Study Of Electrochemical DNA Biosensor Based On Signal Amplification By Gold Nanoparticles For The Detection Of LM

Listeria monocytogenes (LM) exists extensively in food such as meat, dairy products, vegetables, seafood, frozen food and cooked products, which is the fourth food-borne pathogen after pathogenic E.coli, Salmonella and Shigella. Recently foodborne diseases caused by Listeria monocytogenes occur around the world from time to time, so it’s of great importance to study the effective methods of detecting Listeria monocytogenes in food. At the moment, the methods of detecting Listeria monocytogenes mainly include three approaches:traditional isolation, culture and biochemical identification, PCR and immunological detection. Mostly there are some shortcomings about these methods, such as long detecting time, fussy operation, high cost, and so on, which greatly limit their application on on-suit rapid detection and large samples analysising. The electrochemical DNA biosensors which have been developed recently can be used to the detection of food-borne pathogens. In addition, they have some advantages such as fast detection, easy operation, low cost, high sensitivity, etc. In this paper, we develops two kinds of electrochemical DNA biosensors for the detection of hlyA gene of LM using electrochemical analysis containing cyclic voltammetry(CV), electrochemical impedance spectroscopy(EIS) and differential pulse voltammetry(DPV). The main contents of this paper are as follows:1. The well-dispersed gold nanoparticles (AuNPs) which particle size are about15nm were prepared by citrate reduction.1,4-Benzenedimethanethiol (BDMT) was used to be the medium connecting AuNPs with gold electrode. In order to close the blank site on the surface of gold electrode, the ssDNA/AuNPs/BDMT/Au electrode treated with6-Mercapto-l-hexanol (MCH) was prepared by immersing the ssDNA/AuNPs/BDMT/Au electrode in MCH solution for2hours. The modified electrodes were characterized by CV and EIS, indicating that BDMT, AuNPs, ssDNA and MCH were all assembled successfully on the surface of gold electrode.2. The function of MCH to ssDNA/AuNPs/BDMT/Au electrode was studied by immersing the ssDNA/AuNPs/BDMT/Au electrode in MCH solution or not. The results of EIS and DPV tests showed that MCH could remove loosely bound DNA at gold electrode surfaces, reduce nonspecific adsorption, adjust the surface density of gold electrode and thus improve the DNA hybridization rate. The function of AuNPs to electrochemical DNA biosensors was also studied by assembling AuNPs to gold electrode surface or not. It turned out that AuNPs could increase the number of probe DNA on the surface of gold electrode, amplify the hybridization signal, thereby enhance the sensitivity of electrochemical DNA biosensors.3. The electrochemical DNA biosensor using daunomycin (DNR) as hybridization indicator was developed. Then we optimized the hybridization conditions, the results are as follows:the optimal hybridization temperature is40℃, the optimal ionic strength of hybridization solution is15mmol/L, and the optimal hybridization time is40min. The gathering conditions of DNR indicator were also studied:the optimal concentration of DNR solution is0.05mmol/L, the optimal gathering time is15min, and the optimal pH value of DNR solution is6.5. By analysising the peak current of DNR using DPV, we achieved the quantitative detection of target DNA. We found a good linear relationship between the peak current of DNR and the logarithm of the concentration of target DNA in the range from1.0×10-13～1.0×10-8mol/L, and the linear regression equation was I(A)=3.7457+0.17351ogC(mol/L), the linear correlation coefficient R2=0.9958. The detection limit of target DNA was3.78×10-14mol/L. The electrochemical DNA biosensor we developed has been proved to be highly sensitive, stable(the oxidation peak current of DNR dropped about24.8%after a week) and well repeated(RSD=2.44%).4. The electrochemical EIS DNA biosensor without hybridization indicator was developed afterwards. We optimized the immobilization conditions of probe DNA using EIS and the results are as follows:the optimal concentration of probe DNA is1.5×10-6mol/L, the optimal immobilization time is2h and the optimal pH value of probe DNA solution is8.0. The hybridization conditions were also studied:the optimal hybridization temperature is40℃, the optimal hybridization time is40min, and the optimal ionic strength of hybridization solution is15mmol/L. We found a good linear relationship between the hybridization signal△Ret (△Ret=Ret (dsDNA)-Ret (ssDNA)) and the logarithm of the concentration of target DNA in the range from1.0×10-13～1.0×10-7mol/L, and the linear regression equation was△Ret(kΩ)=75.85+5.761ogC(mol/L), the linear correlation coefficient R2=0.9939. The detection limit of target DNA was8.33×10o-14mol/L. The experiment results showed that the electrochemical EIS DNA biosensor we developed had the advantages of high selectivity, excellent stability and reproducibility.