Construction of Novel Electrochemical Biosensors for Disease Markers and Its Applications

Author:Dou Bao Zuo

Supervisor:xiang yun


Degree Year:2019





As important participants in the physiological activities of the organism,biomarkers have important indications for the physiological and pathological processes of organisms.Common biomarkers,including RNA,DNA,small molecules,proteins,amino acids,cells,etc.,are closely related to much diseases and have a wide range of applications in disease prevention,early diagnosis,and drug efficacy evaluation.Taking advantage of their high sensitivity and specificity,rapid response,miniaturization and low cost,electrochemical biosensors are of great significance in the analysis and determination of disease markers.Therefore,aiming to simplify experimental operations,improve sensitivity and accuracy of detection and in situ monitoring,novel electrochemical biosensors have been developed in this research through adopting small molecule epitope and aptamer as novel identification elements;by means of toehold-mediated displacement reaction,enzyme-assisted target recycling amplification and nanomaterial signal amplification techniques and introducing various signal output molecules for multiplexed detection.The obtained results have confirmed the excellent performance of the designed strategies in detection of antibody,screening of single nucleotide polymorphisms and monitoring of nucleic acid,as well as the good specificity and sensitivity in analysis of real samples(such as serum).In addition,the research has realized multiplexed detection of circulating tumor cells in whole blood and in situ monitoring of small molecules secreted by live cells,which has successfully transitioned from in vitro analysis to in vivo detection.And the studied contents are mainly as follows:1.DNA-mediated strand displacement facilitates sensitive electronic detection of antibodies in human serumsWe describe here the development of a sensitive and convenient electronic sensor for the detection of antibodies in human serums.The sensor is constructed by self-assembly formation of a mixed monolayer containing the small molecule epitope conjugated double stranded DNA probes on gold electrode.The target antibody binds the epitope on the dsDNA probe and lowers the melting temperature of the duplex,which facilitates the displacement of the antibody-linked strand of the duplex probe by an invading methylene blue-tagged single stranded DNA(MB-ssDNA)through the strand displacement reaction and leads to the capture of many MB-ssDNA on the sensor surface.Subsequent electrochemical oxidation of the methylene blue labels results in amplified current response for sensitive monitoring of the antibodies.The antibody assay conditions are optimized and the sensor exhibits a linear range between 1.0 and 25.0 nmol/L with a detection limit of 0.67 nmol/L for the target antibody.The sensor is also selective and can be employed to detect the target antibodies in human serum samples.With the advantages of using small molecule epitope as the antibody recognition element over traditional antigen,the versatile manipulability of the DNA probes and the unique properties of the electrochemical transduction technique,the developed sensor thus hold great potential for simple and sensitive detection of different antibodies and other proteins in real samples.2.Target-induced reconfiguration of DNA probes for recycling amplification and signal-on electrochemical detection of hereditary tyrosinemia type I geneBy coupling target DNA-induced reconfiguration of the dsDNA probes with enzyme-assisted target recycling amplification,we describe the development of a signal-on electrochemical sensing approach for sensitive detection of hereditary tyrosinemia type I gene.The dsDNA probes are self-assembled on the sensing electrode,and the addition of the target DNA reconfigures and switches the dsDNA probes into active substrates for exonuclease III,which catalytically digests the probes and leads to cyclic reuse of the target DNA.The target DNA recycling and the removal of one of the ssDNA from the dsDNA probes by exonuclease III result in the formation of many hairpin structures on the sensor surface,which brings the electroactive methylene blue labels into proximity with the electrode and produces a significantly amplified current response for sensitive detection of the target gene down to 0.24 pmol/L.This method is also selective to discriminate single-base mismatch and can be employed to detect the target gene in human serum samples.With the demonstration for the detection of the target gene,we expect the developed method to be a universal sensitive sensing platform for the detection of different nucleic acid sequences.3.Electrochemical screening of single nucleotide polymorphisms with significantly enhanced discrimination factor by an amplified ratiometric sensorThe detection of single nucleotide polymorphisms(SNPs)is of great clinical significance to the diagnosis of various genetic diseases and cancers.In this work,the development of an ultrasensitive ratiometric electrochemical sensor for screening SNP with a significantly enhanced discrimination factor is reported.The ferrocene(Fc)and MB dual-tagged triple helix complex(THC)probes are self-assembled on the gold electrode to construct the sensing interface.The addition of the mutant p53 gene causes the disassembly of the THC probes with the release of the Fc-tagged sequence and the folding of the MB-labeled sequence into a hairpin structure,causing the change in the current response ratio of MB to Fc for monitoring the mutant p53 gene.Such ratio is dramatically enhanced by the toehold-mediated displacement reaction-assisted target recycling amplification with the presence of an assistance hairpin sequence.With the significant signal amplification and the advantageous specificity of the THC probes,sub-femtomolar detection limit and a highly enhanced SNP discrimination factor for the mutant p53 gene can be obtained.Besides,the proof-of-demonstration application of the sensor for diluted real samples has been verified,offering such sensor new opportunities for monitoring various genetic related diseases.4.Aptamer-functionalized and gold nanoparticle array-decorated magnetic graphene nanosheets enable multiplexed and sensitive electrochemical detection of rare circulating tumor cells in whole bloodThe identification and monitoring of circulating tumor cells(CTCs)in human blood has a pivotal role for the convenient diagnosis of different cancers.However,it remains a major challenge to monitor these CTCs because of their extremely low abundances in human blood.Here,we describe the synthesis of a new aptamer-functionalized and gold nanoparticle(AuNP)array-decorated magnetic graphene nanosheet recognition probe to capture and isolate the rare CTCs from human whole blood.In addition,by employing the aptamer/electroactive species-loaded AuNP signal amplification probes,multiplexed electrochemical detection of these low levels of CTCs can be realized.The incubation of the probes with the sample solutions containing the target CTCs can lead to efficient separation of the CTCs and result in the generation of two distinct voltammetric peaks on a screen printed carbon electrode,whose potentials and current intensities,respectively,reflect the identity and number of CTCs for multiplexed detection of the Ramos and CCRF-CEM cells with detection limits down to 4 and 3 cells/mL.With the successful demonstration of the concept,further extension of the developed sensing strategy for the determination of various CTCs in human whole blood for the screening of different cancers can be envisioned in the near future.5.Trimetallic hybrid nanoflower-decorated MoS2 nanosheet sensor for direct in situ monitoring of H2O2 secreted from live cancer cellsIn situ monitoring of hydrogen peroxide(H2O2)secreted from live cells plays a critical role in elucidating many cellular signaling pathways,and it is a significant challenge to selectively detect these low levels of endogenous H2O2.To address this challenge,we report the establishment of a trimetallic hybrid nanoflower-decorated MoS2nanosheet-modified sensor for in situ monitoring of H2O2 secreted from live MCF-7cancer cells.The Au-Pd-Pt nanoflower-dispersed MoS2 nanosheets are synthesized by a simple wet-chemistry method,and the resulting nanosheet composites exhibit significantly enhanced catalytic activity toward electrochemical reduction of H2O2,due to the synergistic effect of the highly dispersed trimetallic hybrid nanoflowers and the MoS2 nanosheets,thereby resulting in ultrasensitive detection of H2O2 with a subnanomolar level detection limit in vitro.Also the immobilization of the laminin glycoproteins on the surface of the nanocomposites increases its biocompatibility for cell adhesion and growth,which enables in situ electrochemical monitoring of H2O2 directly secreted from live cells for potential application of such sensor in cellular biology,clinical diagnosis,and pathophysiology.