Studies On Highly Sensitive Bioaffinity-Type Amperometric Biosensing Based On Metal Labels

For a long time, the innovation of bioanalysis and biosensing methods has been a hot spot of basic research in analytical chemistry and biotechnology fields. To date, a lot of such new methods have been developed to meet diverse analytical needs in the fields such as biomedicine, food safety and environmental analysis. The bioanalysis and biosensing methods based on the high specificity of various bio-affinity behaviors(immune affinity, nucleic acid hybridization, aptamer affinity, sugar-lectin affinity, and etc.) intrinsically own high selectivity, and thus improving the detection sensitivity has long been one of the most important objectives in this research field. Electrochemical analysis methods have the advantages of high sensitivity and selectivity, low limits of detection(LODs), facile operation, simple instrumentation as well as ease of automation and device miniaturization/portability, and thus they are highly promising for rapid and in-situ biomedical analysis. The nanomaterials of metals and their compounds have been frequently used as biolabels for rapid and sensitive bioelectroanalysis(i.e. metal-labeled amperometric bioassay, MLAB), because the metal components can be sensitively determined by anodic stripping voltammetry(ASV) in a very convenient way.From the microcosmic viewpoint, the valence-electron-movement issue is one of the core scientific issues in chemistry and its downstream disciplines such as molecules/ions/atoms-based biology and material sciences. The electron-movement behaviors are microcosmic fundamentals of various chemical reactions and phenomena, including(1) energy-level transitions of valence and inner-shell electrons for various atomic and molecular spectroscopy techniques in chemistry;(2) weak electronic interactions for any known weak inter-/intra-molecular interactions and the interactions-based chromatographic separation/analysis and supramolecular chemistry;(3) strong electronic interactions(electron sharing) for covalent-bond formation and synthetic chemistry; and(4) complete electron gains/losses for electrochemistry, redox chemistry, ion chemistry and mass spectrometry. Hence, in this sense, we may regard chemistry as the science mainly dealing with and making use of extranuclear electrons. We believe that methodology innovation on acquisition and amplification of interfacial electron transfer signals on biosensing electrodes not only can help the development of electroanalytical chemistry and electrochemistry, but also can be of great significance for in-depth understanding of the extranuclear electron movement behavior as a chemical global issue.In this dissertation, the recent advances of electrochemical biosensors and nanobiosensing are reviewed, a series of studies on highly sensitive amperometric biosensing are conducted based on bioaffinity and biolabeling of nanomaterials of metals and their compounds as well as signal amplification, and highly sensitive assays of several tumor biomarkers and myocardial damage biomarkers are realized. The main contents are as follows.1. The Au nanoparticles(Au NPs)-catalyzed redox reaction between HAu Cl4 and NH2OH(gold-label/gold-staining) was employed to notably enlarge the Au NPs-biolabel size at a sandwich-type immuno-electrode with Au NPs-labeled second antibody, followed by simultaneous chemical-dissolution and cathodic-preconcentration of the gold-stained Au NPs biolabels by microliter-droplet HBr-Br2 solution as well as the microliter-droplet ASV analysis of preconcentrated Au directly on the immunoelectrode, thus we realized ultrasensitive electrochemical immunoassay of proteins based on such in situ duple amplification of gold nanoparticle biolabel signals. Under optimized conditions, this electrochemical sandwich immunoassay method can be sensitive to a few protein molecules(human immunoglobulin G or human prostate-specific antigen).2. An ultrasensitive metal-labeled amperometric immunoassay(MLAI) method of proteins is reported, on the basis of multiple gold-label/silver-staining and galvanic replacement reactions(GRRs) at a sandwich-type immuno-electrode with Au NPs-labeled second antibody, followed by simultaneous chemical-dissolution/cathodic-preconcentration of silver for in-situ microliter-droplet anodic stripping voltammetry(ASV) detection on the immunoelectrode. Silver was selectively stained on the catalytic Au NPs surfaces through chemical reduction of Ag+ by hydroquinone(gold-label/silver-staining). Multiple GRRs between HAu Cl4 and the stained silver were used to amplify the signal. Under optimized conditions, this method was used for ultrasensitive quantitative analysis of human immunoglobulin G(Ig G) and human α-fetoprotein(AFP), giving limits of detection(LODs, S/N=3) of 0.2 fg m L-1 for Ig G and of 0.1 fg m L-1 for AFP(equivalent to 5 molecules in the 6 μL samples employed for analysis of both proteins). The theoretical feasibility of such a single-molecule-level amperometric immunoassay is also discussed based on the immunological reaction thermodynamics.3. A new protocol for ultrasensitive electrochemical sandwich-type immunosensing is reported, on the basis of an enlarged signaling by gold-label/copper-staining, galvanic replacement reactions(GRRs), and in situ microliter-droplet anodic stripping voltammetry(ASV) detection on a glassy carbon electrode after an enhanced cathodic preconcentration of copper. Briefly, copper is selectively stained on the catalytic surfaces of second antibody-conjugated Au nanoparticles through Cu SO4-ascorbic acid redox reaction, and the GRRs between HAu Cl4 and the stained copper are used to amplify the quantity of copper, then the corresponding antigen is quantified based on simultaneous chemical-dissolution/cathodic-preconcentration of copper for in-situ ASV analysis directly on the immunoelectrode. Cyclic voltammetry, electrochemical impedance spectroscopy, quartz crystal microbalance and scanning electron microscopy are used for film characterization and/or process monitoring. Under optimized conditions, ultrasensitive analyses of human immunoglobulin G(Ig G) and human carbohydrate antigen 125(CA125) are achieved. The limits of detection are 0.3 fg m L-1(equivalent to 7 Ig G molecules in the 6 μL sample employed) for Ig G(S/N=3) and 1.3 n U m L-1 for CA125(S/N=3), respectively. Satisfactory results were obtained for real serum sample analysis.4. We report a new protocol of metal-labeled amperometric bioanalysis with high sensitivity(MLABhs), on the basis of simultaneous chemical-dissolution/ cathodic-enrichment of the Cd S quantum dots label on the bioelectrode and in-situ anodic stripping voltammetry(ASV) detection. The beforehand exertion of a cathodic potential in air and the use of a small-volume acid can minimize the diffusion-layer thickness to preconcentrate Cd in the labeled Cd S quantum dots on the bioelectrode as entirely as possible, which can notably enhance the subsequent ASV signal. This MLABhs protocol has been used for sandwich-type immunoanalysis and aptamer-based bioanalysis, giving limits of detection of 4.5 fg m L-1 for human immunoglobulin G, 3.0 fg m L-1 for human carcinoembryonic antigen(CEA), 4.9 fg m L-1 for human α-fetoprotein(AFP), and 0.9 f M for thrombin. The simultaneous and sensitive analysis of CEA and AFP at two screen-printed carbon electrodes was also conducted by our MLABhs protocol.5. We report ultrasensitive electrochemical immunoassay of proteins, on the basis of in situ enzymatic formation of Cd S quantum dots(QDs) and simultaneous Cd S chemical-dissolution/cathodic Cd preconcentration for microliter-droplet anodic stripping voltammetry(ASV) detection directly on the immunoelectrode. The antibody 2 with alkaline phosphatase(ALP) and Au nanoframes labeled(Ab2-ALP-Au NFs) can catalyze hydrolysis of ascorbic acid 2-phosphate to form ascorbic acid and inorganic phosphate, and the latter stabilizes Cd S QDs produced in situ through interaction of Cd2+ with S2- ions. The specific interaction of antigen analyte with ALP-Au NFs-labeled antibody can be detected through formation of Cd S QDs, and the use of Au NFs can increase load and activity of the enzyme for improved signaling. The Cd S QDs on the sandwich-type immunoelectrode was dissolved by injection of a 7-μL 0.1 M aqueous HNO3 and the metallic Cd was maximally electrodeposited on the electrode by a beforehand potential "control", followed by ASV detection directly on the electrode. Under optimized conditions, this novel electrochemical method was used for ultrasensitive quantitative analysis of human immunoglobulin G(Ig G) and human cardiopathy biomarkers cardiac troponin I(c Tn I), giving limits of detection(LODs, S/N=3) of 0.6 fg m L-1 for Ig G and of 0.7 fg m L-1 for c Tn I(equivalent to 110 molecules in the 6 μL sample employed for analysis of c Tn I protein). The LOD of Ig G is ca. 3 order of magnitude lower than that of the fluorescent method experimentally obtained by recording the emission spectrum of the Cd S QDs at λex=290 nm and λem=500 nm.6. An ultrasensitive metal-labeled amperometric immunoassay(MLAI) method of proteins is reported, on the basis of labeling the second antibody with zinc oxide nanocrystals decorated multiwalled carbon nanotubes(Zn O-MWCNTs), selective growth of nanocrystalline cadmium sulfide(Cd S) on Zn O and simultaneous chemical-dissolution/cathodic-preconcentration of Cd for in-situ microliter-droplet anodic stripping voltammetry(ASV) detection on the immunoelectrode. This method was successfully used for ultrasensitive quantitative analysis of human immunoglobulin G(Ig G) and human heart-type fatty-acid-binding protein(FABP).