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Benchmark Analysis of Fiber Optics SPR Biosensor - Report Project Work Nanoscience
Benchmark Analysis of Fiber Optics SPR Biosensor - Report Project Work Nanoscience
Benchmark Analysis of Fiber Optics SPR Biosensor - Report Project Work Nanoscience
Benchmark Analysis of Fiber Optics SPR Biosensor - Report Project Work Nanoscience
Benchmark Analysis of Fiber Optics SPR Biosensor - Report Project Work Nanoscience
Benchmark Analysis of Fiber Optics SPR Biosensor - Report Project Work Nanoscience
Benchmark Analysis of Fiber Optics SPR Biosensor - Report Project Work Nanoscience
Benchmark Analysis of Fiber Optics SPR Biosensor - Report Project Work Nanoscience
Benchmark Analysis of Fiber Optics SPR Biosensor - Report Project Work Nanoscience
Benchmark Analysis of Fiber Optics SPR Biosensor - Report Project Work Nanoscience
Benchmark Analysis of Fiber Optics SPR Biosensor - Report Project Work Nanoscience
Benchmark Analysis of Fiber Optics SPR Biosensor - Report Project Work Nanoscience
Benchmark Analysis of Fiber Optics SPR Biosensor - Report Project Work Nanoscience
Benchmark Analysis of Fiber Optics SPR Biosensor - Report Project Work Nanoscience
Benchmark Analysis of Fiber Optics SPR Biosensor - Report Project Work Nanoscience
Benchmark Analysis of Fiber Optics SPR Biosensor - Report Project Work Nanoscience
Benchmark Analysis of Fiber Optics SPR Biosensor - Report Project Work Nanoscience
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Benchmark Analysis of Fiber Optics SPR Biosensor - Report Project Work Nanoscience

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Under a Compulsory course of Nanoscience and Nanotechnology, Project Work Nanoscience. The team of A Zamith Cardoso, PT and A Mitra, India, conducted a 4 month Project under guidance of Prof. Dr. …

Under a Compulsory course of Nanoscience and Nanotechnology, Project Work Nanoscience. The team of A Zamith Cardoso, PT and A Mitra, India, conducted a 4 month Project under guidance of Prof. Dr. Jeroen Lammertyn, BE and PhD-Student Jeroen Pollet, BE at Mebios Group http://www.biosensors.be/ at K.U.L. , Belgium

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  • 1. 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
  • 2. 2 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
  • 3. 3 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.
  • 4. 4 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].
  • 5. 5 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. RIDASCREE 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
  • 6. 6 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
  • 7. 7 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].
  • 8. 8 Figure 9 – EDC linkage and HS 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
  • 9. 9 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).
  • 10. 10 • 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 -5.00 0.00 5.00 10.00 15.00 20.00 25.00 0 20 40 60 80 100 120 140 Time (min) SPRwavelenghtshift(nm) 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
  • 11. 11 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. -2.00 0.00 2.00 4.00 6.00 8.00 10.00 12.00 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 sugar concentration (%) SPRwavelengthshift(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.
  • 12. 12 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. ELISAStandards 0 0.5 1 1.5 2 2.5 0 5 10 15 20 25 Concentration (ppm) Absorbance B. Peanut spiked samples 0 0.5 1 1.5 2 2.5 3 3.5 0 20 40 60 80 100 120 Concentration(ppm) Absorbance
  • 13. 13 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) Absorbance 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
  • 14. 14 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. -30.00 -20.00 -10.00 0.00 10.00 20.00 30.00 40.00 50.00 Time (sec) SPRwavelengthshift(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. egative 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.
  • 15. 15 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. -1.00 0.00 1.00 2.00 3.00 4.00 5.00 -5 0 5 10 15 20 25 Time (min) SPRshift(nm) blank 1 µg/ml 2 µg/ml 6 µg/ml B. 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 0 1 2 3 4 5 6 7 Ara h1 concentration (µg/ml) SPRshift(nm)
  • 16. 16 C. 0 0.5 1 1.5 2 2.5 0 1 2 3 4 5 6 7 Ara h1 concentration (µg/ml ) SPRshift(nm) 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. 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

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