Study On Electrochemiluminescence Immunosensor Constructed By Enzyme And Nanoparticles

Electrochemiluminescence immunosensor, the combination of electrochemiluminescence (ECL) technique with selective biological recognition pattern immunoassays and has inherent advantages such as high selectivity, high sensitivity and low background signal. In order to meet the requirements of quantitative determination of trace protein, using material and biological amplificatory technology to specifically transducer and amplify the signal is an effective method for improving the sensitivity. The in situ generate coreactant could provide new idea for research and develop of immunoassay. Thus, by coupling enzymatic reaction to in situ generate coreactant of ECL reagent with the virtue of nanoparticles has been an important method in constructing immunosensors.Part1Electrochemiluminescence of peroxydisulfate enhanced by L-Cysteine film for sensitive immunoassayIt is the first time that we developed an immunosensor based on the ECL of peroxydisulfate solution without conventional luminescent reagents. Firstly, with L-Cysteine electrodeposited on the bare gold electrode surface, the ECL intensity was greatly enhanced. Then, AuNPs was assembled onto the electrode, which could further improve the ECL intensity and be favorable for the adsorption of antibody. After antibody was linked to the electrode surface via AuNPs, BSA was used to block nonspecific binding sites. In this work, L-Cysteine and AuNPs were amplified the ECL signal simultaneously and improved the detection sensitivity. The ECL intensity decreased linearly with the AFP concentrations in the range from0.01to100ng/mL with a detection limit of3.3fg/mL. The proposed ECL immunosensor provides a rapid, simple, and sensitive immunoassay protocol for protein detection, which might hold a promise for clinical application.Part1Highly amplified electrochemiluminescence of peroxydisulfate using bienzyme functionalized palladium nanoparticles as labels for ultrasensitive immunoassay In a novel peroxydisulfate/O2-based ECL biodetection systems, the coreactant O2is basically from the dissolved oxygen, which suffers from difficulty in labeling and low concentration in the detection solution. By coupling the cooperation of two enzymes to in situ generate coreactant with PdNPs as catalyst for the ECL reaction. PdNPs were previously synthesized and successfully attached to functional carbon nanotubes (FCNTs), which were used to binding the secondary antibody and bienzyme (GOD and HRP). Then the preparedbioconjugates were introduced to the electrode via sandwich immunoreactions. Accordingly, through the ECL response of peroxydisulfate and O2, a dramatically amplified ECL signal was obtained for that H2O2produced by the GOD was subsequently reduced by HRP to in situ generate O2, and PdNPs as catalyst for the ECL reaction. The present immunosensor showed a wide linear range of0.01pg/mL to100ng/mL, with a low detection limit of3.3fg/mL for detecting AFP. This new signal amplification strategy for preparation of ECL immunosensor could be easily realized and has potential application in ultrasensitive bioassays.Part3Highly enhanced electrochemiluminescence based on synergetic catalysis effect of enzyme and Pd nanoparticles for ultrasensitive immunoassayLuminol-H2O2system has been widely used in immunoassay, however, H2O2as coreactant suffering from difficulty in labeling and unstable in the detection solution. In our work, by coupling enzymatic reaction to in situ generate coreactant with ECL reaction could be successfully solve the above disadvantages. For the first, FCNTs-PdNPs@anti-AFP-GOD has been synthesized, which were assembled on electrode surface by sandwich type immunoreactions. When proper amount glucose was added in the detection solution, GOD catalyzed oxidation process of glucose to in situ generate H2O2, which was subsequently catalyzed by PdNPs. The proposed immunosensor showed high sensitivity, wide linear range, good reproducibility and acceptable precision and accuracy. Thus, this work provided a new method for signal amplification of ECL biosensing and would extend the application of luminal in bioanalysis.