Electrochemical Immunosensor Based On DNA Molecular Biotechnology For Amplifying Signal

In this thesis,DNA molecular biotechnologies based amplification strategies were developed in electrochemical field,which could amplify the response signal of biosensor,then improve the sensitivity and decrease the detection limit.Amongst them,hybridization chain reaction(HCR)doesn't require any auxiliary enzyme and the process is simple,which presents more attraction to scientific worker.In addition,the long DNA concatemers from HCR is regarded as an ideal carrier for labeling or loading amount of the electroactive materials(e.g.doxorubicin hydrochloride,ferrocene,methylene blue and copper nanoparticles etc),which could amplify dramatically the electrochemical response signal.The thesis contains the following four chapters:Chapter 1: A review about electrochemical biosensors and the signal amplification technologies.Chapter 2: An ultra-sensitive and label-free immunosensor for detection of C-reactive protein(CRP)was developed by using hybridization chain reaction(HCR)and double strand DNA(dsDNA)-templated copper nanoparticles(Cu NPs).The ds DNA concatemers from HCR were employed as template for synthesizing the Cu NPs,then the concatemers intercalating amounts of Cu NPs were regarded as signal probes in turn.Under optimal conditions,differential pulse voltammetry(DPV)was employed to record the response signal from ds DNA-templated Cu NPs.The signal intensities of Cu NPs was linearly related to the logarithm of the concentration of CRP antigen in the range of 1.0 fg mL-1~100 ng mL-1,and the regression coefficient was 0.996,the limit of detection was 0.33 fg mL-1(at S/N=3).Chapter 3: An electrochemical immunoassay protocol was reported for simultaneous determination of alpha-fetoprotein(AFP)and prostate specific antigen(PSA)based on DNA hybridization chain reaction(HCR)for signal amplification.The immunoassay protocol contained primary antibodies immobilized on gold nanoparticles(Au NPs)modified glassy carbon electrode(GCE),target biomarkers,secondary antibodies bioconjugates and DNA concatemers from HCR of auxiliary probe and signal probe labeled with signal molecules(methylene blue(MB)and ferrocene(Fc)).Under optimal conditions,two well-resolved reduction peaks,one was at-0.35 V(corresponding to MB)and other was at 0.33 V(corresponding to Fc;both vs SCE),were obtained by differential pulse voltammetry(DPV).The peak currents changed were related to the logarithm of biomarkers concentration,the electrochemical immunoassay exhibited a wide linear response range(0.5 pg mL-1~50 ng mL-1)and low detection limits(PSA,0.17 pg mL-1;AFP,0.25 pg mL-1).Chapter 4: We tried coupling the small molecules that could intercalate into DNA double helix with hybridization chain reaction(HCR)technique to fabricate an ultrasensitive multiple immunosensor.Doxorubicin hydrochloride(DXH)and methylene blue(MB)were used as signal molecules.A sandwich immunocomplex was formed among primary antibodies,target biomarkers and secondary antibodies conjugated with DNA primer via specific recognition reaction.Afterwards,DNA concatemers intercalating amounts of DXH or MB were regarded as signal probes,then linked to DNA primer via DNA hybridization reaction to form the ultra-sandwich immunostructure.Under optimal conditions,square wave voltammetry(SWV)was employed to record the response signals from electroactive molecules DXH and MB,and two distinguishable signals were obtained,which peak potentials were at about-0.30 V(corresponding to MB)and-0.70 V(corresponding to DXH,both vs SCE),respectively.The signal intensities of MB and DXH were linearly related to the logarithm of biomarkers concentration in the range of 0.05 pg mL-1~25 ng mL-1,and the limits of detection were 0.03 pg mL-1 for CEA and 0.02 pg mL-1 for AFP(at S/N=3),respectively.