Microcantilevers for the disease detection offer useful advantages including high sensitivity, low cost, low analyte requirement, quick response time and low power requirement. Commonly, the microcantilever surface is modified through a number of chemical steps to immobilize antibodies. In this paper, a biotinylated antibody is conjugated with polyaniline, and the resulting biomaterial is coated on a silicon surface. Field Emission Scanning Electron Microscopy (FE-SEM) and UV-Vis have been used to confirm and characterize the experimental data. This simple approach to immobilize the biomolecules on the microcantilever surface is expected to offer certain advantages like strengthening of the microcantilever by avoiding the chemical overloading, and better signal strength.
During the past few years, immunosensors have been the subject of increasing interest, because of their potentialities in several fields, such as clinical diagnostics and environmental control. Owing to their low cost, small size, possible use in vitro and in vivo, and also their short response time, these devices can compete efficiently with the standard clinical diagnostic methods based on classical immunoasays.
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Typically, antibodies specifically directed against the desired analyte (antigen) are immobilized on the transducer surface as a biofilm. Then, the sensor is exposed to the sample (blood or other biological fluid); if antigen is present, an antigen-antibody complex will be formed . This formation will induce some changes of physicochemical parameters (usually mass, optical, or electrical parameters) of biofilm on the transducer surface, and that change can be subsequently detected. Depending upon the transducer technology used, immunosensors can be divided into three principal classes: optical, piezoelectric, and electrochemical. Regardless of the transduction principle, the coupling of the molecular recognition receptor to the transducer surface is of key importance for the control and the improvement of the characteristics of the derivative sensor.
Conducting polymers possess unusual electronic properties and show great potential for biomolecule immobilization. Several immobilization procedures based on conducting polymers have been used to ensure the coupling of immuno species. They range from an entrapment within the polymer film to an electrostatic binding . Many industrial applications of biosensors demand stable and defined binding of biomolecules to surfaces according easy and reproducible processes. Thus, some methods take advantage of specific interactions between avidin or streptavidin and biotin to immobilize biomolecules modified by methods of molecular biology in a highly oriented way. In this study also, biotinylated antibodies were conjugated with polyaniline using avidin. This biomaterial was coated on a silicon surface. Presence of antigen results in the formation of an antigen-antibody complex over the conducting polymer layer thus generating a potential which was sensed and measured. The potential developed was proportional to the amount of antigen present. For better transduction of the potential generated efforts are being made to obtain self assembled monolayer (SAM) of conducting polymer (polyaniline) over the silicon wafer to manufacture the microcantilevers.
Fig.1. Schematic of a biosensor. A biological sensing element (e.g., enzyme, antibody) detects a specific analyte producing a biochemical signal that is transferred to the transducer (e.g. conducting polymer), which ultimately produces a digital electronic signal that is proportional to the amount of analyte present.
The antibody polyclonal anti-Human IgG developed in goat (Î³-chain specific), the antigen Human IgG reagent grade, from serum and biotin conjugate were purchased from Sigma Aldrich (USA). Aniline, ammonium persulfate, hydrochloric acid (HCl), DMSO - anyhydrous dimethyl sulfoxide, NaHCO3 - sodium bicarbonate, NaCO3 - sodium carbonate, NaCl - sodium chloride, TRIZMA pre-Set crystals 8 (i.e. combination of Tris base and TrisHCl, NaN3 - sodium azide), acetone and avidin from Merck Chemicals, India were used as received. The supporting electrolyte was a phosphate buffer solution (PBS), prepared by dissolving one tablet of phosphate buffer saline (purchased from Sigma) in 200 ml distilled water. The obtained solution consisted of a 10-mM phosphate buffer, 2.7-mM potassium chloride, and 137-mM sodium chloride. The pH of solution at 250C was 7.4. Distilled water which had resistivity of 18mâ„¦ was used.
B. Preparation of antibody
Reaction buffer (100 mM carbonate, pH 8.4) was prepared by dissolution of 84g of NaHCO3 in 1 liter of distilled water. The pH was adjusted to 8.4. The solution was stored at 40C. The antibody (anti-Human IgG) concentration measured after buffer equilibration was 1 mg/ml.
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C. Biotinylation of antibody
10 mg of biotin was dissolved in 1 ml anhydrous DMSO. Biotin (80 Âµg/mg) was added to antibody solution. The tube was wrapped in foil, incubated and rotated at room temperature (250C) for 4 hours. The unreacted biotin was removed by dialyzing with a buffer (10 mM Tris, 150 mM NaCl, 0.1% NaN3, pH 8.2). This buffer was prepared by dissolution of 1.42g TRIZMA 8.0, 8.77g NaCl and 1g NaN3 in 1 liter of distilled water. The pH was adjusted to 8.2.
D. Synthesis of polyaniline
The synthesis of polyaniline was done by oxidative polymerization of aniline with ammonium persulfate as an oxidant. These components were slowly dissolved in 1 M hydrochloric acid as the reaction is very exothermic. The polymer precipitated as small particles and the reaction product was an unstable dispersion with micron scale particulates.
E. Preparation of substrate
Cleaned silicon wafer of size 10 x 10 mm was autoclaved and used for further studies. It was left for drying in vacuum for half an hour. Few drops of diluted polyaniline were spread over the silicon wafer and left for vacuum drying for 36 hours.
The specific binding of avidin to polymer biotin sites was achieved via the incubation of the polyaniline coated silicon wafer with a 2 mg/ml avidin solution, for 30 min at 50C. The obtained substrate was carefully rinsed with PBS. This substrate was then incubated with 1 mg/ml solution of biotinylated antibody (anti-Human IgG) at 50C for 12 hrs. The obtained substrate was then thoroughly rinsed with a PBS solution to remove weakly adsorbed antibodies. Finally, the resulting electrode was exposed to a known concentration of antigen (Human IgG).
III. RESULTS AND DISCUSSION
The polymerization of the aniline was achieved under controlled conditions to avoid the over oxidation of polyaniline which preserves the polymer conductivity.
A. UV-VIS studies
Fig. 2 shows the UV-Visible spectra of antibody (anti-Human IgG) in the range of 200 to 800 nm. The peak was obtained at 230 nm which lies in the UV region.
Figure 3 shows the UV-Visible spectra of polyaniline solution between 200-800 nm.
The antigen concentration was optimized using UV-Visible absorbance spectra. UV-Visible spectra of the interaction of Human IgG and anti-Human IgG was also studied in the range 200 to 400 nm. UV-VIS studies prove that biotinylated antibody (anti-Human IgG) can quantitatively sense the antigen (Human IgG).
B. FE-SEM studies
Fig. 4 shows FE-SEM micrograph of polymerization of aniline to form polyaniline. Surface studies with FE-SEM show that the coating is homogeneous and suitable for use as BIOMEMS.
Fig.2. Absorbance spectra of antibody (anti-Human IgG) at 1 mg/ml concentration
Fig.3. Absorbance spectra of polyaniline
Fig.4. FE-SEM image showing polymerization of aniline to form polyaniline
A biotin-avidin system has been used to immobilize biotinylated antibodies on a silicon wafer functionalized by chemically synthesized biotinylated polyaniline in order to conceive an immunosensor.
Commercial anti-Human IgG, biotin conjugated, was used as a model system. Studies show that this sensor can detect signals during antibody-antigen reaction. Also, surface studies with FE-SEM show that the coating is homogeneous and suitable for use as BIOMEMS.
The work is being continued to obtain signals using piezoelectric effect & optical fibers. In future, efforts will be made to obtain self assembled monolayer (SAM) of the conducting polymer, polyaniline using Langmuir-Blodgett method.