Ultrasensitive Electrochemical Immunoassay Method Based On Functionalized Nanomaterials

Highly sensitive and selective immunoassays capable of detecting protein biomarkers are essential for disease diagnosis, drug screening, and biodefense applications. Electrochemical immunoassay has some intrinsic advantages of good portability, low cost, and high detection sensitivity, resulting in great progress. Recently, Signal amplification has been used to develop ultrasensitive immunosensors for the detection of low-abundance biomarkers by bioconjugating large amount of enzymes on various nanocarriers. However, the practical applications of these report probes are limited due to the denaturation and leakage of enzymes, and the time-consuming and costly preparation and purification process of enzymes. In this dissertation, by combining nanotechnology, host-guest supermolecular assembly and chemical biology, with poly(diallyldimethylammonium chloride) (PDDA) functionalized carbon nanotubes and electrochemically reduced graphene oxide facilitating electron transfer, a series of free enzymes immunoassay were developed. Electrochemiluminescent (ECL) based on quantum dots (QDs) immunoassay strategy was proposed based on hemin biobarcoded nanoparticle tags consumption of ECL coreactant leading to be quenched and low-background nature of ECL. This dissertation includes the following four parts:1. Signal amplification for electrochemical immunosensing by in situ assembly of host-guest linked gold nanorod superstructure on immunocomplexAn amplification strategy for signal tracing was developed by introducing a host-guest binding reaction into the assembly process of gold nanorods (AuNRs) superstructure. The amplification pathway firstly used a thio-β-cyclodextrin (HS-J3-CD) functionalized gold nanoparticles to label signal antibody, and then in situ assembled multi-layer HS-β-CD end-functionalized AuNRs to sandwich immunocomplex on immunosensor surface by using 4,4,4,4-(21H, 23H-porphine-5,10,15,20-tetrayl) tetrakis (benzoic acid) as a bridge to achieve simple and convenient host-guest reaction. The built end-to-end AuNRs superstructure showed excellent performance for the signal amplification in connection with the electrochemical biosensing by preoxidation and then voltammetric analysis of gold element. Using a-fetoprotein as an analyte, the immunosensor was constructed by covalently binding capture antibody to chitosan-carbon nanotubes-poly(diallyldimethylammonium chloride) modified electrode. The superstructure rich in AuNRs brought an enhanced detection sensitivity of protein, which could detect a-fetoprotein in a linear range of 0.5 pg mL-1 to 0.5 ng mL-1 with a detection limit down to 0.032 pg mL-1. The immunoassay exhibited good stability and acceptable reproducibility and accuracy. The in situ superstructure assembly could be extended to other labeled recognition systems, providing a promising novel avenue for signal amplification and potential applications in bioanalysis and clinical diagnostics.2. Triple signal amplification of graphene film, polybead carried gold nanoparticles as tracing tag and silver deposition for ultrasensitive electrochemical immunosensingA triple signal amplification strategy was designed for ultrasensitive immunosensing of cancer biomarker. This strategy was achieved using graphene to modify immunosensor surface for accelerating electron transfer, poly(styrene-co-acrylic acid) microbead (PSA) carried gold nanoparticles (AuNPs) as tracing tag to label signal antibody (Ab2) and AuNPs induced silver deposition for anodic stripping analysis. The immunosensor was constructed by covalently immobilizing capture antibody on chitosan/electrochemically reduced graphene oxide film modified glass carbon electrode. The in situ synthesis of AuNPs led to the loading of numerous AuNPs on PSA surface and convenient labeling of the tag to Ab2. With a sandwich-type immunoreaction, the AuNPs/PSA labeled Ab2 was captured on the surface of immunosensor to further induce a silver deposition process. The electrochemical stripping signal of the deposited silver nanoparticles in KC1 was used to monitor the immunoreaction. The triple signal amplification greatly enhanced the sensitivity for biomarker detection. The proposed method could detect carcinoembryonic antigen with a linear range of 0.5 pg mL-1 to 0.5 ng mL-1 and a detection limit down to 0.12 pg mL-1. The immunosensor exhibited good stability and acceptable reproducibility and accuracy, indicating potential applications in clinical diagnostics.3. Nanogold/mesoporous carbon foam-mediated silver enhancement for graphene-enhanced electrochemical immunosensing of biomarkerNanogold functionalized mesoporous carbon foam (Au/MCF) coupling with a signal amplification by C-Au synergistic silver enhancement was designed for sensitive electrochemical immunosensing of biomarker. The Au/MCF was prepared by in situ growth of nanogold on carboxylated MCF and used as a tracing tag to label signal antibody via the inherent interaction between protein and nanogold. The immunosensor was prepared by covalently immobilizing capture antibody on an electrochemically reduced graphene oxide/chitosan film modified glassy carbon electrode. Through a sandwich-type immunoreaction, AuNP/MCF tags were captured on the immunoconjugates to induce a silver deposition process. The electrochemical stripping signal of the deposited silver was used to monitor the immunoreaction. The Au/MCF-mediated silver enhancement along with the graphene-promoted electron transfer led to high detection sensitivity of carcinoembryonic antigen. Under optimal conditions, the proposed immunoassay method showed wide linear range from 0.1 pg mL-1 to 1 ng mL-1 and a detection limit down to 0.024 pg mL-1. The newly designed amplification strategy holds great potential for ultrasensitive electrochemical biosensing of other analytes.4. Ultrasensitive immunoassay of protein biomarker based on electrochemiluminescent quenching of quantum dots by hemin biobarcoded nanoparticle tagsA hemin biobarcoded nanoparticle probe labeled antibody was designed by the assembly of antibody and alkylthiol-capped bar-code G-quadruplex DNA on gold nanoparticles and the interaction of hemin with the DNA to form a G-quadruplex/hemin biobarcode. An ultrasensitive immunoassay method was developed by combining the labeled antibody with an ECL immunosensor for protein. The ECL immunosensor was constructed by a layer-by-layer modification of carbon nanotubes, CdS quantum dots (QDs) and capture antibody on a glassy carbon electrode. In air-saturated pH 8.0 PBS the immunosensor showed a carbon nanotubes enhanced cathodic ECL emission of QDs. Upon the formation of immunocomplex, the ECL intensity decreased owing to the consumption of ECL coreactant in the biobarcode electrocatalyzed reduction of dissolved oxygen. Using a-fetoprotein as model analyte, the quenched ECL could be used for immunoassay with a linear range of 0.01 pg mL-1 to 1 ng mL-1 and a detection limit of 1.0 fg mL-1. The wide detection range and high sensitivity resulted from the enhanced ECL emission and highly efficient catalysis of the biobarcode. The immunosensor exhibited good stability, acceptable fabrication reproducibility and accuracy, showing great promise for clinical application.