The Application Of Novel Functional Nanomaterials In Electrochemical Biosensor

Nanomaterials offer unique advantages that span several domains, such as a high surface-to-volume ratio, high catalytic activity, powerful adsorption ability, biocompatibility strength. The key points of biosensor are the biosensing interface and the signal amplification. A series of nanomaterials with different morphologies and structures, which included graphene, gold nanomaterial, graphene composite, liposome, magnetic silica nanoparticles and dendrimer, are prepared. The materials are assembled on the surface of electrode, which constructs the biosensing interface. Meanwhile, these nanomaterials used as signal label realize signal amplification. The use of such high-sensitivity sensors can offer the application of clinical diagonosis and environment monitoring.1. Electrochemical Biosensor Based on Graphene Oxide-Au Nanoclusters Composites for L-Cysteine AnalysisA modified Hummers method was utilized to synthesize the oxidized graphite powders. Bovine serum albumin (BSA)-stabilized Au clusters (Au NCs) was prepared by the reduced of NaBH4. A linker-free connected graphene oxide/Au nanocluster (GO-Au NCs) composite was prepared under sonication through electrostatic interactions, and characterized by transmission electron microscope (TEM), atomic force microscope (AFM), ultraviolet-visible (UV-vis) and FT-IR spectrum. The morphological and structural characterizations evidence that the Au NCs can be efficiently decorated on the GO. The electrochemical investigations indicated that GO-AuNCs composite has an important role in the electrocatalytic activity towards the oxidation of L-cysteine (CySH). The GO-Au NCs composite modified electrode shows a large determination range from0.05to20.0μmol/L, a remarkably low detection limit is0.02μmol/L and low oxidation potential (+0.387V). It was found that metal ions, carbohydrates, nucleotide acids and amino acids had no distinct effect on the determination of L-cysteine. In addition, the sensor has some important advantages such as simple preparation, fast response, good stability and high reproducibility. The direct determination of free reduced and total CySH in human urine samples has been successfully carried out without the assistance of any separation techniques.2. Ultrasensitive Electrochemical Immunosensor for CA15-3Using Thionine-Nanoporous Gold-Graphene as Platform and Horseradish Peroxidase-Encapsulated Liposomes as Signal AmplificationThis paper described a novel electrochemical immunosensor using nanoporous gold (NPG)/graphene (GN) hybrid platform combined with horseradish peroxidase (HRP)-encapsulated liposomes as labels for the sensitive detection of cancer antigen15-3(CA15-3). The electrochemical detection was based on the released HRP from HRP-encapsulated liposomes toward the reduction of H2O2with the help of the thionine (TH) electron mediator. In the presence of CA15-3, HRP@liposomes and TH-NPG-GN formed a sandwich-type immunocomplex, and the immunocomplex increased with the increment of the CA15-3concentration in the sample. The more CA15-3antigen in the sample there was, the more HRP@liposomes/anti-CA15-3in the immunocomplex there was. Thus, the catalytic current increased. Under optimized conditions, the linear range of the immunoassay is2×10-5-40U/mL with a detection limit of5×10-6U/mL CA15-3. The CA15-3concentrations of the clinical serum specimens assayed by the developed immunoassay showed consistent results in comparison with those obtained by commercially available electrochemiluminescence assay. This proposed immunoassay system had many desirable merits including sensitivity, accuracy, and little required instrumentation. Significantly, the new protocol may be quite promising, with potentially broad applications for clinical immunoassays.3. Magnetic Silica Nanoparticles Decorated Graphene Oxide by Click Chemistry as Peroxidase-like Labels for a Disposable Enzyme-free Electrochemical Immunosensor A disposable electrochemical immunosensor was established using peroxidase-like magnetic silica nanoparticles/graphene oxide composites as excellent labels and graphene oxide modified screen-printed carbon electrodes for the detection of cancer antigen153(CA153). ZnFe2O4nanoparticles were synthesized by a hydrothermal method, and magnetic silica nanoparticles were prepared with silica-coated ZnFe2O4by the reverse microemulsion method. The magnetic silica nanoparticles/graphene oxide composites were prepared via conjugating azide-functionalized magnetic silica nanoparticles to acetylene-functionalized graphene oxide by click chemistry. Structure characterization was obtained by means of atomic force microscope, X-ray diffraction and transmission electron microscope. The prepared hybrid nanomaterial showed greater peroxidase-like activity and its catalytic enzyme-substrate was used for cancer marker detection. The CA153immunosensor was fabricated by immobilizing a monoclonal anti-CA153antibody on the graphene oxide attached on a screen-printed electrode. This method was simple, inexpensive, highly sensitive, and selective for CA153detection with a linear range from10-3to200U/mL and a detection limit of2.8×10-4U/mL. In addition, the assay was evaluated with CA153spiked serum samples receiving excellent correlation with results from commercially available Ectrochemiluminescent Analyzer.4. Disposable Electrochemical Immunosensor for Simultaneous Assay the Panel of Breast Cancer Tumor MarkersThe multiplexed immunosensor array was fabricated on a carbon electrode array containing three graphite working electrodes, which was prepared with screen-printed technology. This immunosensor was achieved using graphene (GR) to modify immunosensor surface for accelerating electron transfer. The in-situ synthesis of AuNPs on GR modified electrode surface aimed at the immobilization of capture antibody (Ab1). With a sandwich-type immunoreaction, the alkaline phosphatase (ALP)-labeled antibody (Ab2) functionalized Au cluster (AuCs)/GR was captured on the immunosensor surface to catalyze the hydrolysis of3-indoxyl phosphate in substrate, which produced an indoxyl intermediate to reduce Ag ion. The silver deposition process was catalyzed by both ALP and AuCs/GR, which amplified the detection signal. The deposited silver was then measured by anodic linear sweep voltammetric stripping analysis in KCl solution. The increase of stripping peak currents was proportional to the logarithm value of the CA153, CA125and CEA concentration in range from5.0×10-3to50U/mL for CA153,1.O×1O-3to100U/mL for CA125, and4.0×10-3to200ng/mL for CEA with a limit detection of1.5×10-3U/mL for CA153,3.4×10-4U/mL for CA125, and1.2×10-3ng/mL for CEA. The disposable immunosensor array and simple detection method for fast measurement of panel of tumor markers avoided cross talk and the need of deoxygenation for the electrochemical immunoassay, and showed significant clinical value for application in cancer screening and provided great potential for convenient point-of-care testing and commercial application.5. Ultrasensitive Electrochemiluminescence Immunoassay for Protein Specific Detection Based on Dendrimer-Encapsulated Gold Nanoparticles LabelsA sensitivity-enhanced electrochemiluminescence immunoassay (ECLIA) was fabricated by covalently immobilizing a monoclonal prostate specific antigen (PSA) antibody (anti-PSA, Ab2) and a luminophore (luminol) on dendrimer-encapsulated gold nanoparticle (Den/AuNPs) as electrochemiluminescence labels. The primary antibody (Ab1) was immobilized on Fe3O4@SiO2NPs support, and the antibody-loaded Fe3O4@SiO2NPs were placed onto the indium tin oxide working electrode in a home-made electrochemiluminescence (ECL) cell. Then, PSA and Ab2/Luminol/Den/AuNPs were successively injected into the cell, and conjugated to form a sandwich-type immunocomplex. Under the optimized experimental condition, the proposed ECLIA provided a wide linear response range (0.001～100.0ng/mL) with a low detection limit (0.3pg/mL). The assay results of PSA in clinical serum samples were in good agreement with that of the commercially available electrochemiluminescence assay. The as-proposed ECL immunosensor has the advantages of high sensitivity, specificity and stability and could become a promising technique for tumor marker detection.