Construction And Application Of Dual Signal Electrochemical Biosensor Based On Target Cyclic Amplification Technology

Now,30% to 50% cancer can be prevented if early diagnosis and adequate treatment can be achieved,many cancers will have a very high cure rate.However,many cancer patients are often diagnosed only in the middle and late stages of the diseases.DNA,as a carrier of genetic information and coding for proteins,plays a central role in the process of life by transforming genetic codes into proteins that further regulate a variety of cellular functions.The early diagnosis of some diseases by nucleic acid detection is one of the hot spots in analytical chemistry.However,in the early stage of the patient,the content of disease biomarkers to be measured is usually only trace,which makes the early diagnosis more difficult in complex biological environment.Therefore,it is more and more necessary to develop highly sensitive nucleic acid biosensors for rapid and low cost detection of trace target biomolecules using simple methods and portable instruments.Electrochemistry is a kind of surface technology,which provides a tremendous advantage for the detection of biosensors.It is independent of the reaction volume and can be measured using very small sample volumes.Electrochemical DNA sensor combines high sensitivity electrochemical analysis method with high specificity biometric techniques to construct a new biosensor.By combining the nuclear acid layer with the electrochemical transducer,the sensor provides a simple,rapid and inexpensive platform for diagnosis of patients.In this paper,double signal technique was used to enhance the stability and sensitivity of the sensor for quantitative detection of d（CAG）_nand DNA fragments of chronic myeloid leukemia（CML）.The double signal technique improves the detection accuracy of biomarker.This paper focuses on the study of the two kinds of disease biomarkers.（1）A novel double-signal electrochemical sensor was prepared by combining enzyme-assisted recycling amplification target technique and the selective adsorption ability of graphene to single-strand DNA.The method was used for quantitative detection of trinucleotide repeat sequence d（CAG）_n.Using ferrocene and methylene blue labeled hairpin DNA as probes,the probe DNA can hybridize with the target to form a double-stranded structure,and release ferrocene（F） and methylene blue（M）labeled single-stranded DNA fragments and target compounds under the action of exonuclease.The released target participated in the next cycle to produce more single-stranded DNA fragments modified with electrochemical signals.The graphene-modified electrode can selectively adsorb the released report fragments to achieve the detection of double electrochemical signals.In the absence of the target,it was impossible to form a double-stranded structure with the probe DNA,that is to say,it cannot trigger the cleavage of the exonuclease and the release of the labeled single-stranded DNA fragment.And the two electrochemical signals can be used to test repeat concentration with detection limit of 0.22 p M.The signal ratio（F/M）of ferrocene and methylene blue was used to determine accurately repeat length.A linear relationship was found between F/M and numbers of repeats（n）,F/M=0.061n+1.97,with a correlation coefficient of 0.992.Further,the concentration and repeat length of the sample of the human serum matrix can also be detected,and the relative standard deviation is between 2.2%and3.2%.This novel double signal electrochemical sensor provided a reliable and efficient method for the analysis of d（CAG）_n trinucleotide repeat and a potential simplified clinical tool of POCT for neurodegenerative diseases.（2）A double-signal ratio type electrochemical DNA sensor with multiple signal amplification was constructed for the detection of nucleic acid fragment in chronic myelogenous leukemia.One of the signals was Prussian blue film electrodeposited on the surface of glassy carbon electrode,which not only enhances the conductivity of the electrode,but also serves as an electrochemical signal of the sensor.In the exonuclease circulating amplification system,the target DNA released can be recycled,and the single-stranded DNA of the hairpin released due to the enzyme digestion can be captured on the electrode.The nucleic acid on the electrode surface was further increased by hybridization chain reaction for further signal amplification.So,another signal was achieved by the thionine that was adsorbed to the DNA on electrode surface.During this period,due to the weak electron transfer ability and high impedance of nucleic acid itself,the electrochemical signal of Prussian blue decreased with the layer-by-layer modification of the electrode surface.The changes of electrochemical signals of Prussian blue and thionine were measured by square wave voltammetry.A good linear relationship was observed between the electrochemical signal and the BCR/ABL fusion gene concentration in the range of 1 p M to 100 n M,and the detection limit was low to 0.19 p M.It was important that the signal ratio T/P was used as the determination of its concentration with higher sensitivity,more stable and accurate,with the linear equation of lg I_T/P=1.094 lg C+0.1557 and the correlation coefficient of 0.997.Moreover,the detection of samples in human serum matrix was also performed,and exhibited fine sensitivity and accuracy.These results showed that the novel ratio type electrochemical sensor had excellent selectivity and sensitivity,and was simple in operation and low in cost.These indicated that the sensor showed excellent performance in BCR/ABL fusion gene diagnosis and the sensor provided a simple and feasible strategy for the early diagnosis of chronic myeloid leukemia.In this work,we studied the quantitative detection of diseases biomarker including trinucleotide repeats d（CAG）_n and BCR/ABL fusion gene by electrochemical analysis.Simple and convenient double-signal electrochemical DNA sensing systems were constructed by means of amplification techniques such as enzyme-assisted target recycling amplification and hybrid chain reaction.The experimental results showed that all of them have high specificity and excellent linearity,and lower detection limit.