection (pesticide, herbicide, antibody and explosive)

o quantification of medical analytes (from small proteins to viralnd bacterial pathogens) [2–5]. The term biosensor is general andpplies to a variety of techniques which will be discussed in thiseview, especially with regard to detecting viruses. Viruses areiverse in their mechanism of action but usually comprise of airion (10–100 nm in size) containing a DNA or RNA genome sur-ounded by capsid proteins, some of which are used to gain entryo the host cell [6]. All viruses require a host in which to propagateo survive and many are human pathogens [7]. Most, such as theommon cold, are eradicated by the host innate immune response8] but more serious viruses evade this, threatening host survival9,10]. This is especially true in developing countries where malaria,B, pneumonia, influenza and HIV are pandemic. The detection anduantitation of viruses is fundamental for a wide range of appli-ations from sanitation and food production to diagnostics andherapeutics [11,12].

Viral infections often present generic symptoms which doctorsnd hard to use diagnostically. A biosensor would allow a doctoro ascertain the specific virus quickly and prescribe an appropriateourse of treatment. The presence of specific antibodies could alsoe detected using viral components as sensing agents to demon-trate a past history of infection in non-immunocompromisedatients.

Currently, viruses are detected using a variety of time con-uming methods within specialist laboratories. These includerotocols for cell culture over 2–10 days depending on the virusnd then rapid immunoassay procedures. Examples include NAS-As [13–15], REA [16,17] and direct immunofluorescent assays orLISAs aimed at membrane associated viral antigens [18–20]. Fur-her testing of RNA viral cultures using RT-PCR is also common,hereby viral nucleic acids extracted from samples are tran-

cribed into cDNA which is then amplified and detected usinguorescence or luminescence [21–24]. This is quicker than cellulture, taking 24 h compared with a minimum of 4 days, and isenerally more sensitive (Table 1) but can be expensive and com-lex.

Although effective, these methods are limited by sample enrich-ent and purification required prior to analysis, expense and time.biosensor should ideally be rapid, cheap, sensitive, specific and

eproducible, without the need for sample preparation, comparedo established techniques.

PCR 5–100Cell Culture 104

ELISA 105

This review looks at the current state of research and what thefuture holds with respect to detecting viruses efficiently, specif-ically and rapidly. These include optical methods e.g. SPR, fiberoptics, quantum dots; acoustic wave technologies e.g. QCM andelectrochemistry (amperometry, voltammetry and impedance) andnovel nanomaterials [7,26,27].

2. Bioreceptors and immobilisation methods

Bioreceptors are biomolecules selected due to their high speci-ficity to the target analyte and are immobilised onto a transducerto form a functional sensor. The target analyte comprises the anti-genic part of the virus e.g. whole virus, viral proteins (e.g. capsidproteins, fibers or toxins), viral nucleic acids (e.g. RNA or DNAgenomes) or specific antibodies produced by the host in responseto viral infection. In viral sensing the major bioreceptors are wholecells, antibodies, peptides, nucleic acids and aptamers. Antibod-ies are the most common bioreceptor and are produced by thehost in response to the presence of foreign molecules and organ-isms. For research purposes, monoclonal or polyclonal antibodiescan be raised specifically against a protein, another antibody oreven a whole virus and can bind with high affinity (Kd 106–109 M)[28–30]. Peptides are polymeric amino acids that form small pro-tein structures. They can be designed and synthesized, by phagedisplay, to specifically bind viral proteins or antibodies [31–33].Peptides are commonly used in ELISAs and biosensors as they aremore stable, safer to handle and widely available than viral proteinsor whole cells [34–38]. Nucleic acids are single chain oligonu-cleotides (18–40-mer) used to bind viral or bacterial RNA or DNA, bybase pairing [7,39], with high efficiency and specificity in the pres-ence of many different, non-complementary nucleic acids [40–42].Aptamers are similar to nucleic acids in that they are ligands ofDNA or RNA, but are specifically designed (using SELEX) to bind atarget protein and are viewed as a feasible alternative to nucleicacid and antibody bioreceptors. They are cheaper to produce andare more stable than nucleic acid based sensors, but they are moresusceptible to RNA nucleases when deployed in clinical samples[43–46].

Bioreceptors are immobilised onto solid phase transducers toform a functional sensing platform by physioabsorption onto aconducting polymer surface (e.g. polypyrrole or polyaniline) oran mSAM by covalent coupling to a linker molecule via carboxyl,maleimido, amino or thiol groups [7,26,47–51]. The linker moleculeis tethered to the transducer surface, which is often gold, carbon,silicon or hydrogels, using a suitable method including strepta-vidin/biotin affinity, silanisation, protein A or direct attachment[52–55]. The immobilisation of the bioreceptor is fundamental forfunctionality and integrity of the sensor, i.e. the antibody epitopebinding domain must be orientated to facilitate binding to thetarget analyte. Often steric hindrance and denaturation of the biore-ceptor is an issue for reproducibility and sensitivity [52,56–59].Another problem can be cross-specificity between bioreceptors and

non-target molecules. This can be addressed by selecting appropri-ate and highly specific bioreceptors for the viral antigens and byblocking the non-specific binding sites of the sensor surface with aprotein before use.

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0 R.L. Caygill et al. / Analytic

. Electrochemical viral sensors

These are sensors which use electrochemical interrogationncluding current, potential and impedance changes to trans-uce the biological recognition event [29,60]. They were firsteveloped in 1962 with the glucose electrode [1] and combinehe analytical power of techniques such as voltammetry, poten-iometry and conductometry with the specificity of biologicalecognition [60,61]. Early efforts concentrated on sensing smallnalytes e.g. metabolites such as urea or lactate and glucose2,62–64] amperometrically, but in recent years the detection ofarger macromolecules such as proteins has emerged. In this case,etection of the binding event itself via a bioreceptor, using elec-rochemical transduction across the sensor surface or signal, has

ostly been used [47,49,65,66], a method recently applied to viraliosensing. The complex formed from the bioreceptor and the viralntigen binding (typically with a Kd of 106–109 M) at the sensor sur-ace results in a detectable change, converted into a quantitativemperometric, potentiometric or impedimetric signal. Comparedith nearly all other analytical techniques, electrochemical detec-

ion assays have the advantage of being inexpensive, robust andelatively simple to operate [57,67–71].

.1. Voltammetric and amperometric viral sensors

These sensors measure the current–potential relationship inn electrochemical cell; whereby a potential is applied and theesulting current is measured in the case of amperometry. Inoltammetry, the voltage is measured when no current is applied5,72]. For amperometric transducers to detect the analyte bindinghere must be an electroactive product that can undergo a redoxeaction at the electrode. This is usually accomplished by adding aecondary antibody-redox enzyme complex and has been termedn ‘electronic ELISA’.

Amperometry has been applied successfully to detect Epstein-arr virus, cytomegalovirus, HSV and HBV [68] to 3.96 × 10−7 Mf viral DNA using cyclic voltammetry or differential pulsedoltammetry [2]. Authors describe silicon dioxide based microchiprrays with interdigitated gold ultramicroelectode inserts to form

multi-channel array that allowed a multi-channel potentio-tat to simultaneously analyse multiple samples of viral DNA2,73]. Amperometric biosensors often utilise screen printed car-on electrodes because they are stable, inexpensive, disposablend require only a small volume of sample [5]. However, oneroblem faced with amperometric immunosensors, as with alliosensors, is immobilisation of the bioreceptor without denatu-ation or random orientation [74]. Therefore, most amperometricmmunosensors use gold electrode surfaces since they allow for-

ation of self-assembled monolayers (SAMs) of thiol reagents.owever, graphite-based inks can be printed onto a polystyrene

urface to serve as electrode material and allow passive adsorptionf antibodies [72].

An innovative amperometric sensor for West Nile Virus (WNV)as described by Ionescu et al., whereby photochemically immo-

ilised T7 bacteriophage displaying a specific WNV epitopeuccessfully detected WNV antibody dilutions ranging from 10 to06. This demonstrates the potential for amperometric transduc-ion in viral diagnostics [75]. Sensors have also been developedith immobilised ssDNA complimentary to that of the viral DNA

n a glassy carbon solid phase transducer. Upon analyte binding,he newly formed dsDNA transferred electrons along the phos-

hate backbone, detected by differential pulse voltammetry and

inear sweep voltammetry. This method has been used by severalroups in the detection of HBV. Some applications require an elec-roactive mediator, such as hydroxaniline [2,68,73], whilst others

easure the flow of electrons directly through the dsDNA helix,

ica Acta 681 (2010) 8–15

using only cobalt complexes which increased efficiency and sen-sitivity by the planar aromatic ring intercalating with the DNA viahydrogen bond interactions [68,76]. This method used an Ag/AgClelectrode as a reference and is not currently suitable to be used ‘in-field’ as it requires technical interpretation of results with specialistalgorithms. Detection was reached within 2 h without any previousDNA purification with a detection limit of 1.5 mg L−1 and resultswere confirmed with AFM [77]. Problems encountered when usingpotentiometry and amperometry include sensitivity to pH changesand non-specific binding of molecules, leading to false positivedetection. This is particularly true for polypyrrole film sensing sur-faces. The open circuit potentials of partially oxidised (both ion andelectron conducting membrane) polypyrrole are strongly affectedby H+ ions, and although this is an advantage in solid-state pH sen-sors, it is causes severe interferences in other applications, suchas viral biosensing, under both potentiometric and amperometricconditions [78–80].

3.2. Impedance viral sensors

Electrochemical impedance immunosensors are typically con-structed either using an mSAM base layer [47,58,49,57] or aconducting polymer base layer [65,81] as previously described, aschematic of immobilisation can be seen in Fig. 1.

Upon hybridisation between the bioreceptor and the viral anti-gen, there is a measurable response in conductivity across theimmunosensor surface, which is translated into a change in theresistance and/or double-layer capacitance following analyte cap-ture [72]. Detection of the change in capacitance is easier tomeasure as no reference electrode is required and therefore it ismore durable ‘in-field’. However, this technique can be less sen-sitive and non-specific binding can lead to false positive results[29,72]. This can be addressed by ‘blocking’ the non-specific bindingsites of the surface with a protein, such as BSA. A secondary anti-body conjugate or AuNP can also be used to amplify the responseof the binding event [82–84].

EIS has been applied to detect a number of viruses includinginfluenza virus, herpes virus, HBV, HCV, dengue virus, rabies virusand HIV [40,85–93]. Of note was the use of faradaic EIS to detectthe binding mechanisms and kinetics of immobilised HIV antigensto HIV antibodies [29]. This approach used a secondary anti-HIV

Fig. 1. Formation of a viral immunosensor using an antibody tethered to an mSAMon a gold electrode to be interrogated by EIS.

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he detection of HIV, EIS was utilised to count CD4+ cells as quan-itative diagnosis of HIV-infection status in AIDS patients [88]. Thisas a revolutionary approach as it used an array of chemicallyodified electrode pixels to capture individual CD4+ helper cells,hich were then electrochemically interrogated to determine cap-

ure status and indicate the stage of HIV infection. Influenza virusH5N1 serotype) was detected by EIS interrogation of an interdig-tated array electrode comprised of protein A tethered antibodiesgainst the viral surface protein HA [85]. The capture of the virusas confirmed by AFM, with the immunosensor found to have aetection limit of 103 EID50 mL−1 within 2 h, depending on the titref the virus [85]. Although the research to date is promising; EISnterrogation for viral sensing is still in its infancy. Developmentsre continually progressing in sensor surface chemistry to improvelectrode stability, selectivity, sensitivity and speed of responses well as ability to monitor real time dynamic fluctuations. Untileproducibility improves and electrochemical immunosensors cane used by non-specialists there is an issue with marketability.owever, this will be vastly improved as issues are addressed.

. Optical viral sensors

Two of the most popular optical biosensor formats are based onptical fibers and surface plasmon resonance (SPR) surfaces. Opticalbers rely on the total internal reflection of light and the charac-erisation of a surface by the difference in the angle of incidencef reflected light upon sensor surface modification. Optical sensorsetect specific binding between analytes [94], as with other sens-

ng technologies, but do so with no direct electrical connection [95].hey too are fast, reliable and sensitive [95]. However, in generalPR machines are expensive with two of the several commerciallyvailable biosensors to date being the BIAcoreTM system and Texasnstruments’ SpreetaTM [96].

.1. Surface plasmon resonance (SPR)

The use of SPR to interrogate a viral sensor is based on totalnternal reflection in a glass prism onto which a thin metal filmusually gold) is deposited. When total internal reflection of thencident light occurs, an evanescent wave is set up that exerts aeld outside of the prism. This can only occur when the film ishin enough, approximately 50 nm [96] as the delocalised electronsithin the metal cause a surface plasmon to be established at theetal–ambient interface. This causes a wave to propagate along

he surface and therefore reduces the intensity of light reflectedFig. 2).

The angle of incidence at which this occurs is called the SPR anglend is determined by the angular frequency of the light source,ielectric constant of the metal and the geometry of the prism.owever, if these are not changed, then the SPR angle dependsnly on the refractive index, �, of the ambient medium, on the far

ig. 2. Formation of surface plasmon resonance wave for sensor as incidence lightlluminates thin layer of gold causing electrons to be excited and form a surfacelasmon wave at the far side. See Iribe [96] for more detail.

Fig. 3. Principle of 2D-SPR. The film acts as a 2D environment for surface plasmonsand has an adjacent reference area. The reflected light is detected by a charge coupleddevice and forms a pixelated 2D image in real time.

side of the gold film [96–99]. Therefore, if an analyte is passed overan immobilised bioreceptor on the far side of the gold surface, achange in the SPR angle is produced. Coupled with a microfluidicformat both whole virus and bioreceptor binding kinetics as well asbinding affinity can be calculated using minimal amounts of virusor material and with high selectivity and sensitivity [98,100].

The latest development with SPR is 2D-SPR, which allows realtime monitoring of the binding events at the surface using a chargecoupled device (CCD) camera, as shown in (Fig. 3). A CCD camerais used which registers an electric charge upon illumination. Thisallows imaging of the SPR surface to be built up in real time and is avaluable tool in the commercial application of optical immunosen-sors.

To date, SPR has been widely used to observe virus and viral anti-body binding, especially with regards to binding affinity [101,102].Rich and Myszka [103] harnessed SPR to observe of many aspectsof HIV biology. These included binding events such as docking andreplication, budding and maturation as well as epitope mappingand kinetic studies of chemokine receptors. It was concluded thatSPR was a key tool in uncovering many aspects of HIV and using itto develop new anti-HIV drugs.

In 2005, Chung et al. [104] described the use of SPR to detectcaptured human HBV antibodies using a HBV surface antigen inconjunction with a secondary antibody. The detection limit wasfound to be 10 nM HBV antibody. Although this work showed suc-cessful detection of antibodies against HBV, little research has beenpublished since about using SPR specifically as a diagnostic tool.Rather SPR is applied for determination of the binding affinitybetween two molecules in conjunction with other more portableand quick methods. However, it was recently reported that SPRwas used to detect HA proteins of influenza serotypes H1N1 andH3N2 by an inhibition assay to a detection limit of 0.5–10 �g mL−1

[105], which proved more sensitive than the current technique ofquantification of HA by using single-radial immunodiffusion (SRID).SPR has potential for portable rapid in-field viral testing althoughrecent efforts to miniaturise SPR platforms using optical fibers andminiature prism couplers have been limited due to modal noise andpolarization instability [100].

4.2. Optical fiber viral sensors

Optical fiber based biosensors use single optical fibers or bun-dles to detect the presence of an analyte, again using total internalreflection of incidence light. A typical fiber is made of glass or plastic

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nd has two layers. The inner layer is termed the core and the outerayer the cladding. Both are encased in a protective coating against

oisture and damage [94]. Total internal reflection occurs at thenterface between the core and cladding only when the refractivendex of the core is greater than that of the cladding. This propagatesver a very long distance and thousands of these fibers can be fusedogether to form an optical fiber array of 0.2–2 mm in diameter andomprising up to 105 fibers.

The sensing materials used include fluorescently labelled poly-ers, micron sized beads and living cells. When incident light is

ntroduced at the proximal end, these indicators are excited andbsorb the light, allowing fluorescent light to be emitted. This lights transmitted along the fiber to the distal end and the difference in

avelength between incident light and fluorescent light is recordeds a signal. Either absorbance or intensity of the returning light cane measured. These sensor materials are susceptible to bleachingnd leaching of the bioreceptor [95]. However, with the adventf luminometry, which does not have the problems associatedith fluorescence, optical fibers are becoming more of a promisingrospect for infield, cheap and fast diagnosis.

The number of fibers in a bundle is limited due to the outerladding on each fiber adding to the diameter of the overall bun-le. High density arrays of micron and nano scale fibers have beenesigned to overcome this. Each fiber carries an isolated opticalignal and can be used in multiplexed sensors by photodepositingany sensor elements onto the distal face of the imaging fibers

95].Petrosova et al. [106] have developed an immunosensor to

etect antibodies against Ebola strains Zaire and Sudan by immo-ilising Ebola virus antigen onto a photoactivable electrogeneratedolymer film on an optical fiber tip. Upon specific binding of sec-ndary antibodies a change was detected in the luminescence of theptical fiber which correlated to the amount of Ebola virus antibodyound. Authors report that this system was able to detect anti-Ebolantibodies down to 1 ppm [106]. A similar system based on chemi-uminescent detection was also employed by Herrmann et al. [108]o detect anti-West Nile virus IgG and by Atias et al. [107] to detecthe IgM antibody against human Dengue fever [107,108]. Researchas also described using sandwich ELISA based optical fiber sensors

o detect Rift Valley fever virus (RVFV) IgG in sera using immo-ilised RVFV to 95% accuracy [109]. Detection using optical fiber

mmunosensors appears in all cases to be more sensitive than theirnalogous colorimetric and chemiluminescent ELISA counterpartsith detection limits 10-fold and 100-fold lower respectively.

Brossier et al. [71] and Seydack [110] investigated nanoparti-le labels for use in optical sensors to replace gold film. It wasound that lanthanide chelates, colloidal gold, polymer beads, car-on nanoparticles, quantum dots and organic nanoparticles werell practical substitutes for sensor surfaces in optical viral sensors37,38,107,111,112]. Griffin et al. [113] used the nonlinear opti-al properties of gold nanoparticles for the first time to detect anduantify HCV RNA without any modifications using hyper-Rayleighcattering. It was found possible to detect a single pair mismatchnd the detection limit was extremely low at 60 pM [113]. Dengt al. described the simultaneous and independent detection ofwo viruses; H9 avian influenza virus and murine herpesvirus 68sing green and orange CdTe quantum dots [111]. Although notuman virus, this is a very promising multidetection method forther diagnostic applications.

. Piezoelectric detection methods for use in viral sensing

Viral sensors that utilise piezoelectric methods include mass-ased biosensors, such as the quartz crystal microbalance (QCM)nd microcantilever arrays, which are essentially based on atomic

ica Acta 681 (2010) 8–15

force microscopy (AFM), originally developed in 1986 [114]. It isfeasible to apply such electromechanical devices for virus (and bac-terial) detection, owing to the relatively high macromolecular massof these entities.

5.1. Quartz crystal microbalance

The approach of determining mass deposition onto a chip sur-face is very useful for characterising the surface has often been usedin concert with the other techniques to detect virus–antibody bind-ing. QCM is based on the property of an AT cut quartz crystal whichoscillates when an alternating voltage is applied. The quartz surfacecan have a thin gold, or other metal layer, deposited onto it, whichdoubles as a conductor for the AC current flowing and also providesan attachment surface for a bioreceptor against a virus to be immo-bilised. This deposition can be monitored by the frequency decreasecaused by the additional mass on the chip which is related to themass change via the Sauerbrey equation [115]. Once the biorecep-tor is seen to bind to the surface the crystal can then be washedwith buffer before addition of the viral target in suspension whichagain, adds to the mass and reduces the frequency.

Skladal et al. [116] reported the use of QCM to detect HCV virusDNA in serum using biotinylated DNA probes, immobilised onto abiotin tagged SAM surface via streptavidin coupling. Meanwhile,Dell’Atti et al. [117] detected HPV using QCM in combination withPCR, again using DNA probes immobilised onto a SAM layer. Workwas also conducted by Yao et al. [118] into detecting HBV genomicDNA using peptide nucleic acids specific to HBV immobilised ontothe biosensor, with a detection limit using the QCM of 8.6 pg L−1.

QCM is also an invaluable tool for characterising immunosensorchip fabrication and is often used alongside electrochemical tech-niques as the sensor construction can be observed at each stage.However, if the binding analyte is flexible or compressible, theSauerbrey equation does not apply to the data. In this case dis-persion QCM (QCM-D) must be measured [119]. Washing of QCMcrystals can be a potential problem but non-specific binding can bereduced by using a fast flow rate of analyte sample over the sens-ing surface. This sensing technology is not currently suitable forin-field use but is valuable for laboratory based use alongside othermethods such as SPR.

5.2. Atomic force microscopy

Atomic force microscopy (AFM), developed by Binning et al.[121], utilises a sharp tipped oscillating cantilever to characterisethe surface of a sensor (Fig. 4). The lever oscillates at a frequencyclose to that of the surface and a laser is focussed onto the cantileverin such a way that the position of the reflected beam is detected bya CCD chip, thereby allowing tip deflection to be followed.

The tip bends as it encounters an uneven surface, e.g. the biolog-ical component. This can be caused by a whole cell or virus attachedto the tip binding to an antibody immobilised on the sensor surfacefor example. The bend in the cantilever is recorded by the move-ment of the reflected laser [120–122]. The cantilever’s movementis constant because it is mediated by a piezoactuator and the sensorcan be characterised by observing the amplitude, phase and reso-nance frequency of the cantilever. The signal is extrapolated to givea 3D image of the surface, whereby individual molecules are visible.

AFM is highly sensitive and can detect atomic bonding andvan der Waals forces. It does not require the chemical treatments,labelling, staining or fixation that other techniques do, such as scan-

ning electron microscopy. AFM has been substantially developedsince its introduction and is now widely used as an effective toolfor structural determination, identification and characterisation ofmany pathogens [123]. In the last 10 years, more than 100 stud-ies involving AFM have been conducted on proteins and viruses,

R.L. Caygill et al. / Analytica Chimica Acta 681 (2010) 8–15 13

Table 2The biosensing techniques, discussed in this review, which are used to detect and quantify different virus.

Virus Impact on health Characteristics of sensor Detection limit Reference

Dengue Dengue fever, dengue shock syndrome Optical 1 part per million Atias et al. [107]AFM Individual particle ± 10.22 nm Ferreira et al. [120]

Ebola Acute haemorrhagic fever Optical 1 ppm Petrosova et al. [106]HCV Liver inflammation Optical 60 pM Griffin et al. [113]

QCM 200 �g mL−1 Skladal et al. [116]Liver cirrhosis, hepatocellular carcinoma 1.5 mg L−1 Kobayashi et al. [77]

HBV Voltammetry 7.19 × 10−9 M Zhang et al. [76]1.94 × 10−8 M Ding et al. [68]

QCM 8.6 pg L−1 Yao et al. [118]HIV Acquired immunodeficiency syndrome SPR 1 kDa Rich and Myszka [103]

EIS 0.5 �g mL−1 Katz et al. [29]AFM Individual particle ± 11 nm Kuznetsov et al. [127]

HPV Skin and genital warts; cervical, anal, vulvar and penile carcinoma QCM 50 nM Dell’Atti et al. [117]HSV Skin and genital lesions Voltammetry 3.96 × 10−7 M Albers et al. [2]Influenza Fever, gastroenteritis and pneumonia EIS 103 EID50 mL−1 Wang et al. [31]

SPR 0.5–10 �g mL−1 Nilsson et al. [105]AFM 4 virus particles per mL Owen et al. [128]AFMEIS

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Orthopox Smallpox; fever, lesions, encephalitis and erythemaRabies Acute encephalitis and lethargy

emonstrating the potential of this technology [120,122,124]. It hasspatial resolution down to 1 nm and can be used with proteins in

heir natural state such as on the cell membrane [125]. Nanoelec-romechanical systems (NEMs) have increased the sensitivity limiturther [114].

AFM is essentially the basis for microcantilever array deviceshereby the movement of a ‘micro diving board’ can measure

iscosity, density and flow rate by the changes in vibrational fre-uency. The deflection of the microcantilever can be detected, usingapacitor plates, due to molecular adsorption on one side of theicrocantilever (Fig. 4) and the deflection is proportional to the

nalyte concentration [114,126]. One disadvantage of AFM is thathe laser beam can head the surrounding liquid in the cell, poten-ially causing degradation and denaturation of biological samples.

AFM has been used to detect HIV [127], influenza virus [128],rthopox virus [129] and dengue fever virus [120]. Gunter et al.130] have reported use of an embedded piezoresistive microcan-ilever (EPM) sensor to detect vaccinia virus both in solution and

erosols. An EPM is a tiny piezoresistive microcantilever embed-ed in an active sensing material, such as a polymer or hydrogel, tohich an anti-vaccinia polyclonal antibody was immobilised and

erosol vaccinia virus was detected. In conclusion, AFM is a use-

ig. 4. Schematic to show action of microcantilever using AFM topping mode. Aaser is angled to reflect off the micro-cantilever as it passes over the sensor surface.pon virus–antibody interaction, the micro-cantilever bends and causes the anglef the reflected beam to change. This change is then recorded and analysed.

Individual particle ± 10 nm Trindade et al. [129]0.5 �g mL−1 Hnaien [91]

ful tool for confirming the presence of virus in laboratory basedstudies. Although label-free it does rely on sample preparation andwould not be suitable for in-field use due to its extreme vibrationsensitivity.

6. Future perspectives

Detecting viruses and viral components using biosensors is arelatively new concept and for many of the techniques discussed,is still in its infancy. Traditional viral detection methods are timeconsuming and expensive, and consequently, demand for accu-rate viral biosensors by rapid detection systems is increasing. Sincetheir development in the 1990s, AFM and QCM have been the mostresearched techniques to date (Table 2) alongside optical meth-ods, but electrochemical techniques will probably become morepopular as microelectronics and microfluidics also progress.

Some techniques are restricted due to unsuitability in-field andrequirement of highly trained personnel. Many have limitationsinvolving sensor surface formation, reproducibility, renewabilityand necessary sample preparation. More research is needed todevelop this comprehensive library of core techniques into prac-tical diagnostic applications. The general basis of methods todetect binding viral analytes to specific bioreceptors is presentbut requires refinement and in some cases further miniaturiza-tion. With the advent of novel nanomaterials, with optical andelectrical based properties, and nanoparticle based assays it isinevitable the methods described will evolve along with emergingnanotechnologies. Furthermore, bioreceptor design and immobili-sation techniques are progressing to increase both the sensitivity,selectivity and signal amplification of viral sensors. Sensors willbecome smaller and more portable and costs will reduce, as feweror new materials are utilised. This will hopefully lead to the pro-duction of sensitive, cheap, reliable and rapid detection systems foruse in the clinical environment for diagnosis of viral infections.

Acknowledgement

This research was supported by the Biotechnology and Biologi-

cal Sciences Research Council 2010.

References

[1] L.C. Clark, C. Lyons, Ann. N.Y. Acad. Sci. 105 (1962) 20.

1 a Chim

4 R.L. Caygill et al. / Analytic

[2] J. Albers, T. Grunwald, E. Nebling, G. Piechotta, R. Hintsche, Anal. Bioanal.Chem. 377 (2003) 521.

[3] D. Ivnitski, I. Abdel-Hamid, P. Atanasov, E. Wilkins, Biosens. Bioelectron. 14(1999) 599.

[4] C.T. Lin, S.M. Wang, Front. Biosci. 10 (2005) 99.[5] D.H. Yu, B. Blankert, J.C. Vire, J.M. Kauffmann, Anal. Lett. 38 (2005) 1687.[6] K.M. Pepin, J. Domsic, R. McKenna, Infection, Genet. Evol. 8 (2008) 825.[7] X. Munoz-Berbel, N. Godino, O. Laczka, E. Baldrich, F. Xavier Munoz, F. Javier

Del Campo, Impedance-Based Biosensors for Pathogen Detection, Springer,2008.

[8] S. Akira, S. Uematsu, O. Takeuchi, Cell 124 (2006) 783.[9] N. Philpott, G.E. Blair, in: I.K.M.A.J. Zuckerman (Ed.), Perspectives in Medical

Virology, Elsevier, 2001, p. 413.[10] G.E. Blair, M.E. Blair-Zajdel, Adenoviruses: Model and Vectors in Virus–Host

Interactions (Current Topics in Microbiology and Immunology), Springer-Verlag, Berlin, 2004, p. 3.

[11] L. Yang, R. Bashir, Biotechnol. Adv. 26 (2008) 135.[12] F. Ricci, G. Volpe, L. Micheli, G. Palleschi, Anal. Chim. Acta 605 (2007) 111.[13] T. Kievits, B. Vangemen, D. Vanstrijp, R. Schukkink, M. Dircks, H. Adriaanse, L.

Malek, R. Sooknanan, P. Lens, J. Virol. Methods 35 (1991) 273.[14] R.S. Lanciotti, A.J. Kerst, J. Clin. Microbiol. 39 (2001) 4506.[15] J. Saldanha, N. Lelie, A. Heath, W.H.O.C.S. Grp, Vox Sang. 76 (1999) 149.[16] K. Zierenberg, R. Raue, H. Muller, Avian Pathol. 30 (2001) 55.[17] V. Gouvea, C.H. Hoke, B.L. Innis, J. Virol. Methods 70 (1998) 71.[18] A.C.M. Boon, A.M.F. French, D.M. Fleming, M.C. Zambon, J. Med. Virol. 65

(2001) 163.[19] A. Voller, A. Bartlett, D.E. Bidwell, M.F. Clark, A.N. Adams, J. Gen. Virol. 33

(1976) 165.[20] A. Gadano, O. Galdame, S. Marciano, Ann. Hepatol. 9 (2010) S34.[21] A. Judd, J. Parry, M. Hickman, T. McDonald, L. Jordan, K. Lewis, M. Contreras,

G. Dusheiko, G. Foster, N. Gill, K. Kemp, J. Main, L. Murray-Lyon, M. Nelson, J.Med. Virol. 71 (2003) 49.

[22] S.A. Bustin, J. Mol. Endocrinol. 25 (2000) 169.[23] W.N. Burnette, Anal. Biochem. 112 (1981) 195.[24] R. Pauwels, J. Balzarini, M. Baba, R. Snoeck, D. Schols, P. Herdewijn, J. Desmyter,

E. Declercq, J. Virol. Methods 20 (1988) 309.[25] H. Kohler, K.N. Masihi, W. Lange, O. Knorn, Zentralbl. Bakteriol. Mikrobiol.

Hygiene Series a – Med. Microbiol. Infect. Dis. Virol. Parasitol. 246 (1980)474.

[26] A.W. Wark, J. Lee, S. Kim, S.N. Faisal, H.J. Lee, J. Ind. Eng. Chem. 16 (2010) 169.[27] J. Heo, S.Z. Hua, Sensors 9 (2009) 4483.[28] P.J. Conroy, S. Hearty, P. Leonard, R.J. O’Kennedy, Semin. Cell Dev. Biol. 20

(2009) 10.[29] E. Katz, I. Willner, Electroanalysis 15 (2003) 913.[30] P. Holliger, P.J. Hudson, Nat. Biotechnol. 23 (2005) 1126.[31] S.Q. Wang, E.S. Humphreys, S.Y. Chung, D.F. Delduco, S.R. Lustig, H. Wang,

K.N. Parker, N.W. Rizzo, S. Subramoney, Y.M. Chiang, A. Jagota, Nat. Mater. 2(2003) 196.

[32] M. Yemini, M. Reches, E. Gazit, J. Rishpon, Anal. Chem. 77 (2005) 5155.[33] P. Samuelson, E. Gunneriusson, P.A. Nygren, S. Stahl, J. Biotechnol. 96 (2002)

129.[34] H. Aoki, P. Buhlmann, Y. Umezawa, Electroanalysis 12 (2000) 1272.[35] K. Kerman, D. Ozkan, P. Kara, A. Erdem, B. Meric, P.E. Nielsen, M. Ozsoz, Elec-

troanalysis 15 (2003) 667.[36] M. Zhou, I. Ghosh, Biopolymers 88 (2007) 325.[37] K.E. Sapsford, J.B. Blanco-Canosa, P.E. Dawson, I.L. Medintz, Bioconjug. Chem.

21 (2010) 393.[38] K.E. Sapsford, T. Pons, I.L. Medintz, S. Higashiya, F.M. Brunel, P.E. Dawson, H.

Mattoussi, J. Phys. Chem. C 111 (2007) 11528.[39] E. Palecek, M. Fojta, Anal. Chem. 73 (2001) 74A.[40] F. Lucarelli, G. Marrazza, A.P.F. Turner, M. Mascini, Biosens. Bioelectron. 19

(2004) 515.[41] J. Wang, Nucleic Acids Res. 28 (2000) 3011.[42] J. Wang, Anal. Chim. Acta 469 (2002) 63.[43] A.A. Rowe, E.A. Miller, K.W. Plaxco, Anal. Chem. 82 (2010) 7090.[44] M. Liss, B. Petersen, H. Wolf, E. Prohaska, Anal. Chem. 74 (2002) 4488.[45] S. Tombelli, A. Minunni, A. Mascini, Biosens. Bioelectron. 20 (2005) 2424.[46] I. Willner, M. Zayats, Angew. Chem.-Int. Ed. 46 (2007) 6408.[47] M. Billah, H.C.W. Hays, P.A. Millner, Microchim. Acta 160 (2008) 447.[48] A.A. Karyakin, G.V. Presnova, M.Y. Rubtsova, A.M. Egorov, Anal. Chem. 72

(2000) 3805.[49] H.C.W. Hays, P.A. Millner, M.I. Prodromidis, Sens. Actuators B: Chem. 114

(2006) 1064.[50] S. Weiss, P. Millner, A. Nelson, Electrochim. Acta 50 (2005) 4248.[51] J.J. Gooding, F. Mearns, W.R. Yang, J.Q. Liu, Electroanalysis 15 (2003) 81.[52] B. Prieto-Simon, M. Campas, J.L. Marty, Protein Pept. Lett. 15 (2008)

757.[53] E. Brynda, M. Houska, J. Skvor, J.J. Ramsden, Biosens. Bioelectron. 13 (1998)

165.[54] Y. Liebes, L. Amir, R.S. Marks, M. Banai, Talanta 80 (2009) 338.

[55] S. Zheng, D.K. Kim, T.J. Park, S.J. Lee, S.Y. Lee, Talanta 82 (2010) 803.[56] K. Bonroy, F. Frederix, G. Reekmans, E. Dewolf, R. De Palma, G. Borghs, P.

Declerck, B. Goddeeris, J. Immunol. Methods 312 (2006) 167.[57] P.A. Millner, H.C.W. Hays, A. Vakurov, N.A. Pchelintsev, M.M. Billah, M.A.

Rodgers, Semin. Cell Dev. Biol. 20 (2009) 34.[58] N.K. Chaki, K. Vijayamohanan, Biosens. Bioelectron. 17 (2002) 1.

ica Acta 681 (2010) 8–15

[59] S.F. Chen, L.Y. Liu, J. Zhou, S.Y. Jiang, Langmuir 19 (2003) 2859.[60] J. Wang, Analytical Electrochemistry, Wiley-VCH, New Jersey, 2006.[61] A.J. Bard, L.R. Faulkner, Electrochemical Methods, 2nd ed., John Wiley and

Sons, NewYork, 2001.[62] S.B. Adeloju, S.J. Shaw, G.G. Wallace, Anal. Chim. Acta 323 (1996) 107.[63] F. Mizutani, S. Yabuki, Y. Sato, T. Sawaguchi, S. Iijima, Electrochim. Acta 45

(2000) 2945.[64] S.B. Adeloju, S.J. Shaw, G.G. Wallace, Anal. Chim. Acta 341 (1997) 155.[65] A.C. Barton, S.D. Collyer, F. Davis, G.-Z. Garifallou, G. Tsekenis, E. Tully, R.

O’Kennedy, T. Gibson, P.A. Millner, S.P.J. Higson, Biosens. Bioelectron. 24(2009) 1090.

[66] T. Hianik, V. Gajdos, R. Krivanek, T. Oretskaya, V. Metelev, E. Volkov, P.Vadgama, Bioelectrochemistry 53 (2001) 199.

[67] S. Hahn, S. Mergenthaler, B. Zimmermann, W. Holzgreve, Bioelectrochemistry67 (2005) 151.

[68] C.F. Ding, F. Zhao, M.L. Zhang, S.S. Zhang, Bioelectrochemistry 72 (2008) 28.[69] M. Pumera, S. Sanchez, I. Ichinose, J. Tang, Sens. Actuators B: Chem. 123 (2007)

1195.[70] K. Kerman, M. Kobayashi, E. Tamiya, Meas. Sci. Technol. 15 (2004) R1.[71] P. Brossier, G. Jaouen, B. Limoges, M. Salmain, A. Vessieres-Jaouen, J.P. Yvert,

Ann. Biol. Clin. 59 (2001) 677.[72] Wijayawardhana, in: N.A. Wai Tak Law, M. Arthur, Usmani (Eds.), Biomedical

Diagnostic Science and Technology, Marcel Dekker, New York, 2002.[73] E. Nebling, T. Grunwald, J. Albers, P. Schafer, R. Hintsche, Anal. Chem. 76 (2004)

689.[74] Y.W. Jung, H.J. Kang, J.M. Lee, S.O. Jung, W.S. Yun, S.J. Chung, B.H. Chung, Anal.

Biochem. 374 (2008) 99.[75] R.E. Ionescu, S. Cosnier, G. Herzog, K. Gorgy, B. Leshem, S. Herrmann, R.S.

Marks, Enzyme Microb. Technol. 40 (2007) 403.[76] S. Zhang, Q. Tan, F. Li, X. Zhang, Sens. Actuators B: Chem. 124 (2007) 290.[77] M. Kobayashi, T. Kusakawa, M. Saito, S. Kaji, M. Oomura, S. Iwabuchi, Y. Morita,

Q. Hasan, E. Tamiya, Electrochem. Commun. 6 (2004) 337.[78] A. Michalska, K. Maksymiuk, Microchim. Acta 143 (2003) 163.[79] E. Larraz, M.I. Redondo, M.J. González-Tejera, M.A. Raso, J. Tortajada, E.

Sánchez de la Blanca, M.V. García, Synth. Met. 122 (2001) 413.[80] A. Sezai Sarac, S. Sezgin, M. Ates, M.C. Turhan, Adv. Polym. Technol. 28 (2009)

120.[81] A.C. Barton, F. Davis, S.P.J. Higson, Anal. Chem. 80 (2008) 9411.[82] C.-C. Lin, L.-C. Chen, C.-H. Huang, S.-J. Ding, C.-C. Chang, H.-C. Chang, J. Elec-

troanal. Chem. 619–620 (2008) 39.[83] S. Guo, E. Wang, Anal. Chim. Acta 598 (2007) 181.[84] L.-D. Li, Z.-B. Chen, H.-T. Zhao, L. Guo, X. Mu, Sens. Actuators B: Chem. 149

(2010) 110.[85] R. Wang, Y. Wang, K. Lassiter, Y. Li, B. Hargis, S. Tung, L. Berghman, W. Bottje,

Talanta 79 (2009) 159.[86] Y. Amano, Q. Cheng, Anal. Bioanal. Chem. 381 (2005) 156.[87] N.J. Montalto, Am. Family Physician 67 (2003) 111.[88] X.Q. Jiang, M.G. Spencer, Biosens. Bioelectron. 25 (2010) 1622.[89] D. Zhang, Y. Peng, H. Qi, Q. Gao, C. Zhang, Biosens. Bioelectron. 25 (2010)

1088.[90] X.Q. Fang, O.K. Tan, M.S. Tse, E.E. Ooi, Biosens. Bioelectron. 25 (2010) 1137.[91] M. Hnaien, M.F. Diouani, S. Helali, I. Hafaid, W.M. Hassen, N.J. Renault, A.

Ghram, A. Abdelghani, Biochem. Eng. J. 39 (2008) 443.[92] M.S. Hejazi, M.H. Pournaghi-Azar, F. Ahour, Anal. Biochem. 399 (2010) 118.[93] P.D. Tam, M.A. Tuan, T.Q. Huy, A.-T. Le, N.V. Hieu, Mater. Sci. Eng.: C 30 (2010)

1145.[94] R.G.a.D.R. Walt, in: R.S. Marks (Ed.), Handbook of Biosensors and Biochips,

John Wiley and Sons, 2007.[95] K.J. Albert, in: N.A. Wai Tak Law, M. Arthur, Usmani (Eds.), Biomedi-

cal Diagnostic Science and Technology, Marcel Dekker, New York, 2002,p. 121.

[96] M.S.a.Y. Iribe, in: R.S. Marks (Ed.), Handbook of Biosensors and Biochips, JohnWiley and Sons, 2007.

[97] J. Homola, S.S. Yee, G. Gauglitz, Sens. Actuators B: Chem. 54 (1999) 3.[98] J. Mitchell, Sensors 10 (2010) 7323.[99] W.L. Barnes, A. Dereux, T.W. Ebbesen, Nature 424 (2003) 824.

[100] J. Homola, Anal. Bioanal. Chem. 377 (2003) 528.[101] H. Baac, J.P. Hajos, J. Lee, D. Kim, S.J. Kim, M.L. Shuler, Biotechnol. Bioeng. 94

(2006) 815.[102] B.N. Feltis, B.A. Sexton, F.L. Glenn, M.J. Best, M. Wilkins, T.J. Davis, Biosens.

Bioelectron. 23 (2008) 1131.[103] R.L. Rich, D.G. Myszka, Trends Microbiol. 11 (2003) 124.[104] J.W. Chung, S.D. Kim, R. Bernhardt, J.C. Pyun, Sens. Actuators B: Chem. 111–112

(2005) 416.[105] C. Nilsson, S. Abbas, M. Bennemo, A. Larsson, M.D. Hämäläinen, Å. Frostell-

Karlsson, Vaccine 28 (2010) 759.[106] A. Petrosova, T. Konry, S. Cosnier, I. Trakht, J. Lutwama, E. Rwaguma, A. Chep-

urnov, E. Mühlberger, L. Lobel, R.S. Marks, Sens. Actuators B: Chem. 122 (2007)578.

[107] D. Atias, Y. Liebes, V. Chalifa-Caspi, L. Bremand, L. Lobel, R.S. Marks, P. Dussart,

Sens. Actuators B: Chem. 140 (2009) 206.

[108] S. Herrmann, B. Leshem, S. Landes, B. Rager-Zisman, R.S. Marks, Talanta 66(2005) 6.

[109] A. Sobarzo, J.T. Paweska, S. Herrmann, T. Amir, R.S. Marks, L. Lobel, J. Virol.Methods 146 (2007) 327.

[110] M. Seydack, Biosens. Bioelectron. 20 (2005) 2454.

a Chim

(2007) 691.

R.L. Caygill et al. / Analytic

[111] Z.T. Deng, Y. Zhang, J.C. Yue, F.Q. Tang, Q. Wei, J. Phys. Chem. B 111 (2007)12024.

[112] T.J. Baek, P.Y. Park, K.N. Han, H.T. Kwon, G.H. Seong, Anal. Bioanal. Chem. 390(2008) 1373.

[113] J. Griffin, A.K. Singh, D. Senapati, E. Lee, K. Gaylor, J. Jones-Boone, P.C. Ray,Small 5 (2009) 839.

[114] A.B. Haefliger, in: R.S. Marks (Ed.), Handbook of Biosensors and Biochips, vol.2, Wiley and Sons, 2007, p. 949.

[115] G. Sauerbrey, Z. Phys. 155 (1959) 206.[116] P. Skladal, C.D. Riccardi, H. Yamanaka, P.I. da Costa, J. Virol. Methods 117

(2004) 145.[117] D. Dell’Atti, M. Zavaglia, S. Tombelli, G. Bertacca, A.O. Cavazzana, G. Bevilacqua,

M. Minunni, M. Mascini, Clin. Chim. Acta 383 (2007) 140.[118] C. Yao, T. Zhu, J. Tang, R. Wu, Q. Chen, M. Chen, B. Zhang, J. Huang, W. Fu,

Biosens. Bioelectron. 23 (2008) 879.[119] M. Rodahl, B. Kasemo, Rev. Sci. Instrum. 67 (1996) 3238.[120] G.P. Ferreira, G.S. Trindade, J.M.C. Vilela, M.I.N. Da Silva, M.S. Andrade, E.G.

Kroon, J. Microsc.-Oxford 231 (2008) 180.

ica Acta 681 (2010) 8–15 15

[121] G. Binning, C.F. Quate, C. Gerber, Phys. Rev. Lett. 56 (1986) 930.[122] N.C. Santos, M.A.R.B. Castanho, Biophys. Chem. 107 (2004) 133.[123] A. McPherson, Y.G. Kuznetsov, M. Plomp, A. Malkin, Biophys. J. 86 (2004)

152A.[124] C.-K. Lee, Y.-M. Wang, L.-S. Huang, S. Lin, Micron 38 (2007) 446.[125] D.J. Muller, Biochemistry 47 (2008) 7986.[126] R. Raiteri, M. Grattarola, H.J. Butt, P. Skladal, Sens. Actuators B: Chem. 79

(2001) 115.[127] Y.G. Kuznetsov, J.G. Victoria, W.E. Robinson Jr., A. McPherson, J. Virol. 77

(2003) 11896.[128] T.W. Owen, R.O. Al-Kaysi, C.J. Bardeen, Q. Cheng, Sens. Actuators B: Chem. 126

[129] G.S. Trindade, J.M.C. Vilela, J.M.S. Ferreira, P.H.N. Aguiar, J.A. Leite, M.I.M.C.Guedes, Z.I.P. Lobato, M.C. Madureira, M.I.N. da Silva, F.G. da Fonseca, E.G.Kroon, M.S. Andrade, J. Virol. Methods 141 (2007) 198.

[130] R.L. Gunter, W.G. Delinger, K. Manygoats, A. Kooser, T.L. Porter, Sens. ActuatorsA-Phys. 107 (2003) 219.