A Benchmark analysis – SPR fiber optic biosensor Report of the Project Work Nanoscience

31 of May 2010

Program Coordinator: Professor Andre Stesmans

Promoter: Professor Jeroen Lammertyn

Supervisor: Dr. Jeroen Pollet

Workgroup:

Andre Joao Zamith Cardoso

Aniruddha Mitra

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In the frame of the course of Project Work Nanoscience, we chose a topic in the field

bionanotechnology. We were introduced to the Mechatronics, Biostatistics and Sensors (MeBioS)

research group to have hands-on experience in one of their latest developments, a Fiber Optic SPR

biosensor. This proved to be an interdisciplinary research project, with optics, chemistry and

bioengineering. This biosensors group is part of the Department of Biosystems at the K.U.Leuven

and is headed by Prof. Ir. Jeroen Lammertyn. We had the opportunity to work close with a PhD

student who has development this biosensor during his thesis, Ir. Jeroen Pollet was our monitor and

kindly guided our experiments.

1. Introduction

In the context of their research in bionanotechnology, they focused on developing new bio-

molecular detection concepts for integration in miniaturized analysis systems. The Fiber optic SPR

biosensor takes advantage of Surface Plasmon Resonance (SPR) effect probably the best-known

method for online detection of biological binding reactions. Although it could give valuable

information for industrial applications, current SPR is rarely used outside research centers, because

most commercial available systems are expensive and require specialized staff. In their search for

more convenient SPR-sensors, the traditional prismbased systems have been replaced by fiber optic

designs. In their research, they introduce among other technologies, a low-cost fiber-SPR-sensor. In

the research for a versatile, compact and low cost biosensor, the fiber-optic SPR-probes have proven

to be very useful for remote sensing, continuous analysis and in situ monitoring. In the diagnostics

market, there is currently a very well established biochemical technique used for detection purposes

called enzyme-linked immunosorbent assay (ELISA). This commercial detection mechanism is

adapted and sold in various kits to detect different substances. Our scientific objective in with this

project was to benchmark the fiber-optic SPR biosensor with the sandwich enzyme immunoassay

(ELISA) for the same test, a peanut allergy problem.

1.1. The Peanut allergy problem

Peanut (Arachis hypogeae) is a very nutritious fruit among the legume family. Unfortunately

peanuts belong to the most allergenic foods. Nearly 7 - 10 % of the total protein content (about 25 %)

consists of allergic proteins (i.e. Arachine, Conarachine). Even consumption of few milligrams of

peanut can induce allergic reactions in highly allergic persons. Symptoms range from allergic skin

reactions up to death due to anaphylactic shock. Since peanut allergy is persistent during life and

treatment of this allergy is not possible, avoiding peanuts is extremely important for these patients.

Involuntary exposure to peanut in foods poses heal risks for peanut allergic individuals that can be

reduced by improving detection systems for all allergen contaminants in food products and

manufacturing processes. Detection of traces of peanut in food, cosmetics, or drinks is difficult.

Chocolate is one of those foods, which has proven to be difficult [1]

.

For the protection of consumers against peanut contaminations, methods like rocket immuno

electrophoresis (RIE), FPLC and enzyme immunoassay (ELISA) have been established. The

diagnostics by the patient or doctor are done a posteriori, based on signs and symptoms. These

methods may contribute to increased safety of products for peanut allergic patients.

It represents a major importance for people with allergies to have a prevention mechanism to

have a healthier life. In the case of peanuts in food, they are sometimes added to give a special taste,

and many times, their quantities and presence is not mentioned in the labels of the products. Hence it

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is necessary to develop hands on tools to detect presence of peanut allergens in any kind of food

product. The current technology is tedious, costly and not practical for large scale detection. Hence

cheaper and more efficient methods need to be designed. The fiber optics biosensor is one such

technique. It makes use of the phenomenon of surface plasmon resonance to detect allergens.

1.2. Surface Plasmon Resonance

Surface Plasmon Resonance, by definition, is an electron charge density wave phenomenon

that arises at the surface of a metallic film when light is reflected at the film under specified

conditions.

When light propagating in a medium of higher refractive index meets an interface at a

medium of lower refractive index at an angle of incidence above the critical angle, the light is totally

reflected back and propagates in the high refractive index medium. If the total internal reflection

(TIR) phenomenon occurs in an interface coated with a conducting medium, such as a gold layer, an

evanescent wave penetrates the metal and interacts with the electron. In this case, there is a transfer

of energy from the evanescent wave

to electrons in the metal generating

surface plasmon waves (electron

oscillations). The resonance is a

result of energy and momentum

being transformed from incident

photons into surface plasmons, and

is sensitive to the refractive index of

the medium on the opposite side of

the film from the reflected light.

The surface plasmon resonance, is a

resonance effect which takes place

when the wave vector (characterized

by a direction and magnitude) of

the incident photons is equal to the

wave vector of the plasmons

(oscilations of the electrons in the lattice of metal atoms) ksp=kx (figure 1).

Given that, the wave vector of the plasmon wave is bound to the conductor surface, it is the wave

vector of the component of the incident photon, which is parallel to the conductor surface that must

be equal to the wavevector of the surface plasmons. So, to have surface Plasmon resonance, it is

required to tune the properties of the incident light wave vector to match the plasmon wave vector.

This can be done by varying the wavelength of light or by varying the angle of incidence. [2,3]

1.3. SPR Biosensing Systems

Most of the commercially available SPR biosensing systems, such as Biacore, make use of a

prism and a thin highly reflecting metal layer deposited upon the prism base, in which the wave

vector of the light is tuned by changing the angle of incidence.

Figure 1 - The phenomenon of surface plasmon resonance occurring at the

glass gold interface.

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Figure 2 - The Biacore system. A shift in the SPR dip in the reflection intensity curve is observed because of change in the

resonance conditions, when antigens bind to the antibodies on the gold surface.

The major pitfall of such a system, as mentioned earlier, is that it is highly expensive and thus

cannot be used for wide scale applications.

In the search for a more cost-effective SPR sensor, an alternative arrangement can be made in

which it is possible to measure the resonance spectrum by the method of fixing the angle and

modulating the excitation wavelength. This is the fundamental basis for the development of the SPR

fiber-optic sensor[5]

. In this system, the cladding from the tip of the optic fiber is stripped and a gold

layer is coated on the glass surface. White light is introduced into the fiber. The resonant wavelength

induces surface plasmon resonance on the gold surface, which can be observed as a dip in the

reflection intensity spectrum.

Any changes on the surface of

the gold layer changes the refractive

index. This changes the resonance

conditions (value of ksp), which is

observed as a shift in the SPR dip in the

reflected spectrum (Fig.3). The

sensitivity of this device is remarkable.

It is sensitive to changes of 10-5

-10-6

refractive index (RI) units within

approximately 200 nm of the SPR

sensor/sample interface. Thus, a

submonolayer of adsorbed protein-like

substance (RI 1.4) from an aqueous

solution (RI 1.3) can easily be

observed[6].

This is the key principle

utilized by the fiber optic SPR sensor in

detecting biomolecules. The gold

surface of the biosensor can be

functionalized with a biorecognition Figure 3. An increase in Refractive index leads to a blue-shift in the SPR

wavelength. Graph taken from [4].

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layer for a particular antigen. Specific binding of antigen molecules to the surface bio recognition

layer is detected by the shift in the SPR wavelength. The greater the concentration of antigens the

greater is the shift in the SPR wavelength.

In this case, the SPR fiber optic sensor is used to detect Ara h1, the major peanut allergen.

The surface of the fiber optic probe is functionalized with a monolayer of antibodies specific to the

Ara h1 protein

1.4. ELISA technique

The most commonly used technique to detect peanut

allergens at present is the ELISA assay procedure.

This technique has been used not only as a diagnostic tool

in medicine, and plant pathology, as well as a quality control check

in various industries. In the basic process of ELISA, this technique

is used to detect the presence of an unknown antigen in the sample

using antibodies. Antigens are molecules recognized by the

immune system, and they are specifically detecting antigens. In

general terms, an unknown amount of antigen is fixed to the surface and then a specific antibody is

applied to the surface, binding to the antigen in a specific way. This antibody is linked to an enzyme,

and in the final step a substance is added that the enzyme can convert to some detectable signal. This

signal is proportional to the antigen concentration in the sample, making it a useful technique for

biochemical detection.

1.4.1. RIDASCREE4 FAST Peanut kit and microtiter plate spectrometer

The RIDASCREEN FAST Peanut is a sandwich

ELISA for quantitative analysis of peanuts or parts of

peanuts in food, either ingredient component or

contamination in raw and cooked food.

The basis of the test is the antigen-antibody

reaction. The wells of the microtiter strips (figure 4) are

coated with specific antibodies against peanut proteins

(figure 5(1)). By adding the sample solution with

unknown amount of peanut allergens to the wells, present

peanut protein will

bind to the specific

capture antibodies

and an antibody-antigen-complex is formed (figure

5(2)).

In a washing step components not bound are removed.

Then antibody conjugated to peroxidase (enzyme conjugate) is

added. This antibody conjugate is bound to the antibody-antigen-

complex. An antibody-antigen-antibody-complex (sandwich) is

formed (figure 5(3)). Any unbound conjugate is then removed in

a washing step. The detection of peanut protein takes place by

adding substrate/chromogen solution. The enzyme conjugate

Figure 4 - ELISA microtiter plate

Figure 5. – A sandwich ELISA procedure. (1) Plate is

coated with a capture antibody; (2) sample is added,

and any antigen present binds to capture antibody;

(3) enzyme linked capture antibody used as detecting

antibody is added, and binds to antigen; (4) substrate

is added, and is converted by enzyme to detectable

Figure 6 - Microtiter plate spectrometer

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converts the chromogen into a blue product (figure 5(4)). The addition of the stop solution leads to a

color change from blue to yellow. The measurement is made photometrically at 450 nm using a

Microtiter plate spectrometer (figure 6). The absorbance is proportional to the peanut protein

concentration in the sample, so that we have a detection mechanism.[7]

This ELISA kit is used as a benchmark for the fiber optic SPR.

2. Materials and Method

2.1. Optic fiber SPR probe preparation

The optic fiber probes used had a diameter of 400µm and a numerical aperture of 0.39. The

fibers were cut into 3 cm pieces and fixed inside a syringe needle. A 1 cm SPR-sensitive tip was

constructed at the tip of the fiber. This was done by stripping the polymer cladding carefully with

acetone. After this the stripped part of the fiber was cleaned in 30:70 (v/v) solution of 30% hydrogen

peroxide and concentrated sulphuric acid. The cleaned fibers were then coated in a gold sputter

coater for 1 min to deposit an approximately 50nm thick gold layer. The layer deposited was made as

smooth as possible.[4]

Figure 7- The SPR system. A.The optic fiber sensor probe. B. The system setup mounted on a computer controlled

positioning robot[4].

2.2. Apparatus and system setup

The replaceable sensing probes are fixed at the end of an optic fiber which guides white light

from a LS-1 tungsten halogen light source into the probes. The light that is reflected at the gold tip of

the probe is collected by a spectrometer, with detection range from 350 to 1100nm and gives a

typical spectral resonance SPR-dip as a result. This particular system, basically, measures wavelength

modulations of the SPR dip in the reflection intensity curve obtained. The angle range at which the

light ray is incident on the gold layer is assumed to be constant. The resonance wavelength, at which

there is maximum coupling between the incident wave and surface plasmons, is taken as the sensor

output. Any binding event on the gold layer changes the resonance conditions shifting the resonance

wavelength allowing real time monitoring of the surface chemistry on the gold surface. To make it a

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fully automated system the fiber probe is linked to a pre-programmed computer controlled robotic

arm. The robotic arm can be given instructions to position the SPR probe in 96-well microplates and

standard 1ml glass vials. This allows repeatable, fast and easy surface immobilization procedures and

measurements. [4]

The program is simple and makes use of three commands to maneuver the whole procedure.

Commands Operation

PA x y gives the x,y position where the arm should move

@Za z gives the z position (guides up down movement)

@Wt t gives the time for which the arm needs to wait at a certain position

2.3. Data Processing

The data was recorded in software called Spectrasuite and further processed using Matlab.

Each SPR spectrum is the ratio of the ‘spectrum of the sample’ and the air-based reference spectrum’.

The raw data were filtered with a Savitsky-Golay filter. The resonance wavelength which is at the

minima in the SPR dip curve is calculated using the Minimum Hunt Method. In this method a second

order polynomial is fit to the SPR-dip to determine the local minimum. In the resulting sensograms

the SPR wavelength shift (nm) is plotted versus time. The data of the different washing steps are

removed from the plot, as they are not important in data interpretation. [4]

2.4. Surface Chemistry

Both, the optic fiber sensor probe and the magnetic bead surface was functionalized by layer

of Ara h 1 binding antibodies. In the first step a self assembled monolayer of 5% carboxylic acid-

capped hexa (ethylene glycol) undecanethiol and 95% (1-mercapto-11-undecyl)tri(ethylene glycol)

was used as a prime layer (Figure 8). This layer has a high hydrophilic character and high chain

flexibility. Water molecules get tightly bound around PEG molecules preventing uncontrolled protein

absorption. Also, compression of PEG chains causes steric repulsion. All these factors significantly

reduce non specific adsorption.

Now, to prepare the fiber, it was dipped in a solution of 95µl MES 100mM 2-(Nmorpholino)

ethanesulfonic acid (MES) buffer with 5µl of EDC (0.5mM) and 5µl of NHS for 30 minutes. EDC

links to the COOH capped PEG molecules on the surface of the fiber. NHS stabilizes the layer by

interconnecting adjacent EDC molecules (figure 9). After this, the fiber was dipped into a 100ml

solution (98µl MES and 2µl Ara h1 binding antibody concentrate) for another 30 minutes. Antibodies

were immobilized to the PEG through the zero length linker EDC. This two step procedure allowed

formation of a single layer of antibodies on the optic fiber sensor probe.

Figure 8 – Self assembly of PEG molecules on the gold surface. Only the carboxylic acid capped PEG molecules are

represented in the figure[8].

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Figure 9 – EDC linkage and 4HS stabilization[8]

Figure 10 – Antibody immobilization in the PEG [8]

In case of the magnetic beads the results are the same whether a single layer or multiple layers

of antibodies are formed on the system. Hence to prepare the magnetic bead, they were mixed in the

MES buffer with both EDC and antibodies mixed at the same time, leading to the formation of a

multilayer of antibodies. This was also done for at least 30 minutes. The magnetic beads were then

washed in PBS to remove free antibodies. This step was carried out thrice.

2.5. Sample Preparation

The dilution series was prepared with the following standard protocol.

2.5.1. Peanut spiked samples:

Liquid nitrogen was used to powder the peanuts and the chocolate samples (peanut free).

0.02g peanut, 0.98g of sample and 1g of skimmed milk was weighed and mixed in a falcon with

20ml preheated extraction buffer to create a 20ppt stock. Then, a dilution series (of 1ml) was made

with the 20ppt stock sample: 100ppm, 20ppm, 10ppm, 5ppm, 2.5ppm. Also a blank sample was

made. The prepared series was then shaken gently for 10 minutes at 600

C and centrifuges for 10

minutes at 40

C.

2.5.2. Ara h1 spiked samples:

0.1g of sample and 0.1g of skimmed milk powder was weighed and mixed in a falcon with

2ml preheated extraction buffer and 6.7µl Ara h1(1.8 mg/ml) to generate a 6µg/ml stock. Then a

dilution series was made with the 6µg/ml stock sample: 6, 2,1,0.5,0.25µg/ml in 1 ml preheated

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extraction buffer. Also a blank sample was made. The prepared series was then shaken gently for 10

mins at 600

C and centrifuges for 10 mins at 40

C.

2.6. Experiment Procedures:

2.6.1. Sucrose Experiment

A dilution series of sucrose in water was made ranging from 0-4%.The SPR system was set up

and the dilution series was placed in the sample rack in the ascending order. The robotic arm was

programmed with the following steps:

• The fiber optic probe was dipped in the first sample for 5 minutes.

• The probe was washed for 5 minutes in the washing buffer.

• The probe was dipped into the next sample.

In this way the programme was carried out thrice over the dilution series.

2.6.2. Peanut and Ara h1 Experiments

Again, the SPR system was set up and dilution series of peanut spiked and Ara h1 spiked

samples was placed on the rack for either experiments. The robotic arm was programmed with the

following steps.

• The fiber optic probe was dipped in the first sample for 20 minutes, for Ara h1 to attach to

the antibodies functionalized on probe surface.

• The probe was washed three times for 5 minutes each in the washing buffer.

• The probe was then dipped in magnetic beads solution for 20 minutes. The magnetic beads

bind with the Ara h1 bound to the antibodies.

• The probe was washed in the washing buffer for 5 minutes, followed by a 5 minute washing

step in glycine solution and then a 5 minute washing step in NaOH solution. This elaborate

step removes the Ara h1 linked to the antibodies by change of pH

• The cleaned sensor probe was then dipped into the next sample.

This process was carried out for all the samples and the SPR wavelength was traced in real time.

2.6.3. ELISA Experiment

The standard ELISA protocols were followed using a commercially available ELISA toolkit

(RIDASCREEN FAST Peanut). The protocol in this part used is the following:

• A sufficient number of wells were inserted into the microwell holder for all standards and

samples to be run. Standard and sample positions were recorded.

• 100 µl of each standard solution or prepared sample was added to separate wells and

incubated for 10 min at room temperature (20 - 25 °C / 68 - 77 °F).

• The liquid was poured out of the wells and the microwell holder was tapped upside down

vigorously (three times in a row) against absorbent paper to ensure complete removal of

liquid from the wells. All the wells were filled with 250 µl washing buffer and the liquid was

poured out again. This was repeated two more times.

• 100 µl of the enzyme conjugate was added to each well. The samples were mixed gently by

shaking the plate manually and incubated for 10 min at room temperature (20-25°C/68-77

°F).

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• The liquid was poured out of the wells and the microwell holder was tapped upside down

vigorously (three times in a row) against absorbent paper to ensure complete removal of

liquid from the wells. All the wells were filled with 250 µl washing buffer and the liquid was

poured out again. This was repeated two more times.

• 100 µl of the reddish substrate/chromogen solution was added to each well. The samples

were mixed gently by shaking the plate manually and incubated for 10 min at room

temperature (20 - 25 °C / 68 - 77 °F) in the dark.

• 100 µl of the stop solution was added to each well. The samples were mixed gently by

shaking the plate manually and the absorbance was measured at 450 nm. Reading was made

within 10 minutes after addition of stop solution.

3. Discussion and Results

3.1. Fiber Testing

The optical probe is highly sensitive. Every batch of optic probe is slightly different due to

the processing steps involved. One way of proving the accuracy of the sensor for our experiments,

check for its sensitivity and the reproducibility of the experiments is by validation with known

sucrose concentrations. Evaluation of the state of the optic probe and the process is possible with a

simple set of sucrose dilutions in water, solutions that we set to be with concentrations ranging from

0% to 4%. During measurements, the SPR probe was dipped in each solution for 5 minutes from the

lowest concentration to the highest. Between two subsequent dips, the SPR probe went through a

washing step, which is reflected in Figure 11 by the abrupt change in signal intensity between two

subsequent sucrose solutions.

SUGAR measurements

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0 20 40 60 80 100 120 140

Time (min)

SP

R w

av

ele

ng

ht

shif

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Figure 11 - SPR wavelength shift as function of time for three consecutive detection steps on a set of sucrose solutions

containing 0-4% sucrose.

It is clear from figure 11 that the sensing experiment is reproducible. We can see that a linear

relationship (Figure. 12) is well fitted in the data between the SPR wavelength shift and the

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concentration of sucrose in solution. We can see a small deviation between each observation. The

little differences seen between the three sets could be contributed to the noise in the light signal

intensity. Also, only a single washing step was carried out between subsequent measurements, which

might not be good enough to remove all the sucrose attached on to the probe, varying the results

slightly for the subsequent measurements. However, it was clear from this data that the SPR fiber

was adequate to be used for further experiments with peanut allergens.

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sugar concentration (%)

SPR wavelength shift (nm)

Figure 12 - Linear calibration curve for concentration of sucrose in water solution.

3.2 ELISA Results

The ELISA test was carried with standard ELISA samples, peanut spiked samples and Ara h1

spiked samples using the ELISA toolkit, using the standard ELISA protocol. We use it as a

benchmark for the results obtained with the SPR probe. Table 1 – ELISA microtiter plate configuration for each reservoir/concentration. The yellow refers to

the ELISA standards for calibration; the green refers to the peanut spiked samples; and the blue refers

to Ara h 1 spiked samples.

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Table 2 – Absorbance obtained from each of the reservoir of the microtiter plate spectrophotometer

(Left) and corresponding picture of the microtiter plate (Right).

0,0432 1,6598 1,0657 3,1846 2,9519 3,2935

0,2394 0,2855 1,7682 0,0429 3,2383 2,0896

0,5516 0,8591 3,2364 0,3683 3,3423 3,0184

0,9138 1,373 0,0868 1,0033 3,3142 3,1602

1,7694 2,4739 0,4427 1,2316 2,0465 3,2336

0,2789 0,0799 0,9766 2,0231 2,936 3,2075

0,49 0,4363 1,1753 3,1416 3,2502 0,0448

0,9279 1,0417 1,9172 1,9774 3,1424 0,0466

In table 1 it is shown that the ELISA microtiter plate configuration for different

concentrations of standard, peanut spiked and Ara h1 spiked samples. The graphs in figure 13 were

obtained by processing the absorbance data from table 2.

By the first two graphs presented in Figure 13, we can see that as expected, the points

obtained with the standard ELISA samples (Figure 13A) adjust to the peanut spiked samples (Fig.

13B). The absorbance curves for Ara H1 samples (Fig. 13C) also look fine. This gives us an

indication that the samples prepared by us were reasonably accurate and could be used for the SPR

experiments. One interesting thing that can be noticed from the graphs is that the absorbance has a

linear relationship with the concentration of peanut allergen only to a certain concentration limit,

after which the signal saturates. The signal saturates at approximately 40 ppm in the case of peanut

spiked samples and at approximately 1 µg/ml in the case of Ara h 1 spiked samples.

A.

ELISA Standards

0

0.5

1

1.5

2

2.5

0 5 10 15 20 25

Concentration (ppm)

Absorb

ance

B.

Peanut spiked samples

0

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1.5

2

2.5

3

3.5

0 20 40 60 80 100 120

Concentration(ppm)

Absorb

ance

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C.

Ara H1 Spiked samples

0

0.5

1

1.5

2

2.5

3

3.5

0 1 2 3 4 5 6 7

Concentration (g/ml)

Absorb

ance

Figure 13. Points representing the absorbance with respect to concentration of A. ELISA standard samples, B. Peanut

spiked samples, C. Ara H1 spiked samples.

3.3 Interpretation of a SPR wavelength shift graph

The fiber optic SPR biosensing process is carried out on a sample monitoring the SPR wavelength

shift during the whole experiment. The sequence of steps as seen in SPR wavelength shift graph is:

1. The optic fiber was first dipped into the samples for 20 minutes for specific interaction to take

place between the antibodies and the allergens. The shift is high giving a large peak in the

graph. This is because along with the specific interaction, the non specific interactions due to

sugar and other components in the background of the solution greatly increases the refractive

index, thus contributing to a large shift surface plasmon resonance wavelength.

2. Now, the fiber goes through an intensive washing step so that all the non specific interactions

are removed and the change in refractive index is only due to the binding of the antibody to

the allergen, which is very small. Therefore, there is a sudden drop in the graph, giving an

almost zero signal. This signal thus needs to be enhanced.

3. The fiber is now dipped into the solution of magnetic beads (linked to antibodies). The

magnetic beads attach to the allergen through the antibody. Being very heavy, the magnetic

beads change the refractive index. Thus, effectively, the magnetic beads magnify the SPR

wavelength shift caused by the antibody-allergen binding. This is the critical part of the graph

which indicates the concentration of the allergen that was present in the sample.

4. Now, the fiber goes through an elaborate washing step. Also the specific interactions between

the antibody and allergen are broken by changing the pH. This is seen in the graph as a series

of random fluctuations. Once this step is over the optic fiber is ready to be dipped into the

next sample and the whole process is repeated again.

3.4 The ‘Failed’ Experiment

Now, after the ELISA test was performed, we refrigerated the peanut spiked and Ara h1

spiked dilution series over a period of two days before we started our experiments with the SPR

probe. The fiber optic SPR biosensing process was carried out on a dilution series of peanut spiked

samples (100 ppm, 20 ppm, 10 ppm, 5 ppm, 2.5 ppm and a blank). The SPR wavelength shift was

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monitored during the whole experiment. The results obtained were negative. On dipping the fiber

into the sample solutions, there was a large peak due to interaction of the optic fiber with the sugar

and other components in the solution. However, after the washing step, it became clear that there was

no antibody-allergen interaction taking place. There was zero shift seen in case of each samples

(figure 14). There are two possible explanations for this to occur. In one hand, it is possible that the

optic fiber was not functionalized with antibodies properly. In this case it would not be possible for

the allergen molecules in the sample to bind to the optic fiber, leading to no change in the refractive

index. Hence we can see no shift in the SPR wavelength. In the other hand, keeping the samples

refrigerated for two days could have led to the flocculation of the allergen molecules at the bottom of

the sample solutions and hence antibody-allergen interaction was not possible on dipping the fiber

into the solution.

To search for the issue that led to negative results, two control solutions, one spiked with

excess of peanuts and one spiked with excess of Ara h1 were freshly prepared. The SPR probe was

dipped into both these solutions. In both the case a large shift in SPR wavelength was observed on

dipping the fiber into the magnetic beads solution (Figure 14). This made it clear that the optic fiber

was properly functionalized with antibodies. So, the problem was caused due to the flocculation of

allergens in the sample because it was kept refrigerated for a long time. This was an interesting

development, as initially we had assumed that refrigerating the samples over a period of time does

not have any effect.

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Time (sec)

SPR wavelength shift (nm)

samples 0-2.5-5-10-20 ppm

Ara h1

Control

Peanut

Control

Negative Results Highly positive results

Figure 14. . The SPR wavelength shift monitored throughout the experiment. 4egative results are obtained with the

samples 0-2.5-5-10-20 ppm while higly positive results are obtained with the Ara h1 and peanut control samples.

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3.5 Ara h1 Results

Again, the fiber optic SPR biosensing process was carried out, this time on a dilution series of

Ara h1 spiked samples (6, 2, 1, 0.5, 0.25 µg/ml and a blank). This set of sample was also refrigerated

for 2 days. The SPR wavelength shift was monitored during the whole experiment. Unlike the case of

the peanut spiked samples, this set of samples gave us good results. This indicates that Ara h1 spiked

samples can be refrigerated for a longer time without undergoing any flocculation of the Ara h1 in

the solution. Fig.15A shows us the sections of SPR wavelength shift curve where the magnetic beads

attach to the optic fiber, for different concentrations. On taking an average value of the shift, in the

case of each concentration, we can plot a calibration curve plotting SPR wavelength shift versus

concentration of Ara h1 in the solution (Fig 15B).

Now, we also wanted to check how the results varied with a different batch of optic fiber. A

new batch of fiber was thus obtained. The refrigerated Ara h1 samples were properly centrifuged to

remove any doubts of flocculation having taken place. The fiber optic SPR biosensing process was

carried out all over again. Positive results were obtained again. Fig. 15C shows the calibration curve

of the SPR wavelength shift versus concentration of Ara h1 for the new batch.

A.

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B.

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t (n

m)

Figure 15 - A. SPR wavelength shift for different concentrations of Ara h1. B. calibration curve of SPR wavelength

shift versus the concentration of Ara h1. . C. Calibration curve of SPR wavelength shift versus the concentration

of Ara h1 for a different batch of fiber

It is clear from the Fig. 15B and 15C that the two batches of fibers are different, giving

different results. This maybe due to slight differences in thickness of the gold layer deposited on the

fiber or differences in the density of the antibodies functionalized on the surface of the optic fiber.

There is also a possibility that the concentrations of the Ara h1 samples had slight variations when

they were measured by the different fibers.

However in both the cases, it was observed that the calibration curve was linear initially and

saturated at very high concentration of Ara h1. The saturation occurs somewhere between 3-5 µg/ml,

in both the cases as compared to 1 µg/ml in the case of the ELISA technique.

4. Conclusions

The ‘failed’ experiment indicates that the samples to be tested should ideally not be

refrigerated for a long period of time. Fresh samples should be used or the refrigerated samples being

used should be properly centrifuged before the detection process to avoid any form of sedimentation.

This applies both in the case of ELISA technique and the SPR biosensing technique.

On comparing the results of the ELISA test and the fiber optic SPR measurement technique, it

was apparent that the range over which the calibration curve remains linear is much larger in the case

of SPR biosensing. This gives the SPR biosensing technique of measuring an edge over the ELISA

test in precise measurement of higher concentrations of peanut allergen in an unknown solution. For

clinical purposes this might not be very important, as the concentration does not matter as long as the

allergen is detected. But for any application for which one needs to measure the high concentration of

allergen in a solution accurately, the ELISA technique fails. The SPR biosensor technique can be

useful in such cases.

It is also observed, that the sensitivity of the SPR technique is as good as the ELISA

technique. Also, the ELISA kit is highly expensive and the protocol involves more steps. Also, the

steps have to be carried out manually, they are more tedious (for example, it involves a few shaking

steps which needs to be carried out efficiently to obtain proper results) and are much more time

consuming. On the other hand, the SPR biosensing technique is cheap, less time consuming and can

17

be easily carried out by a programmed robot. It is also possible to make the testing device portable.

Thus, if the SPR biosensing technique is properly developed it can be the clear winner.

To conclude, we would like to say that we gained hands-on experience during the course of

the project work, which we felt prepared us to some extent for independent work during the master

thesis in the second year. Along with other aspects, we feel that we developed research skills, while

working with the ELISA biosensing kit and the fiber optic kit. The rigorous and methodological way

of following specific procedures as well as critical analysis of results obtained from the experiments

provided us experience to work in a highly experimental research oriented environment. Finally we

would like to thank the MeBios Biosensors group for this opportunity to work on this project.

References

[1] Peanut allergen (Ara h1) detection in foods containing chocolate, A. Pomés at al. 2003

[2] Chapter 6: Optical Transducers, Biosensor Technology and Bioelectronics, Jeroen Lammertyn,

2010

[3]Chapter 14: Molecular nanopatterns on surfaces, Chemistry at Nanometre Scale, Steven De

Feyter, 2010

[4] Fiber optic SPR biosensing of DNA hybridization and DNA–protein interactions,

Jeroen Pollet et al.2009

[5] A fiber optic chemical sensor based on surface plasmon resonance, R. C. Jorgenson and S.S.Yee,

1992

[6] Tuning Dynamic Range and Sensitivity of White-Light, Multimode, Fiber-Optic Surface Plasmon

Resonance Sensors , Obando et al. 1999

[7] RIDASCREEN®FAST Peanut protocol

[8] Presentation:Label free apta-sensing with fiber optic SPR, Jeroen Pollet