| Pathogenic Escherichia coli(E.coli)is one of the main pathogenic bacteria causing foodborne diseases,which widely exists in nature.For example,E.coli O157: H7 can rapidly reproduce in the suitable environment,cause pollution to the environment,and infect other organisms in the environment with water,food,air and other media,to clinical medicine and animal husbandry to bring serious crisis,causing a variety of diseases and even death.And it also causes a lot of economic losses.E.coli O157: H7 is also easy to enter the human body through water,air,food and so on.And the E.coli O157: H7 release a strong toxin,which can cause hemorrhagic diarrhea,fever,abdominal pain and vomiting.In severe cases,it may lead to kidney disease,which may be fatal if not treated properly.The prevention and control of foodborne disease have attracted worldwide attention.Rapid detection of pathogenic bacteria is critical in industrial settings,clinical and hospital diagnostics,and water and environmental quality controls as well as in resource-limited settings to reduce outbreaks of food-and water-borne diseases.At present,the rapid detection methods of foodborne pathogenic bacteria mainly include analytical immunological methods and molecular biological detection,such as enzyme-linked immunosorbent assay(ELISA),polymerase chain reaction(PCR).However,these methods also have their own limitations,such as time-consuming,insensitive,prone to false positives,or require large instruments and trained operators.And their application scope is limited.In addition,the traditional culture method is often used in clinical biological detection,and the analysis time needs at least 2-3 days and can be even longer for slow-growing bacteria.This significant delay in microbiological analysis leads to the use of broad-spectrum antibiotics,resulting in unnecessary treatment,ineffective antibiotic selection,and the potential for poor clinical outcomes.Therefore,there is an urgent need for new technologies that can quickly identify pathogenic bacteria and their drug resistance analysis,and be used for clinical medication guidance,which is of great significance in the fields of food safety and medical detection.Microfluidic technology is a new technology that manipulates and controls liquids in micro-scale channels.Compared with the defects of existing technology,microfluidic chip technology has attracted extensive attracted wide attention because of its high integration,miniaturization,diversification of analysis methods,fast analysis speed,high accuracy and low cost.The microfluidic chip based on polydimethylsiloxane(PDMS)has been widely used in field of microfluidic due to its good light transmittance,easy processing,low cost and good biocompatibility.In this work,a microfluidic platform that based on tetrahedral DNA nanostructures(TDNs)is introduced to the specific capture,release,bacterial enrichment,growth,and antimicrobial susceptibility testing(AST)of E.coli O157: H7.Firstly,through literature review,the reasonable structure of the microfluidic chip was designed,including the herringbone structures and microcavity structures.According to the literature report,determine the way of production,seal and drive.PDMS prepolymer was poured on SU-8 mold to fabricate chip.PDMS layer with pattern and PDMS substrate without pattern were bonded after processing by plasma cleaning machine for 60 s.The PDMS chip has no cracking and leakage.It can successfully realize the sample injection by the negative pressure driving method without any external valves or pumps and other complex injection methods.The TDNs can precisely regulate the distance between the probes,effectively improve the uniformity of the probe distribution on the surface.The TDNs as a rigid scaffold ensure the direction of aptamer and avoids the entanglement of aptamer.And the TDNs can effectively improve the efficiency of aptamer specific capturing E.coli O157: H7 cells.First of all,the successful self-assembly of TDNs was verified by agarose gel electrophoresis and atomic force microscopy(AFM).Each TDN carries a biotin group at one vertex and three acrylamide groups at the other three vertices.These can be firmly anchored on the thiol-modified PDMS surface via a Michael addition and the surface roughness of the microchannel is improved.Biotin-labeled aptamers are connected to TDNs via biotin-streptavidin linkage,and a rapid detection platform for E.coli O157: H7 was established.The PDMS can complete the detection of six samples at the same time in about 2 hours.The detection of bacteria with a concentration of 101 CFU/m L was realized.The specificity of the system was verified by using four other bacteria as negative control.And the recovery rates were 88.3% ~ 108.3% measured in the milk and orange juice.After enzymatic digestion,92.8% of the released bacteria were enriched in microcavities for subsequent AST.Six different antibiotics were used to develop the microcavity AST approach and determine their minimum inhibitory concentration(MIC)within 5 hours.These results are identical to those from broth dilution methods used in 96-well plates.In conclusion,this paper designed a PDMS chip that integrates herringbone structures,DNA tetrahedrons,aptamers,and microcavity structures is introduced to the rapid and effective detection,in vitro culture and AST of E.coli O157: H7.The results showed that under optimized experimental conditions,the microfluidic platform can greatly improve the detection sensitivity of E.coli O157:H7.It is a promising tool that is simple and fast.And it provides a new detection method for the detection,diagnosis and treatment of foodborne diseases. |
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