Design Of Signal Amplification Technology For The Fabrication Of Electrochemical Immunosensor

In this thesis,a series of electrochemical immunosensors were developed based on signal amplification technologies including nanomaterial signal amplification,molecular biological amplification and so on,which could improve the sensitivity and decrease the detection limit of single biomarkers of the immunosensor.Hence,numerous of biomarkers containing hormone,alpha fetoprotein,carcinoembryonic antigen were effectively detected by those electrochemical immunosensors.Furthermore,we investigated the practical application of these methods,which provide the possibility for making portable and affordable devices for online detection to meet the requirement of modern medical diagnostics and biomedical research application.There are six chapters in this thesis,and main contents are listed as follows:In the first chapter,a general introduction of immunoassay and immunosensor was made.Then,the detection principle,categories and application of the electrochemical immunosensor were described in detail,particularly,the several signal amplification strategies.Meanwhile,the research purpose and signification of this thesis were summarized.In the second chapter,cadmium ion-doped magnetic poly(styrene-acrylic acid)nanospheres(Cd-MPSA)was synthesized by polymerization.The prepration and washing were easy for the nanomaterials,which could be designed as molecular tags.A typical sandwich-type immunoassay protocol was built for determination of LH on monoclonal mouse anti-human luteinizing hormone antibody(MAb1)-functionalized gold electrode.The assay was carried out by square wave voltammetry(SWV).Under optimal conditions,the currents increased with the increasing LH level in the sample,and exhibited a linear range from 0.25 mIU mL-1 to 240 mIU mL-1 with a detection limit of 0.08 mlU mL-1.In the third chapter,self-assembly CdS quantum dot hollow microspheres(QDHMS)were designed by layer-by-layer assembled technology.UV-Vis absorption spectra and zeta potential were employed to characterize the preparation process of QDHMS.This nanomaterial owned large specific surface area due to quantum dots adsorbed on the surface of CaC03,which could crosslinking numbers of antibodies.A sandwich-type immuno-assay format was developed on the magnetic beads to detection of IgG1.Under optimal conditions,anodic-stripping voltammetric was carried out and the dynamic concentration range spanned from 1.0 fg mL-1 to 1.0 mg mL-1 of IgG1 with a detection limit of 0.1 fg mL"1.In the fourth chapter,we constructed sandwich-type immunoassay protocol based on PbS nanolabels in a transparent polystyrene microplate.The carried nanolabel can release numerous metal ions by acid,which could activate the corresponding DNAzyme,and make the substrate RNA chain hydrolysis,thus resulting in the change of electrochemical signal.The signal amplification was usetized for determination of low-abundance protein(free prostate-specific antigen,PSA,used as a model)with high sensitivity and high selectivity by coupling metal sulfide(PbS)-based nanolabels with cleavage of the corresponding lead ion-induced DNAzymes.Under optimal conditions,the immunoassay was carried out by DPV and presented an obvious electrochemical response in the range of 0.1 pg mL-1～20 ng mL-1,and allows detection of PSA at a concentration as low as 0.1 pg mL-1.In the fifth chapter,on the basis of the above experiments,to realize the signal amplification of electrochemical immunosensors,we further combined rolling circle amplification(RCA)strategy.That is,when lead ions reacted with DNAzyme immobilized on the electrode,the residual single-stranded DNA on the electrode could be used as the primer to produce numerous repeated oligonucleotide sequences via the RCA reaction for the hybridization with many AgNC-labeled detection probes,resulting in the amplification of the electronic signal due to the unique properties of silver nanoclusters.Under optimal conditions,the developed immunoassay was carried out by DPV and exhibited high sensitivity for the detection of AFP with a dynamic range of 0.001 ng mL-1～200 ng mL-1 and a detection limit(LOD)of 0.8 pg mL-1.In the sixth chapter,we prepared nanogold-labeled signal probes conjugated with the DNA initiator strands and antibody to develop immuno-HCR assay on the magnetic immunosensing probes.In the presence of target IgG,the sandwiched immunocomplex can be formed between the immobilized antibodies on the magnetic beads and the signal antibodies on the gold nanoparticles.The carried DNA initiator strands open the hairpin structures in sequence and propagate a chain reaction of hybridization events between two alternating hairpins to form a nicked double-helix.Numerous ferrocene molecules are formed on the neighboring probe,and obtained an enhaced electrochemical signal.Under optimal conditions,the immuno-HCR assay was carried out by SWV and presented good electrochemical responses for determination of target IgG at the range of 0.1 fg mL-1～100 ng mL-1 with the limit determination of 0.1 fg mL-1.