Study On The Novel Biosensing Methods Based On Multiple Signal Amplification Strategies

As biosensor which possesses unique advantages such as high selectivity,good sensitivity and low sample consumption can be conveniently used for the cheap,rapid and accurate detection of various analytes in complicated environments,it has been widely studied and applied in the field of analytical chemistry in recent years.Meanwhile,due to the increasing requirements on the accurate detection of trace biomolecules in the clinical diagnosis field,more and more researchers are working on the construction of various new biosensing methods and the improvement of their analytical sensitivity.With the rapid development of nanotechnology and the emergence of various new biological techniques as well as the integration of different disciplines,a large variety of nanomaterials and enzymes based signal amplification strategies have been adopted in the bioassay and biosensing fields in order to effectively improve their analytical sensitivity.Therefore,based on the multiple nanomaterials and biological techniques based signal amplification strategies,in this thesis I have carried out three researches on the development of new biosensing methods,which are described in details as follows:1.Enzyme-triggered polydopamine deposition and signal inhibition to prussian blue for ultrasensitive electrochemical immunoassayThis work utilizes the unique property of chitosan(CS)to realize the facile and controllable synthesis of a chitosan-Prussian blue(CS-PB)nanocomposite,and combines it with the urease-triggered polydopamine(PDA)deposition and signal inhibition to PB to successfully develop a novel electrochemical immunoassay method.The electrode modification of CS-PB provides an ideal platform for the successive assembly of gold nanoparticles and capture antibody to construct the immunosensor.When the nanoprobe prepared by covalently linking signal antibody and high-content urease on silica nanosphere is used for sandwich immunoreaction,the quantitative captured nanoprobes cause obvious electrochemical impedance increase.Moreover,the enzymatic reaction of urease releases numerous OH~-to destroy the PB structure and trigger the PDA deposition on the immunosensor surface,resulting in drastic electrochemical signal decrease of PB.This amplified signal inhibition mechanism enables the successful construction of an ultrasensitive signal transduction strategy for the electrochemical immunoassay.Using carcinoembryonic antigen as a model protein analyte,excellent performance including a wide linear range of six-order of magnitude and a very low detection limit of 0.042 pg mL~-11 are obtained(S/N=3).2.Hybridization chain reaction-catalyzed hairpin assembly for ultrasensitive colorimetric biosensing of proteinAs an isothermal signal amplification technique with the unique advantages like simple operation,enzyme-free and high amplification efficiency,hybridization chain reaction(HCR)has attracted considerable interests in recent years.Based on the self-assembly of hairpin DNA to conduct a HCR,this work successfully develops an ultrasensitive homogeneous protein biosensing methods.First,this work designs four unlabeled hairpin probes which are designated as H1,H2,H3 and H4,respectively.Upon the biorecognition of the target analyte of thrombin,H1 is opened followed by the hybridization with H2 to form a DNA duplex structure.Meanwhile,the thrombin target is released during this process to participate the target recycling reaction,and the resultant duplex DNA further induces the successive HCRs with H3 and H4.As this result,a bidirectional branched structure containing numerous G-quadruplexes is spontaneously formed.Finally,through specifically binding of hemin by the G-quadruplexes,the peroxidase-mimicking DNAzymes are thus produced for the high-efficiency catalytic color reactions and sensitive colorimetric signal transduction.As the bidirectional HCRs lead to the spontaneous formation of a large amount of DNAzyme for the catalytic color reactions,and the target recycling reactions further amplify the signal response,an ultrahigh sensitivity is obtained for this method.Under the optimal experimental conditions,the method has a wide linear range of more than three orders of magnitude with a detection limit of 0.82 pM(S/N=3).3.Target-biorecognition stimulated release of G-quadruplex DNAzymes for the colorimetric biosensing of chloramphenicolBased on target-biorecognition to stimulate the release of G-quadruplex DNAzymes from a Y-scaffold DNA backbone structure,a new colorimetric homogeneous biosensing method is successfully developed for the convenient and accurate detection of chloramphenicol antibiotic.The Y-scaffold DNA backbone is synthesized through the DNA hybridizations among three oligonucleotides,which ends containing the bases sequences of the G-quadruplex DNAzymes.As the mixing of the chloramphenicol-aptamer strand with these Y-scaffold DNA backbones can induce the DNA hybridization reactions among them,the activity of the DNAzymes exposes on the termini of the Y-scaffold DNA backbones is well inhibited.Once the target chloramphenicol is added,the specific binding between chloramphenicol and its aptamer strand will lead to the corresponding release of the DNAzyme sequences,and thus produce sensitive colorimetric signal readout through its enzymatic chromogenic reaction.Based on this signal transduction mechanism,this work successfully developed a new biosensing method for the homogenous colorimetric detection of chloramphenicol.Under the optimum conditions,this method can be used for the convenient detection of chloramphenicol in a wide concentration range from 20 nM to1.0?M with a detection limit of 13 nM(S/N=3).