The Construction Of "Signal-on" Electrochemical Biosensor And Detection Of Biological Molecules

Electrochemical biosensor is a kind of device that specifically recognizes the target based on bio-recognition element,then produces a series of physical and chemical changes and converts these changes into electrochemical signals in the form of current,potential,resistance or impedance through signal conversion elements.Electrochemical biosensors,which have attracted widespread attention,provide fast,accurate and sensitive responses through an economical and efficient manner.Electrochemical biosensors improve the sensitivity of sensing strategy based on different types of nanomaterials,including metal nanomaterials,metal oxide nanomaterials,carbon nanomaterials,etc.,and introduce the auxiliary strategies of host-guest recognition and DNA enzyme assisted target circulation to achieve the purpose of signal amplification.Until now,electrochemical biosensors have been used to detect biomarkers of infectious diseases,tumors and neurodegenerative diseases.The purpose of this paper is to improve the sensitivity of electrochemical biosensors.We construct three new "signal-on" electrochemical biosensor methods,and apply them to the detection of disease-related biomarkers.The main work contents are as follows:1.Electrochemical aptasensor for 17β-estradiol using disposable laser scribed graphene electrodes17β-Estradiol(E2),the strongest of the three major physiological estrogens in females,is an important factor in the female reproductive system.The abnormal level of E2 causes health issues,such as weak bones,urinary tract infections and even depression.Here,we present a novel,sensitive and selective,electrochemical aptasensor for detection of 17β-estradiol(E2).The E2 recognition aptamer was split into two fragments:the first fragment,functionalised with adamantane,is attached to poly(β-cyclodextrin)(poly(β-CD))-modified electrode surface through host-guest interactions between the adamantane and poly(β-CD).The second fragment,labelled with gold nanoparticles,forms the stem-loop structure with the first fragment only in the presence of E2.That specific recognition process triggers the change in the electrochemical signal(a change in the peak current from reduction of AuNPs),recorded by means of differential pulse voltammetry(DPV).The feasibility of the sensing design was firstly investigated on the commercially available glass carbon electrodes(GCE),with achieved a linear detection range of 1.0×10-13 to 1.0×10-8 M and a limit of detection(LOD)0.7 fM.The sensing methodology was then translated onto single-use,disposable,laser-scribed graphene electrodes(LSGE)on a plastic substrate.The dynamic sensing range of E2 on LSGE was found to be 1.0 ×10-13 to 1.0 × 10-9 M,with a LOD of 63.1 fM,comparable to these of GCE.The successful translation of the developed E2 aptasensor from GCE to low-cost,disposable LSGE highlights a potential of this sensing design in commercial,portable sensing detection systems for E2 and similar targets of biological interest.2.Novel electrochemical insulin aptasensor based on laser scribed graphene electrodesInsulin,a peptide hormone secreted by pancreatic β cells,affects the development of diabetes and associated complications.Herein,we design an electrochemical aptasensor for sensitive detection of insulin using Exonuclease I(Exo I)and Gold nanoparticles(AuNPs).We deposite gold nanoparticles on bare glassy carbon electrodes(GCE)by potentiostatic method to increase the conductivity and specific surface area of the electrode,and self-assemble the thiolated aptamer to the electrode surface by Au-S bond.The aptasensor is based on using Exo I that catalyses the hydrolysis of single-stranded aptamers attached to the electrode surface.However,the hydrolysis does not occur if the insulin is bound to the aptamer.Therefore,the unbound aptamers are cleaved by Exo I while insulin-bound aptamers remain on the electrode surface.In the next step,the gold nanoparticle-aptamer(AuNPs-Apt)probes are introduced to the electrode surface to form a ’sandwich’ structure with the insulin on the surface-attached aptamer.The redox probe,methylene blue(MB),intercalates into the aptamers’ guanine bases and the sandwich structure of AuNPs-Apt/insulin/surface-bound aptamer amplifies electrochemical signal from MBs.The signal can be well-correlated to the concentrations of insulin.The feasibility of the sensing design was firstly investigated on the commercially available glass carbon electrodes(GCE),with achieved a linear detection range of 1.0×10-13 to 1.0×10-8 M and a limit of detection(LOD)0.7 fM.Firstly,the development and verification of sensor experiments on glassy carbon electrode(GCE)were carried out.The dynamic sensing range of insulin on GCE was found to be 0.1 pM to 1.0 μM,with a LOD of 9.8 fM.The sensing methodology was then translated onto single-use,disposable,laser-scribed graphene electrodes(LSGE).The dynamic sensing range of insulin on LSGE was found to be 0.1 pM to 0.1 μM,with a LOD of 22.7 fM.The results show that a sensitive and disposable LSGE sensor for insulin detection is successfully constructed.3."Signal-on" electrochemical biosensor for the detection of BACE1 based on 2-pyridinecarboxaldehyde and peptideβ-secretase(the β-site amyloid precursor protein cleaving enzyme 1,BACE1)in the brain of Alzheimer’s disease(AD)is a key hydrolytic enzyme in the pathway of β-amyloid(Aβ)production,so BACE1 is considered a promising therapeutic target for early AD.In this work,Fc-Probe was synthesized through 2-pyridinaldehyde(2PCA),which could specifically recognize the N-terminal of the peptide without binding to the amino side chain.We designed a simple and sensitive signal-on electrochemical method to detect the activity of BACE1 based on the special properties of Fc-Probe.Firstly,a layer of gold nanoparticles(AuNPs)was deposited on the glassy carbon electrode(GCE).The N-terminal of acetylated substrate peptide were incubated with the electrode.The C-terminal cysteine residues of peptide were bonded to the AuNPs/GCE surface by Au-S bond.After blocking the unoccupied Au sites with MCH,BACE1 shears the N-terminal of acetylated substrate peptide,resulting in the exposure of peptide’s N-terminal.Fc-probe probe specifically recognizes N-terminal and introduces electrochemical signal.Ferrocene electrochemical signal becomes larger with the increase of BACE1 concentration,finally achieving quantitative detection of BACE1 activity.The detection range of the sensor is 0.02 U/mL-50 U/mL,and the detection limit is 8.0 mU/mL.The advantages of this method are simple,sensitive and low-cost,and it is suitable for the detection of BACE1 activity.