Addressable Electrochemical Array Immunosensor

Electrochemical sensor array has received much attention in clinical diagnoses, pharmaceutical analysis, food safety and environmental monitoring because of its following features:easy miniaturization, low power consumption and cost, insusceptible to optical path and sample turbidity, especially compatibility with machining process. Electrochemical immunosensor array combining the high-throughput of electrochemical array technology with high selectivity of immunoassay has been widely applied in clinical analysis. Although considerable efforts have been made in the current research in electrochemical immunosensor array, some problems such as preparation of sample, synthesis of probe, immobilization of molecular recognition, analytical performance and date reading have not been addressed.This research work was supported by National Natural Science Foundation of China (NO.20805028). Our long-term goal is to fabricate simple, sensitive, simultaneous multianalyte immunoassays electrochemical immunosensor array. The aim of our work is to develop a addressable immobilization method in multi-electrode array in order to resolve the difficult result from the miniaturization of array; and develop a biological compatibility sensor interface to construct a sensitive electrochemical immunoassay method.The major contents in this thesis are described as follows:First part, it involves the general introduction of immunoassay, immunosensor and immunosensor array. The fabrication of electrochemical immunosensor array and its classification were presented in detail. The application of nanoparticles in electrochemical immunosensor array was partly reviewed.Second part, Highly sensitive amperometric immunosensor array for tumor markers incorporating electrochemically addressing and one signal antibody.A novel electrochemical immunosensor array with high sensitivity and operational simplicity for the determination of multiple tumor markers was developed. Two important tumor markers, carcinoembryonic antigen (CEA) and a-fetoprotein (AFP), were utilized as model analytes while a horseradish peroxidas-labeled antibody was employed as the signal antibody. A base electrode array consisting of six carbon paste working electrodes, one carbon paste auxiliary electrode and one Ag/AgCl reference electrode was designed and fabricated by screen-printing technology. The immunosensor in the array was fabricated in sequence by covalently coupling the capture antibody onto the surface of the working electrode which was electrochemically addressable grafted with aminophenyl group by reduction of in situ generated aminophenyl diazonium cation generated from p-phenylenediamine, which allowed selectively immobilization of different antibodies at desired positions on a single array via electrochemical operation. The immunoassay in sandwich model was performed by specifically binding the targets, second antibodies and one signal antibody to the immunosensor array, and then measuring the amperometric response at-0.25 V (vs. Ag/AgCl) in the presence of hydroquinone and hydrogen peroxide. The result showed that the obtained steady current density was directly proportional to the logarithm of concentration of target AFP/CEA in the range from 0.10 to 50 ng/mL with the much low detection limits of 0.03 ng/mL for CEA and 0.05 ng/mL for AFP (S/N=3), respectively. The immunosensor array fabricated has attractive advantages such as simple operation, negligible cross-talk, fine reproducibility, good storage stability, and successful application to determining CEA and AFP in serum samples. This work demonstrates that electrochemically addressing for the fabrication of the electrochemical immunosensor array and one signal antibody for the determination of multiple tumor markers are promising approaches.Third part, Electro-click immobilization of antigen on carbon surface for selective and reproducible immunoassay of anti-IgG.An electro-click method based on azide photoligation and Cu-catalyzed azide-alkyne cycloaddition has been evaluated for the immobilization of antigen to electrode. The biomolecular recognition properties of the electrode have been investigated with regard to the electrochemical immunoassay for the determination of anti-IgG. As a proof-of-concept, a newly designed alkyne functionalized IgG was used as molecular recognition and HRP labeled anti-IgG was used as label for the determination of anti-IgG. Glassy carbon electrode was firstly grafted with azido group by the electrochemical reduction of phenylazide diazonium salts and then alkyne functionalized IgG was immobilized onto the azide group modified electrode in the presence of active Cu(Ⅰ) catalyst to form immunosensor. The immunosensor was used to determine anti-IgG and the current response is linear with the logarithm of concentration of anti-IgG from 0.1 ng/mL to 10 ng/mL with a detection limit of 0.03 ng/mL. The functionalized surfaces were also evaluated in electrochemical addressable immobilization of protein onto multi-electrode. The method proved to be versatile and efficient immobilization for the fabrication of immunosensor with high and predictable selectivity.Fourth part, An addressable amperometric immunosensor array based on click chemistry with the functionalization of single-walled carbon nanotubes as sensing platform.Covalent functionalization of azide decorated SWNTs with alkyne modified protein was accomplished by the Cu(Ⅰ)-catalyzed [3+2] Huisgen cycloaddition. FT-IR spectroscopy, Raman spectroscopy, scanning electron microscopy, and a transmission electron micrograph were used to characterize the functionalized SWNTs. The functionalized SWNTs showed excellent dispersion in water and kept good biocompatibility with conjugated with protein. As a model proof-of-concept, antibody functionalized SWNTs were cast onto glassy carbon electrode interface as the immunosensing platform and a sensitive electrochemical immunoassay was developed for the determination of anti-immunoglobulin (anti-IgG) using horseradish peroxidase (HRP) as redox label. Compared with directly immobilization of anti-IgG onto glass electrode, SWNTs as immobilization platform showed a better sensitizing effect, a detection limit of 0.03 ng/mL (S/N=3) was obtained for anti-IgG. An addressable immunosensor array by electro-click chemistry on the azido-functionalized SWNTs was fabricated for the determination of AFP, CEA and DCP. The ability to independently immolization of different antibody onto each electrode in an array by electrical addressing was evualated. The result showed that the obtained steady current was directly proportional to the concentration of target AFP/CEA/DCP in the range from 0.01 to 0.1 ng/mL with the much low detection limits of 3 pg/mL for CEA,5 pg/mL for AFP and 3 pg/mL DCP (S/N=3), respectively. The proposed strategy provided a biocompatible immobilization and sensitized recognition platform for analytes and demonstrated that the click coupling of SWNTs with protein was effective for controlling the material properties and achieving high performance materials with high selectivity and sensitivity.This thesis proposed two addressable immobilization methods for the immobilization of different recognition molecular onto multi-electrode array; established two addressable immunosensor arrays for simultaneous detecting two or three tumor marker. It provided a good analytical method and an apparatus for the detection and preliminary screening test of tumor marker and offered some basical references for early diagnosis of tumor.