Study On The Novel Electrochemical Biosensors Based On Aptamers And Nano Materials

Adenosines and proteins etc. are important materials in composing and maintaining life activities. It is significant to analyze and examine these materials. Among so many detecting methods, electrochemical biosensor technology based on molecular electrochemical characters presents unique advantages such as high sensitivity, speediness, low cost, portability of measuring equipments, low consumption and easy microminiaturization as well as integration. Using such latest researching results as the high selectivity and affinity of aptamers as well as the amplification effect of nano materials, this thesis has developed a series of novel electrochemical biosensors for highly sensitive detection. The details are summarized as follows:A new adenosine biosensor based on aptamer probe was introduced (chapater 2). An amino-labeled aptamer probe is immobilized on the gold electrode modified with an o-phenylenediamine electropolymerized film using glutaraldehyde (GA) as the cross-linker. When an adenosine binds specifically to the aptamer probe, the interface of the biosensor is changed, resulting in the decrement of the peak current. Cyclic voltammetry (CV) and AC impedance are employed to record the process in which adenosine binds to the aptamer probe. The peak current is detected by alternating current (AC) voltammetry, the decrease of which is proportional to the amount of adenosine added. The used electrode can be easily regenerated in a heated water bath at 80°C. Under the optimized experimental conditions, the presented work exhibits a nice specificity towards adenosine.An aptamer-based electrochemical sensing platform for the direct protein detection has been developed using immunoglobulin E (IgE) and a specifically designed oligonucleotide strand with hairpin structure that has the standard aptamer segment as the model analyte and probe sequence, respectively (chapater 3). In the absence of IgE, the aptamer immobilized on an electrode surface forms a large hairpin due to the hybridization of the two complementary arm sequences, and peak currents of redox species dissolved in solution can be achieved. However, the target protein-binding can not only cause the increase of the dielectric layer but also trigger the significant conformational switching of the aptamer due to the opening of the designed hairpin structure that pushes the biomolecule layer/electrolyte interface away from the electrode surface, suppressing substantially the electron transfer (eT) and resulting in a strong detection signal. The proposed aptamer-based sensing system can exhibit an impressive sensitivity, selectivity and a wide response range without any amplifier. Owing to this surface-confined aptamer sequence, the limitations of the conventional electrochemical aptasensors have been overcome.A highly sensitive electrochemical immunoassay strategy based on the combination of ferrocene (Fc) label and poly(o-phenylenediamine) (PPD) film/gold nanoparticle (GNP) amplification for the detection of immunospecies was proposed using human IgG as the model analyte (chapater 4). A gold electrode is firstly modified with an electropolymerized film of poly(o-phenylenediamine), which provides a stable matrix with abundant amino groups for the fabrication of sensing interface. Using glutaraldehyde as a cross-linker, cystamine is coupled onto the modified electrode. Subsequently, gold nanoparticle monolayer is assembled onto the resulting surface. Antibodies can be self-assembled onto the surface-confined gold nanoparticles via amine-Au affinity with a high loading amount and reserve high immunological activity. After the introduction of model analyte, the ferrocene (Fc)-labeled antibody is immobilized on the sensing interface by antibody-antigen specific reaction, resulting in a redox current signal. The peak current is proportional to the amount of the analyte. Under the optimized experimental conditions, the proposed sensing strategy provides a wide linear dynamic range. In addition, good reproducibility, high selectivity and stability can be achieved.A kind of ordered 3D Au nano-prickle clusters by directly electrodeposited on glassy carbon electrode utilizing the spatial obstruction/direction of the polycarbonate (PC) membrane can be employed to fabricate electrochemical immunosensor (chapater 5). Due to large surface area of the 3D nanoclusters, the proposed nanoclusters might be effective for the immobilization of biomolecules. With IgG as a model analyte, a sandwich-type complex of goat anti-hIgG/IgG/HRP labeled goat anti-hIgG was formed. The content of IgG can be calculated via the decrease of reductive current caused by catalyzing enzymatic substrates. The deposited Au nanoclusters are stable with good biocompatibility, large specific surface area, and high electron exchange capability. The developed immunosensor based on Au nano-prickle clusters possesses advantages such as fast response, low detection limit, wide linear range and easy regeneration.