Rapid Detection Of Salmonella By Bacteria Imprinted Polymer-based Electrochemical Sensor

According to the report by European Center of Disease Prevention and Control（ECDC）and the World Health Organization（WHO）,Salmonella is second common causes of food poisoning in the world,with about 1.3 billion cases of acute gastroenteritis/diarrhea caused by Salmonella each year,resulting in about 3 million deaths.It is urgent to develop a sensitive and rapid salmonella detection method because of the low limit of Salmonella infection and the rapid progress of the disease,which seriously threaten human health and public health.Electrochemical sensors based on molecularly imprinted polymers（MIPs）have been widely used in the detection of small organic molecules,but it has few applications and faces some challenges in bacterial detection.On the one hand,the bacteria are large in size and embedded deeply in the polymer,so it is difficult to remove the template.In addition,the deep holes make it difficult for the template bacteria to combine with the recognition site.On the other hand,the bacterial surface chemical composition is very complex,so it is a challenge to design suitable functional monomers.In view of the existence of some functional groups on the surface of bacteria（such as hydroxyl,carboxyl,amide,phosphoryl,etc.）,we choose the monomers which can produce hydrogen bond with bacteria,an in-situ bacterial imprinting film（BIF）was prepared on the electrode surface using the surface imprinting strategy-electrochemical polymerization,and the thickness of the film was controlled by changing the polymerization conditions,so as to realize rapid elution of templates and rapid recognition of bacteria.The main contents are as follows:First of all,3-thiophene ethanol（TE）was selected as the functional monomer,and Salmonella Paratyphi B（S.Paratyphi B）was used as the template bacteria,and the bacterial imprinted film was prepared in situ on the surface of the glass carbon electrode（GCE）by cyclic voltammetry（CV）.This film structure is novel and no cavity-like imprinting site,so as to achieve rapid capture and label-free detection of Salmonella.The preparation process and morphology of the film were characterized by means of CV,electrochemical impedance spectroscopy（EIS）,scanning electron microscope and atomic force microscope（AFM）.In order to obtain the best sensing performance,the preparation and recognition conditions of the imprinted film were optimized.Under the best experimental conditions,the linear range of this sensor is as wide as 6 orders of magnitude（10¹～10⁶CFU/m L）and the detection limit is as low as2.0 CFU/m L.In addition,the sensor has good selectivity,reproducibility and a certain degree of regeneration.The method was successfully applied to the rapid detection of Salmonella in drinking water and no-fat milk powder,and achieved the ideal recovery rate（93%～104.9%）.Secondly,the bacterial imprinting film was prepared by constant charge method,and the best experimental conditions were obtained by optimizing the polymerization potential,the polymerization charge,the p H of electrolyte and the ion concentration of electrolyte.Then,compared the bacterial imprinting effects of four thiophene functional monomers,3-thiophene methylamine（TA）,3-thiophene boric acid（TBA）,3-thiophene acetic acid（TAA）and 3-thiophene ethanol（TE）.The results showed that all these four monomers could be used as ideal monomers for the preparation of bacterial imprinted film and TE had the best imprinting effect.In addition,under the same polymerization conditions,different side chain substituent have different binding ability to the bacterial surface,which leads to different recognition efficiency and non-specific adsorption of bacterial imprinting.This phenomenon indicates that it is very important to select suitable functional monomers for the preparation of bacterial imprinted film.Finally,the chemical recognition mechanism of bacterial imprinting was preliminarily explored.The bacteria were treated with the complexing agent-ethylene diamine tetraacetic acid（EDTA）and the cationic polymer-polydiallyldimethylammonium chloride（PDDA）to destroy the surface structure or change the surface charge of the bacteria,respectively.The results showed that EDTA and PDDA had significant inhibitory effect on the recognition efficiency of bacterial imprinting,and the inhibitory effect became stronger with the increase of concentration/incubation time.It is concluded that the interaction between O-antigen and static electricity plays an important role in the chemical recognition of bacterial imprinting.Based on the above work,we proposed to adopt two labeling strategies to amplify the signal:Metal-Organic Frame structure（MOFs）and DNA hydrogels,respectively.On the one hand,nanoparticles（Au NPs）are prepared in situ on MOFs carrier,and Salmonella aptamers are assembled on the surface of the composite material through the Au-S bond to form the recognition material,and then,a sandwich electrochemical sensor is constructed.Because the synthesized MOFs materials have peroxidase activity,they can catalyze the oxidation of H₂O₂-containing3,3,5,5-tetramethylbenziline（TMB）.The detection of S.Paratyphi B was achieved by calculating the S.Paratyphi B concentration with the resulting DPV current response of TMB/H₂O₂.On the other hand,dozens or even hundreds of double-stranded DNA hydrogels were prepared in situ by rolling circle amplification（RCA）using primers and two partially complementary ring templates based on bacterial imprinting.Methylene blue（MB）as a dye can be embedded in the DNA duplex through both electrostatic adsorption and intercalation binding.Due to its redox activity,electrons travel along the double strands of DNA to produce a strong current response,thereby implementing the labeling strategy.Unfortunately,both labeling strategies are in the preliminary exploration stage,our research group will continue to carry out related work.