Sandwich Electrochemical Sensor Based On Dual Recognition Of Bacteria-imprinted Polymer Film-aptamer For The Detection Of Foodborne Pathogenic Bacteria

Infectious diseases caused by pathogenic bacteria are usually characterized by rapid onset,rapid progression of infection symptoms,and extremely low pathogenicity limits,posing a serious threat to human safety and health.Therefore,the development of rapid and sensitive detection methods for pathogenic bacteria is of great research significance for timely and effective prevention and control of infectious disease outbreaks and epidemics,food safety,and public health.In this paper,we propose a dual recognition sandwich-type electrochemical sensor based on bacteria-imprinted polymer-aptamer to detect pathogenic bacteria.The two recognition elements can be synthesized rapidly customized.Using 6-（Ferrocenyl）hexanethiol（Fc）,Anthraquinone-2-Carboxylic Acid（AQ）and Methylene Blue（MB）as redox molecules,three different redox potentials were explored for signal amplification nanoprobes.They were then combined with single-template Bacteria-imprinted polymer film（BIF）for bacterial detection.It is hoped that by synthesizing multiple templates of BIF and altering the Aptamer,it will be possible to build a platform for the detection of a wide range of bacteria.Gram-positive bacteria,Staphylococcus aureus（S.aureus）,and Gram-negative bacteria,Salmonella typhimurium（S.typhimurium）,were selected as template bacteria.The feasibility of sandwich electrochemical sensing was verified and various sensing performances were evaluated.The main tasks are as follows.Firstly,based on the group’s previous work,3-thiopheneethanol,which showed the best recognition effect of the imprinted sensor,was selected as the functional monomer.S.aureus was used as a template for the in situ preparation of the capture probe BIF by electropolymerization on the electrode surface within 15 min.The functionalized gold nanoparticles（Au@Fc-Apt）were obtained by successively modifying the S.aureus sulfhydryl groups Aptamer and Fc onto the surface of Au NPs through Au―S bonding.Once the target bacteria are immobilized on the BIF-modified electrode,Au@Fc-Apt further binds specifically to the bacteria,generating an enhanced current signal for ultra-sensitive detection of S.aureus.Individual cells can be detected in phosphate buffered solution.The sensor can detect S.aureus down to 10 CFU m L^-1even in a 10-fold dilution of a milk sample.In addition,the current response of the sensor to the target bacteria is virtually unaffected by the simultaneous presence of multiple interfering bacteria,which is 30 times greater than the number of target bacteria,showing excellent selectivity.The method is more sensitive and faster than most reported sandwich-type electrochemical sensors,and the sensing interface takes less time to construct（1.5 h）.Preparatory work was carried out to combine bacterially imprinted polymers of the three bacteria in order to achieve simultaneous electrochemical detection of the three bacteria.Two other redox signal probes with different potentials were explored with AQ and MB electroactive molecules,respectively.The signal nanoprobe Au@AQ-Apt with a reduction current potential at-0.49 V.The electrochemical signal probe was prepared using a microwave dry heating method by rapidly attaching a bacterial capture Aptamer on the surface of Au NPs and an oligonucleotide sequence S1 modified with an amino group at the end.AQ was attached by EDS/NHS.BIF was rapidly prepared as a capture probe using 3-thiopheneethanol as the functional monomer and S.aureus as the template.The feasibility of this sandwich sensor for S.aureus detection was verified.Furthermore,to improve the sensitivity of the sandwich sensor,the ratio of Aptamer in the signal nanoprobe for capturing bacteria and oligonucleotide S1 for attaching the electroactive molecule AQ was optimized.Although,the signal probe was applied to the BIF-based sandwich sensor for the detection of S.aureus with some feasibility,unfortunately,the difference in current response between the experimental group and the control group was 1.5-fold.Using MB as the electroactive molecule,a poly（methylene blue）-nanogold composite was prepared by a one-pot synthesis method,followed by the synthesis of a signal probe（Au@PMB-Apt2）with an oxidation potential at-0.30 V by attaching a S.typhimurium capture aptamer via an Au―S bond.Subsequently,Salmonella BIF was rapidly prepared as a capture receptor under the same preparative conditions.A sandwich-type electrochemical biosensor consisting of the signal probe Au@PMB-Apt2,S.typhimurium,and the capture probe BIF was used to detect S.typhimurium.The synthesised polymethylene blue-nanogold composite was in the form of a rod-like composite and demonstrated the feasibility of the sandwich electrochemical sensing strategy with a signal ratio of 2.7 for the experimental group to the control group.