Research on the Construction and Performance of Electrochemical Biosensor for Breast Cancer Tumor Markers of MicroRNA and Circulating Tumor Cells

Author:Tian Liang

Supervisor:wang zuo hong


Degree Year:2018





Circulating microRNA-21 and circulating tumor cells MCF-7 are typical biomarkers for breast cancer.The sensitive,rapid and accurate analysis and detection of these cancer biomarkers play a key role in obtaining cancer information to provide timely treatment and follow-up assessment.In this dissertation,a highly sensitive and highly selective electrochemical biosensor in vitro detection method was constructed by means of signal amplification strategies such as substrate signal amplification,double enzyme synergistic catalysis and hybridization chain reaction.The specific content is divided into the following four parts:Firstly,a nanoparticles superlattice as supporting material was formed by self-assembly of two size gold nanoparticles(AuNPs)coated with conductive polymer polypyrrole.Small molecule dye such as toluidine blue(TB)was used as a redox indicator.A label-free,simple electrochemical microRNA biosensor was constructed.The tight alignment of the superlattice assembly and the large specific surface area formed by the nanoparticles result in efficient amplification of the signal.TB with efficient signal amplification was first applied to microRNA biosensors.Electrochemical cyclic voltammetry(CV)methods as well as differential pulse voltammetry(DPV)technique were used to determine the oxidation peak current of TB under optimal condition.By employing this strategy,microRNA can be detected in a range from 100 aM to 1 nM with a relatively low detection limit of 78 aM,equivalent to the similar microRNA biosensors.Alongside the outstanding sensitivity and selectivity,this nanobiosensor had great reproducibility and showed a remarkable response in the real sample analysis with serum samples.In conclusion,the proposed electrochemical nanobiosensor could be constructed easily without sample preparation,RNA extraction or amplification.Secondly,on the basis of planar intercalation molecules,we report a label-free bioassay for ultrasensitive electronic determination of microRNA at an ultralow concentration based on target-triggered long-range self-assembly DNA-based hybridization chain reaction(HCR)protocol coupled with bienzyme mimics synergistic catalysis strategy.In this work,a planar intercalation molecule,copper(II)complex,is applied for the first time as a small molecule enzyme mimic as well as intercalation molecule in microRNA biosensor for signal amplification.Fe3O4nanozyme were used as a separate and enriched target under magnetic field,and also in combination with HCR protocol detected in 3,3’,5,5’-tetramethylbenzidine and hydrogen peroxide(TMB+H2O2)system to improve the sensitivity of the biosensor.Under optimal conditions,these strategies present good electrochemical behaviors for the detection of microRNA with a wide range from 100 aM to 100 nM and at relatively low detection limit of 33 aM,lower than other microRNA biosensors.This remarkable sensitivity can make this proposed approach a promising scheme for development of next-generation microRNA sensors without the need of enzyme labeling or fluorophore labeling.Thirdly,an ultrasensitive electrochemical detection method was developed to detect CTCs by using reduced graphene oxide/gold nanoparticles composites(rGO/AuNPs composites)as a support material with CuO nanozyme as a catalyst.MCF-7circulating tumor cells were detected by an electrochemical cytosensor with effective surface recognition between specific mucin 1 protein(MUC-1)over-expressed on the MCF-7 cell membranes and MUC-1 aptamer.The CuO nanozyme is used as a signal-amplifying nanoprobe in an ultrasensitive electrochemical cytosensor to detect CTCs for the first time.Under the optimized experimental conditions,the proposed cytosensor exhibited significant analytical performance for the determination of MCF-7 circulating tumor cells.A wide detection range from 50 to 7×103 cells mL-11 on the condition of well selectivity and reproducibility.Furthermore,the cytosensor can easily distinguish CTCs from the real serum sample due to the specific combination of MUC-1 and MUC-1 aptamer.In another part,an ultrasensitive electrochemical CTCs detection strategy was developed based on magnetic field-induced,targeted separation and enrichment,and reduced graphene oxide/molybdenum disulfide(rGO/MoS2)composites and Fe3O4NPsbinanozymesynergisticcatalysisforsignalamplification.Immunomagnetic beads(Fe3O4NPs)act as both separation and enrichment CTCs and as enzyme mimics with rGO/MoS2 synergistic catalysis for signal amplification in cytosensors for the first time,effectively increases detection sensitivity,MCF-7detecting down to 6 cells mL-11 with a linear range from 15 to 45 cells mL-1,lower than other types of cytosensors.The utilization of rGO/MoS2 and Fe3O4NPs as electrochemical signal indicator and enhancer to fabricate biosensor could avoid the need for additional redox mediator for detection sensitivity amplification.The immunomagnetic beads were coated on the magnetic glassy carbon electrode(MGCE)surface by inserting a magnet for electrochemical cytosensing fabrication.Furthermore,this cytosensor could be regenerated by simply pulling out the magnet.This strategy possesses the advantages of high efficiency,high sensitivity,low cost and versatility,thus holds great potential for other low-abundance circulating tumor cells detection.