SRCA-based Ratiometric Electrochemical Biosensor For Rapid Detection Of Salmonella Typhimurium In Food

Salmonella typhimurium is a zoonotic foodborne pathogen,which causes a fearful threat to public health in the world.It can cause gastroenteritis,typhoid fever and septicemia,particularly the elderly,children and people with low immune function.Therefore,it is necessary to establish a specific and accurate detection method for Salmonella typhimurium.Ratiometric electrochemical biosensor is an electrochemical technology based on dual signal strategy,which can overcome the internal system error caused by the difference of background current and environmental or personal factors.The reproducibility and stability of the electrochemical sensor can be greatly improved.SRCA is a novel method of nucleic acid isothermal amplification developed by our research team in recent years.The aim of this reseach is to construct a ratiometric electrochemical biosensor based on SRCA reaction and dual signal readout for the detection of Salmonella typhimurium.In order to construct the ratiometric electrochemical biosensor,firstly,the primers were modified with ferrocene(Fc),and a lot of amplified products with ferrocene(Fc)were obtained by SRCA reaction to achieve the purpose of amplification detection.Secondly,glassy carbon electrode was modified with gold nanoparticles and the mercapto modified β-cyclodextrin β-CD)was immobilized on the electrode surface by combining with gold nanoparticles to form Au-S bond.The amplification products could bind to β-CD through host-guest interactions to achieve the function of immobilizing the amplification products.Then the methylene blue(MB)signals are introduced,because MB can be embedded between DNA double strands through electrostatic interaction,and finally form a dual signal detection strategy with Fc.In order to verify the feasibility of the sensor,positive and blank tests were designed.It was found that MB signal(IMB)of positive test group was greater than Fc signal(IFc),while IMB of blank test group was less than IFc.After considering the factors that have great influence on the experiment,the concentration of Mg2+and Bst DNA polymerase in SRCA system were 3 mM and 8 U,respectively;the amplification time was 60 min and the amplification temperature was 62℃;the concentration of β-cyclodextrin on the electrode was 1 mM and the incubation time with SRAC product was 60 min.Under the optimal conditions,a linear relationship was found between the ratio of IMB/IFc and the logarithm of Salmonella typhimurium concentration.The linear range was 30 fg/μL～30 ng/μL,and the detection limit was 15.8 fg/μL(S/N=3).The correlation coefficient is 0.993.Compared with other nucleic acid amplification methods combined with electrochemical biosensors,the linear range and detection limit were expanded by 10～100 times.In addition,the specificity,stability and reproducibility of the biosensor were tested.It was found that the biosensor had high specificity for the detection of Salmonella typhimurium,and the stability and reproducibility were better than single signal electrochemical biosensor.In order to evaluate its practical application value,the ratio electrochemical biosensor was used to detect food samples contaminated by Salmonella typhimurium.It was found that the results of the ratio electrochemical biosensor were consistent with the real-time fluorescent quantitative PCR(RT-qPCR).In conclusion,the electrochemical biosensor based on SRCA can improve the sensitivity and expand the linear range of Salmonella typhimurium detection.The ratiometric electrochemical biosensor provides a reliable and accurate strategy for detection of Salmonella typhimurium.It is expected to be a common platform for detecting various food borne pathogens,and has broad application prospects in the field of food safety.