Novel Aptamer-based Electrochemical Sensors For Cocaine Detection

Electrochemical Aptamer(EA)sensor conbine the properties of aptamers and biosensors,electrochemical sensors with aptamers modified at the end of the signal molecule as the recognition probe has the advantages of fast,low cost,easy operation,high spectificity,high selectivity,sensitivity and reliability.It is the frontier and development direction of food satety analysis and testing research,with important research significance and broad application potential.In view of the shortcomings of low sensitivity and slow response of conventional EA sensors,this paper takes the idea of rationally designing aptamer-cDNA(complementary DNA)duplex probe as the starting point to explore new solutions to the problem.By virtue of the concept"cooperative effect",designed the aptamer-cDNA probe for rapid recognition of the target molecule cocaine under the displacement-based EA sensors.On the basis of"cooperative effect"extended for cooperative sandwich recognition of an appositive probe of aptamer cocaine.Based on the aptamer probe,a novel electrochemical sensor with high sensitivity,selectivity and specificity constructed for the quantitative analysis of the target cocaine.The main research contents and results are as follows:(1)Design and application of a displacement double-stranded cooperative EA sensor for cocaine.In the second part of the paper,the influence of the structure of the double-stranded coordinated aptamer probe(the length and complementary position of the complementary strand)on the detection performance of the displacement type EA sensor for cocaine was investigated in detail.The results showed that the complementary strycture of the complementary strand with the additional base added at the bottom of the aptamer probe reduced the steric hindrance of the binding of the aptamer with the cocaine,which enabled the target molecule to rapidly unravel the double strand and produce strand displacement reaction,effectively improving the response speed of the EA sensor.With the decrease of complementary length(5+7,5+6,5+5 base pairs),the background inhibition on the sensing interfance is weakened,the response signal to the cocaine is enhanced,and the response time decreases in turn.However,when the length of the complementary strand(5+4 base pairs)is reduced,the response signal generated by the sensing interface to the target molecule cocaine is also reduced due to the weak binding fastness of the double-stranded collaborative structure.On the basis of the above results,a double-strand EA sensor with complementary length of"5+5"base pairs was designed,and the influence of the concentration of complementary strand,the concentration of salt ions in the best buffer solution and the square wave voltammentry test frequency on the signal gain of the sensor was further investigated.The detection performance of the sensor under optimal experimental conditions:the response time of the sensor to the target molecular cocaine was 2 minutes,and the linear equation of the sensor for the quantitative analysis of cocaine was Y=1.5821C+2.6336(R~2=0.9934),with a linear range of 100 times(0.5~50?M).In the complex mixture,the sensor has the specific recognition function to the target molecule,and in the actual sample,the measured recovery rate is as high as 96%.The experimental results fully show that the design scheme of double-stranded coordinated cocaine EA sensor can effectively improve the sensitivity and response speed of the appositive sensor,and has the accuracy for the detection of cocaine in accrual samples.(2)Design and application of an electrochemical sensor for co-sandwich recognition of aptamer-cocaine appositive.In the third part of the paper,a new design scheme for the probe structure of collaborative aptamer-cocaine was proposed.The original aptamer-cocaine were cut into two segments,and then part of the bases were copied at the end of the two segments to form two new aphadites.Due to aptamer-cocaine unique conformation,to fix a strand on the electrode surface,another free in solution,two strand combination sensor interface can produce very low background suppression,cocaine in join formed coordination and stable hairpin conformation,produce high signal gain,the sensor signal sensitivity enhancement.Based on these results,the influence of the concentration of synergistic complementary strand,the frequency of square wave voltammetry and the concentration of salt ions in the buffer solution on the sensor signal sensitivity was further investigated.The detection performance of sensor under optimal experimental conditions:the linear equation of the sensor for the quantitative analysis of cocaine is Y=0.3359C+21.75(R~2=0.9968),the linear range of cocaine detection is 5 nM~7.5?M,and the minimum detection limit is 5 nM.In the complex mixture,the sensor has the specific recognition function to the target molecule,and in the actual sample,the measured recovery rate is as high as 96%.The experimental results fully show that the design of the cored electrochemical sensor can effectively improve the sensitivity of the cored sensor,and is accurate for the detection of cocaine in actual samples.