Study On Electrochemical Biosensors Based On Multiplex Signal Labels

Electrochemical biosensor has been widely applied in clinical diagnosis,environmental monitoring,food analysis and pathogenic microorganisms’ research because of its inherent advantages such as simplicity,low cost and high sensitivity.To improve the performance of electrochemical biosensors,new methods and new technology has been constructed based on various signal labels,combining multi-funcational nanomaterials and effective signal amplification strategies,which has very important sicentific significance and practical value for specific and sensitive detection of various biological molecules.This thesis focus on using various signal lables,multi-functional nanomaterials and amplification strategies to prepare electrochemical biosensors with easier operation,lower cost and stronger practicability.The details are mainly as follows:1 High throughput immunosenor based on multi-label strategy and a novel array electrode Accurate prediction of a particular cancer can be achieved by measuring multiplex biomarkers.Traditional methods for multi-biomarkers detection are either multi-spots assay with chip or multi-label assay with one detection spot.However,the detection throughput of these two approaches is limited by the substrate area and the numbers of available label respectively,which may further restrain the widespread use of multi-biomarkers detection.To solve this problem,in the present study,a high throughput immunosensor was firstly prepared by combining multi-label strategy and multi-spot assay with a novel array electrode for simultaneous detection of six biomarkers of hepatocellular carcinoma（HCC）.Firstly,two different capture antibodies were immobilized on the each detection spot,and the corresponding detection antibodies were labeled with the redox probes of thionine（Thi）and anthraquinone-2-carboxylic acid（AQ）,respectively.Based on the sandwiched immunoreactions,each individual protein could be detected with the distinct voltammetric peaks,whose position and size reflected the identity and level of the corresponding antigen.In addition,both of the graphene nanosheet/Pt Pd bimetallic nanocomposites（Pt Pd-GS NPs）and horseradish peroxidase（HRP）also labeled on the detection antibodies for a signal amplification to improve the sensitivity of the immunoassay.Therefore,this detection model could serves as the starting point for high throughput of multianalyte assay.2 Highly effective protein converting strategy for ultrasensitive electrochemical assay of Cystatin C So far,antibodies are still main recognition elements in the protein detection.In this study,a highly effective protein converting strategy based on immunoreaction-induced DNA strand displacement and T7 Exonuclease（T7 Exo）-assisted protein cyclic enzymatic amplification for ultrasensitive detection of Cystatin C was described.Through such a system,each input Cystatin C could induce more than one output DNA,enhancing detection sensitivity.Moreover,the output DNA triggered a hybridization chain reaction（HCR）on biosensor surface,resulting in ds DNA polymers.Then,a large mount of thionine（Thi）as electron mediator was embed into ds DNA polymers to produce detection signal,which could further enhance detection sensitivity.With above design,nucleic acid technologies including enzyme-assisted protein cyclic enzymatic amplification and HCR were successfully applied in protein detetion.3 Cu,Mn double-doped CeO₂ nanocomposites as novel redox label and signal amplifier for electrochemical detection of procalcitonin Up to date,the source of current signal in electrochemical biosensor was mainly depended on traditional electron mediator.Using nanomaterials as electron mediator for the construction of electrochemical biosensor are rarely reported.In this work,an simple signal-amplified electrochemical immunosensor was developed using Cu2+ and Mn2+ double-doped CeO₂ nanocomposites（CuMn-CeO₂）as redox label,signal amplifier and matrix for sensitive detection of procalcitonin（PCT）.CuMn-CeO₂ could implement as matrix for immobilizing amounts of Ab2 by forming ester-like bridging between carboxylic groups of Ab2 and CeO₂ without extra chemical modification.Due to the Ce3+ on the surface of CuMn-CeO₂ could be easily electrocatalytically oxidized to Ce4+ in phosphate buffer,CuMn-CeO₂ could directly act as redox probe for electrochemical signal readout.Moreover,with superior catalytic activity of CuMn-CeO₂,in the presence of H2O2,the electrochemical signal could be greatly amplified by CuMn-CeO₂-catalyzed oxidation of H2O2 with improving electron transfer from Ce3+ to Ce4+.Importantly,CuMn-CeO₂ exhibited more excellent catalytic performance than pure CeO₂ owing to increased Ce3+/Ce4+ ratio.This method demonstrates that CuMn-CeO₂ can simultaneously implement as redox label,signal amplifier and matrix for the construction of immunosensor,which would greatly simplify the preparative steps and reduce the detection time.4 An amplified thrombin aptasensor based on alkaline phosphatase and hemin/G-quadruplex-catalyzed oxidation of 1-naphthol Alkaline phosphatase（ALP）-based biosensor can in situ generate electroactive product by enzymatic hydrolysis of inactive substrates.To obtain a higher signal-to-background ratio,chemical redox cycling signal-amplified strategies based on the addition of strong reducing agent often are applied in the construction of ALP-based biosensors.However,the strong reducing agent not only affects the activity of ALP but also readily react with dissolve oxygen,leading to inaccurate results.In this work,a new signal-amplified strategy for thrombin（TB）aptasensor based on directly catalytic oxidation of ALP-generated products,1-naphthol（NP）,using hemin/G-quadruplexs DNAzymes was reported.The amplified signal was carried out in two steps:（i）ALP catalyzed inactive substrates,1-naphthyl phosphate（NPP）,to in situ produce NP on the surface of electrode.（ii）NP as a new reactant,on the one hand,could be directly electrooxidized and output electrochemical signal.On the other hand,NP could be oxidized by hemin/G-quadruplex in the present of H2O2,resulting in amplification of the electrochemical signal.To further amplify current signal,Au nanoparticles decorated ZnO nanoflowers（Au-ZnO）used as matrix for immobilizing biomoleculars,providing a good micro-environment for electronic transmission.The proposed TB aptasensor achieved a linear range of 1 p M to 30 n M with a detection limit of 0.37 p M（defined as S/N = 3）.5 An amplified impedimetric aptasensor combining target-induce DNA Hydrogel formation with p H-stimulated signal amplification for heparanase assay The conventional impedimetric sensing based on target itself nonconductive property,suffering from low sensitivity.If the target can induce formation of some less conductive material on the electrode surface,it will significantly improve the detection sensitivity.Herein,a novel electrochemical impedimetric biosensor for heparanase（HPA）assay is developed based on target protein-induced DNA hydrogel formation and p H-stimulated the hydrogel density increase for signal amplification.The method involves the synthesis of two different copolymer chains consisting two cooperatively functioning cross-linking elements,where one element is associated with HPA-response and another one is p H-responsive.In the presence of HPA,the HPA-responsive element binding to HPA induce DNA hybridization between two copolymer chains and capture DNA,giving rise to formation of a low-density polymer hydrogel film on electrode surface and obtaining an obvious impedimetric response.Significant signal enhancement was observed when changing the p H of hydrogel film to 5.0,which is ascribed to that p H-responsive element can fold into four stranded i-motif structure at p H 5.0,leading to the increment of density of hydrogel film.By implementing DNA hydrogel to induce impedimetric response change,analytical sensitivity of this impedimetric bioensor was successfully improved.