Study Biosensors Based On Biomolecule Immobilized On Nanoparticles

Biosensors are widely used for the analysis of biological analytes. Biosensors become a very active research topic, owing to the advantages of their high sensitivity, fast analytical time, simple measurement, low-cost and high selectivity. It has been reported that the crucial aspect in the fabrication of a biosensor is the immobilization of biorecognition molecule in high amounts with retention of their bioactivity. For these reasons, we explore and study the electrochemical assays based on different immobilization methods and different transducers for analytes determination.Part I investigated the novel immunosensor based on HRP instead of BSA as blocking agent1. A highly hydrophilic, non-toxic and conductive TiO₂ nanoparticles/gold nanoparticlesbilayer films as immobilization matrix via self-assembly (SA) and deposition method was prepared on a gold electrode. Subsequently, positively charged horseradish peroxidase (HRP) was assembled onto the bilayer films, which provided an interface to assemble gold nanoparticles for immobilization of carcinoembryonic antibody (anti-CEA). Finally, HRP was used to block sites against non-specific binding. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were applied to characterize the electrochemical properties of the SA process. The CVs reduction current of the immunosensor decreases linearly in two concentrations ranges of CEA from 0.3 to 10 and from 10 to 80 ng/mL with a detection limit of 0.2 ng/mL in presence of 0.7 mmol/LH₂O₂ in analyte solution. Effects of deposition time, pH of working buffer, temperature and incubation time were also explored for optimum analytical performance by using the amperometric method. Moreover, the proposed immunosensor exhibited good accuracy, high sensitivity, long-term stability and made it to determine CEA in serum samples with satisfactory results. 2. A novel amperometric immunosensor with amplified sensitivity for the determination of carcinoembryonic antigen (CEA) has been developed based on the assembly of {Thi⁺/nano-Au}_n layer-by-layer films by alternate adsorption of negatively charged gold nanoparticles (nano-Au) and positively charged thionine (Thi+) on Titania nanoparticles/gold nanoparticles composite film formed previously on the electrode via self-assembly and deposition method, which provided an interface to assemble carcinoembryonic antibody (anti-CEA). Subsequently, HRP was backing-filled into the CEA-modified electrode surface to amplify the response of the antigen-antibody interactions. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were used to monitor and confirm the films growth. Assay conditions were optimized including the incubation temperature and time, the pH of working buffer, the concentration of the H₂O₂ and the electrodeposition time of nano-Au, etc. The reduction current of the immunosensor decreases linearly in the range of CEA from 0.2 to 80.0 ng/mL with a detection limit of 0.07 ng/mL in presence of 0.55 mmol/LH₂O₂ in working solution. Moreover, the immunosensor showed acceptable reproducibility, high sensitivity and long-term stability. Clinical serum samples were assayed with this method and the results were in acceptable agreement with those obtained from the enzyme-linked immunosorbent assays (ELISAs). Therefore, the platform that combines the advantages of nanostructured materials with those of the layer-by-layer self-assembling technique opens the doors to the new and exciting possibilities for the development of immunosensor using different transduction modes.Part II investigated the hydrogen peroxide biosensor.1. An amperometric biosensor for the determination of H₂O₂ has been developed by means ofimmobilizing horseradish peroxidase onto a platinum electrode based on Au colloids and poly(thionine) as matrixes. Thionine, as a mediator, was first polymerized on a platinum electrode surface, then gold nanoparticles were chemisorbed onto ploy(thionine), furthermore, horseradish peroxidase (HRP) was absorbed on the electrode surface through the gold nanoparticles conjugation. Finally, HRP and Au colloids were firmly immobilized on the electrode surface by the entrapping of polyvinyl butyral (PVB). The performance and factors influencing the performance of the biosensor were studied. The biosensor exhibited a dynamic range of 2.15×10^-6 -1.43×10^-2 mol/L to H₂O₂ with a detection limit of 2.00×10^-7 mol/L and a correlation coefficient of 0.998. Moreover, the biosensor exhibited high sensitivity, good repeatability and long-time stability.2. A novel amperometric biosensor for the detection of hydrogen peroxide (H₂O₂) has been developed by means of self-assembly and opposite-charged adsorption to immobilize horseradish peroxidase (HRP) on titania nanoparticles (nano-TiO₂) and gold nanoparticles (nano-Au) bilayer films modified Au electrode. The immobilized HRP could transfer electron directly between the bilayer films and the Au electrode, the number of electron transport (n) and the electron transfer rate constant k_s were calculated as 1.13 and 0.86 s^-1,respectively. Cyclic voltammetry and chronoamperometry were employed to demonstrate the direct electron transfer between immobilized HRP and the Au electrode. The performance and factors influencing the performance of the resulting biosensor such as sensor fabrication, assay process, pH, temperature and ionic strength were optimized. The biosensor displayed a direct electrocatalytic response to the reduction of H₂O₂, provided a wide linear range from 0.70μmol/L to 6.89 m mol/L with a detection limit of 0.30μmol/L at a signal-to-noise ratio of 3 and a correlation coefficient of 0.999. The response showed a Michaelis-Menten behavior at higher H₂O₂ concentration.The apparentMichaelis-Menten constant K_M^app for biosensor was calculated to be 1.56 m mol/L, showing a high affinity. Moreover, the biosensor exhibited high sensitivity, good reproducibility and long-time stability.