Fabrication and Performance Study of Enhanced-sensitivity Electrochemical Kanamycin Aptasensor

Author:Li Feng Qin

Supervisor:zhang gui ling yu zhi gang

Database:Doctor

Degree Year:2018

Download:102

Pages:142

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Recent years,with a growing global fervent concern on the residual event of veterinary drug for food and environment,various of new detection technologies have been developed rapidly.Among these technologies,the electrochemical sensor based on electrode-bound redox-modified aptamer shows a lot of unique advantages,for example,simple operation,no exogenous reagent,good reusability,incomparable anti-fouling ability,being compatible with biochip,easy-achieving miniaturization and on-line monitoring.Compared with other detection technologies,through the performance improvement of basic functional parameters,this technology with high development value is more conducive to practical applications,which has been one of future development directions.In this paper,using the specificity reaction of DNA aptamer-kanamycin A as the model target studying system,five electrochemical sensors with different structures were directionally designed and successfully fabricated through changing the probing self-assembled monolayer structures.These 5 sensors are based on 3 different classes of 1,2 and 3-probe systems separately.The effect of sensor structure change on improving the sensitivity was systematically investigated.The goal was successfully achieved with controlling and effectively improving the signal enhancement(SE%)and absolute detection signal value(DS)and the parameter performance of some basic functions,for example,detection limit,sensing speed and selectivity etc.The main works are as follows:1.A 1-probe sensor(S-1)based on the signaling mechanism of target-induced conformation change was fabricated.Four key parameters affecting the efficiency of sensor fabrication and working were optimized through designing proper experiments.These parameters are the probe coverage,the length of supporting diluent chain,the ion strength of working media and the interrogation temperature.Under the optimized conditions,when used for the detection of target KMY-A,226.1±8.0% of SE% was obtained at the adding concentration of 2.0 m M.A good linear relationship is obtained between detection signal and the target concentration(log c/M)within a range from 1.0 μM to 1.0 m M with the detection limit of 0.33 μM.When used for discriminating other interferents,the proposed sensor shows good discrimination for other 5 different kinds of antibiotic molecules with the discrimination coefficients of-0.002~0.019.However,the discrimination for the analogue,KMY-B,is not good with the discrimination coefficient of 0.90.Besides,this sensor also shows a super-fast sensing speed with only ~15 s of response time.When used in three real-life samples(50% milk,50% tap water and 50% calf serum)in which the target residue is usually found,the sensor method performs well with the SE% of 116.2±7.2%,172.7±8.7% and 81.7±5.8%.2.For S-1’s weekness of high background signal resulted from the free elastic oscillation of CSP-MB,a two-probe sensor(S-2)based on the signaling mechanism of target-induced displacement reaction was designed and successfully fabricated through introducing the supporting assistant probe(AP)in sensor construction.With the help of assistant duplex,this sensor restricts the free elastic oscillation of CSP effectively,thereby lowing the background signal and improving the detection sensitivity relatively.Four key parameters affecting the efficiency of sensor fabrication and working were optimized.These parameters are the ratio between AP and CSP,the mixed probe concentration,the location of AP/CSP duplex and the AP length.Under the optimized conditions,when used for the detection of KMY-A,502.1±26.0% of SE% was obtained at the adding concentration of 2.0 m M.A good linear relationship is obtained between detection signal and the target concentration(log c/M)within a range from 5.0 n M to 10.0 μM with the detection limit of 1.7 n M.When used for discriminating other interferents,the proposed sensor shows good discrimination for other 5 different kinds of antibiotic molecules with the discrimination coefficients of-0.002~0.009.However,the discrimination for the analogue,KMY-B,is not good with the discrimination coefficient of 0.84.Besides,this sensor also shows a fast sensing speed with ~240 s of response time.When used in 3 real-life samples(50% milk,50% tap water and 50% calf serum)in which the target residue is usually found,the sensor method performs good with the SE% of 176.5±15.2%,407.4±21.0% and 126.9±14.0%.Except the response speed,this sensor generally performs better than S-1.3.It can be predicted that the stem loop conformation change should performs better than the conformation change of changing from CSP to be CSP/KMY-A complex for the efficiency of driving MB labels.This is helpful for enhancing SE%,further improving the sensitivity.Following this idea,another two-probe sensor(S-3)was designed and successfully fabricated based on the signaling mechanism of target-induced “open and cut” of stem loop using the aptamer as CP and a stem-loop DNA as ASP.Four key parameters affecting the efficiency of sensor fabrication and working were optimized.These parameters are the ratio between CP and ASP,the mixed probe concentration,the length of CP/ASP duplex and the interrogation temperature.Under the optimized conditions,when used for the detection of target KMY-A,801.1±32.0% of SE% was obtained at the adding concentration of 2.0 m M.A good linear relationship is obtained between detection signal and the target concentration(log c/M)within a range from 2.5 n M to 10.0 μM with the detection limit of 0.83 n M.When used for discriminating other interferents,the proposed sensor shows good discrimination for other 5 different kinds of antibiotic molecules with the discrimination coefficients of 0.0005~0.0071.However,the discrimination for the analogue,KMY-B,is not good with the discrimination coefficient of 0.64.Besides,this sensor also shows a fast sensing speed with ~60 s of response time the same as S-2.When used in three real-life samples(50% tap water,50% milk and 50% calf serum)in which the target residue is usually found,the sensor method performs well with the SE% of 342.9±15.2%,665.4±21.0% and 260.3±18.2%.4.The above 3 sensors all use the electrode-bound MB-modified probe.This is not easy to further lower the background signal effectively because the MB labels are confined within the certain range of electrode surface.For solving this problem,a “double brace” three-probe sensor(S-4)was designed and successfully fabricated based on the signaling mechanism of target-induced SP shifting following the idea of using free SP.In this sensor,both CP and AP are co-immobilized on the electrode surface with CP/AP duplex state in a “double brace” way,the SP is free in the detection solution.Five key parameters affecting the efficiency of sensor fabrication and working were optimized.These parameters are the ratio between CP and AP,the mixed probe concentration,the SP concentration,the AP length and the interrogation temperature.Under the optimized conditions,when used for the detection of target KMY-A,0.81±0.08 μA of DS value with SE% of 35000±6250% was obtained at the adding concentration of 2.0 m M.A good linear relationship is obtained between detection signal and the target concentration(log c/M)within a range from 100 p M to 1.0 μM with the detection limit of 30 p M.When used for discriminating other interferents,the proposed sensor shows good discrimination for other 5 different kinds of antibiotic molecules with the discrimination coefficients of-0.004~0.0021.However,the discrimination for the analogue,KMY-B,is acceptable with the discrimination coefficient of 0.49.Besides,this sensor also shows a fast sensing speed with ~15 min.When used in three real-life samples(50% milk,50% tap water and 50% calf serum)in which the target residue is usually found,the sensor method performs well with 44.4±3.6%,48.1±4.2% and 26.2±2.8% of DS values accounting for the total DS value obtained from 100% pure buffer.5.Based on S-4,we redesign and fabricate another sensor(S-5)with a slight difference,in which the CP/AP duplex is immobilized on the electrode surface in a “single brace” way with only AP immobilized.Such change in design results in the hybridization resistance decreasing way opposite to S-4 for this sensor,further improving the comprehensive sensing performances.Under the optimized conditions,when used for the detection of target KMY-A,1.98±0.12 μA of DS value with SE% of 70000±8850% was obtained at the adding concentration of 2.0 m M.A good linear relationship is obtained between detection signal and the target concentration(log c/M)within a range from 10 p M to 1.0 μM with the detection limit of 3.3 p M.When used for discriminating other interferents,the proposed sensor shows good discrimination for other 5 different kinds of antibiotic molecules with the discrimination coefficients of-0.0012~0.0005.However,the discrimination for the analogue,KMY-B,is acceptable with the discrimination coefficient of 0.47.Besides,this sensor also shows a fast sensing speed with ~9 min faster than S-4.When used in three real-life samples(50% milk,50% tap water and 50% calf serum)in which the target residue is usually found,the sensor method performs well with 46.2±3.2%、65.0±4.0% and 40.1±2.8% of DS values accounting for the total DS value obtained from 100% pure buffer.