Studies On Novel Amperometric Immunosensor And Aptasensor Based On Nano-materials

A biosensor is a device for the detection of an analyte that combines a biological component (eg. enzymes, antibodies, nucleic acids, cell, etc) with a physicochemical detector component. Ameperometric biosensor is based on the change in the amperometric response before and after biochemical reaction to detect the concentration of biomolecule. So, enhancing current responses sigle and developing of sensitive bioassay technology is one of the important tasks in the study of ameperometric biosensor. In recent years, nano-materials (eg. Nanoparticles,nanowires,nanotubes etc), exhibiting large specific surface area, good biocompatibility and strong adsorption capacity, are favourable for constructing biosensors, which not only retain the biological activities of biomolecules, but also enhance the sensitivity and service life of the sensors.The electrochemical immunosensor uses specificity of the reaction of antigen and antibody to combine traditional method for immunoassay with the technology of modern sensor, which both has the high specificity of the immunochemical system and has the high sensitivity of electrochemical analysis and has been broadly applied in clinical diagnosis. Electrochemical aptasensors, which are based on the specificity of aptamer-target recognition with electrochemical transduction for analytical purposes, have received particular attention in recent years.In this paper, the nano-technology, bionsensing technology and electrochemical analytical techniques are combined for immunoassay and aptamer analysis. It focuses on designing and fabricating novel biomimetic interface combining some kinds of nanoparticles with conducting polymers, carbon nanotubes, platinum nanoparticles, and gold nanoparticles for the immobilization of biomolecules. Transmission electron microscopy, Scanning electron microscopy, Atomic force microscopy, UV-Vis spectra and electrochemical techniques are used as tools for studying the properties of interface. We discussed their applications in bioanalysis domain. This research focuses on the details as follow:1. Amperometric Immunosensor for Carcinoembryonic Antigen Based on poly (2,6-pyridinediamine) and Gold Nanoparticles A new highly sensitive amperometric immunosensor for the determination of carcinoembryonic antigen (CEA) had been constructed by employing a novel conducting polymer film coupled with gold nanoparticles. The poly 2,6-pyridinediamine (pPA) film was formed on the glassy carbon electrode by utilizing electropolymerization, which yielded an interface with amine groups, good conducting and stable nanowire construction. On the struction, thionine (Thi) were easily immobilized through covalent conjugation using glutaraldehyde, which built an interface for the assembly of gold nanoparticles (GNPs). Then, the carcinoembryonic antibody (anti-CEA) was chemiadsorbed onto the surface of modified electrode. Under optimal conditions, the resulting immunosensor displayed a high sensitivity for the detection of CEA, and exhibited high selectivity, long-term stability and good reproducibility.2. Amperometric Immunosensor for a-1-Fetoprotein Based on Gold Nanoparticles and Multi-wall Carbon Nanotube-Thionine CompositeA new sensitivity amperometric immunosensor for the detection ofα-1-fetoprotien (AFP) was developed based on multi-wall cabon nanotube (MWNTs)-thionine(Thi) nano-composite membrane. Carbon nanotube, as the carrier and condonctor of Thi, permit an important decrease in the overvoltage for the redox of chemicals and a dramatic improvement in the reversibility of the redox behavior of biomolecules. The high surface area is propitious to the immobilization of Thi and promote electron transfer between redox center of Thi and electrode surface, which will improve the sensitivity and stability of biosensor. At the sametime, the modified MWNTs created an interface with amine groups, good biocompatibility, which made nanoparticles and biomolecules can be easily immobilized. GNPs was immobilized on the electrode according to Au-NH2 covalent bond, and then the immunosensor was prepared by immobilizing antibody ofα-1-fetoprotien (AFP) which can make bond with GNPs. The immunosensor had good stability with a wide linear range of 0.20～120 ng/mL and a detection limit of 0.12 ng/mL (S/N=3).3. An ultrasensitive amplified amperometric thrombin aptasensor taking PtNPs and HRP labeled adivin-biotin sysytem in sandwich mannerAn amplified amperometric aptasensor for sensitive detection of ubiquitous protein thrombin was developed based on PtNPs-enzyme labeled thrombin aptamer. Adopting sandwich format, gold nanoparticles (GNPs) were electrodeposited onto a glassy carbon electrode (GCE) to create a nanomaterial platform for immobilization of the thiolated aptamer (TBA I). After capturing the target thrombin, signal aptamers tagged with Pt nanoparticles (PtNPs) and avidin or biotin labeled HRP (PtNPs-enzyme labeled-TBAⅡ) were attached to the assembled electrode surface for amplifying current signal. The use of TBAⅡlabeled with PtNPs, HRP-avidin, HRP-biotin, and the consumption-regeneration cycle of H2Q could significantly amplify the current signal and increase the sensitivity of aptasensor. The GNPs electrodeposited on GCE used as a platform for immobilization of the thiolated aptamer can enhance the immobilization amount of aptamer and improve the sensitivity of an amperometric biosensor for the determination of protein. A signal-on sandwich sensing manner is developed to monitor the current increase caused by PtNPs-enzyme labeled TBAⅡ, which would greatly avoid the sensing scheme because of a negative readout signal.Under optimized conditions, the aptasensor based on PtNPs and HRP as enhancer exhibited low detection limit of 0.8 pM with the linear range from 2.3 pM-1.0 nM thrombin. The developed method showed high sensitivity, acceptable stability and reproducibility, and could be used for detection of various protein targets.