DNA-based Hydrogel Biosensing For The Detection Of Escherichia Coli

Foodborne pathogenic bacteria,as the main problem facing food safety risk assessment,have become the main transmitting pathogens of food and water,which can cause human death in severe cases.Due to the widespread and uncertainty of pathogen transmission,human beings are facing serious food safety risk challenges,and it is necessary to develop and explore rapid portable,economical and efficient pathogen detection technologies to meet the strict detection requirements of today.In recent years,aptamer biosensors have attracted great attention.DNA hydrogels,a three-dimensional reticulated polymeric material formed mainly by the participation of DNA,have the advantages of high-water content,large drug loading space,and good biocompatibility,and the perfect integration of its retained DNA biological properties with the mechanical properties of its own backbone makes it one of the most popular emerging functional polymeric materials in recent years.However,there are still some limitations of DNA hydrogels constructed by DNA and other hybrid materials,and further attention is needed to expand the scope using these materials.First,in order to make DNA more widely used,a large amount of added raw material concentration is required to increase its gel-forming effect increasing the preparation cost.Second,DNA hydrogels are constructed from polymers and other materials that are less biocompatible and exhibit some biotoxicity in clinical medical bioassays,making them difficult to degrade.With the growing concern for personalized healthcare,there is an increasing need for miniaturized and easy-to-implement devices for even detection,and instant detection technologies have emerged,mainly for biosensing and clinical medical testing.Therefore,in this paper,based on the above background,a DNA hydrogel was prepared,and a DNA hydrogel-based portable glucose meter biosensor was constructed for rapid visual detection of foodborne pathogenic bacteria by rolling loop amplification technique.The paper focuses on the physicochemical properties of DNA hydrogels,classification strategies,and further reviews the research on the application of DNA hydrogels in the field of biosensing.The DNA hydrogel biosensor was constructed by the good surface modification properties and superparamagnetic properties of magnetic beads.Firstly,DNA hydrogels with non-chemical bonds as cross-linking sites were amplified by rational design of the loop-forming sequences and primers.The DNA hydrogels synthesized by this method are white flocculent and have good mechanical properties.It exhibits liquid-like properties in liquid and solid-like properties after removal of aqueous medium.The DNA hydrogel can be synthesized in a short time and at low cost.120μL of DNA hydrogel can be amplified using only 8μL of the loop template sequence,and it has the ability to be edited and modified,and it can be used for colorimetric studies because of its striking color when combined with nucleic acid dyes,etc.It is also biocompatible and can be naturally degraded at room temperature and has high bio-affinity for clinical testing.Meanwhile,the encapsulation performance of DNA hydrogel is tested based on its microscopic morphology,and the combination with glucose meter can realize fast signal output.Based on the construction of a DNA hydrogel biosensor,a representative foodborne pathogenic bacterium E.coli O157:H7(Escherichia coli O157:H7,E.coli O157:H7)was selected as the model bacterium,and a DNA hydrogel biosensor was designed with aldehyde-based magnetic beads as the aptamer carrier and rolling loop amplification as the main technology.The addition of functional nucleic acids,bio-functional molecules and nanomaterials to the DNA hydrogel enabled the hydrogel to have excellent properties such as biocompatibility and biodegradability,while demonstrating the superior detection performance of this biosensor.When E.coli O157:H7 was present in the test article,the aptamer bound on the magnetic beads specifically captured E.coli and competitively released the complementary sequence that hybridized with the aptamer This sequence is used as a primer for pre-loop formation and RCA.After magnetic separation,Phi29 DNA polymerase and d NTPs are added,and amplification can be achieved at 37℃under enzyme catalysis to generate DNA hydrogels.Sucrase as well as nanogold-sucrase complexes are added,and the supernatant is collected for reading in a portable glucose meter.When the system contains more E.coli O157:H7,the more primers are released,the higher the efficiency of RCA generates more DNA hydrogels,and thus the more sucrase or nanogold-sucrase complexes are encapsulated,and then sucrose solution is added,the lower the glucose meter readings,and vice versa.Using this method,the DNA flocculent hydrogel visible to the naked eye can be amplified,and the detection limit of this biosensor can reach1×10~3 CFU/m L when encapsulating sucrase,and the detection limit can be increased to1×10~2 CFU/m L when encapsulating nanogold-sucrase complex,while colorimetric study can be realized based on the color of nanogold,which is an observable phenomenon and provides a basis for the visual detection method This is an observable phenomenon,which provides a reference for the fielded assay.In summary,the biosensor prepared in this study greatly shortens the food safety detection time,saves the preparation cost,and enables efficient synthesis of DNA hydrogels in a short time.Compared with other current detection methods,the present method has simple operation steps,high detection limits,and can be directly observed by the naked eye to achieve qualitative and quantitative detection results.Moreover,the method has good assay versatility and can be used to detect any pathogenic target by simply changing the aptamer sequence.This method combined with rapid detection equipment provides a new idea and attempt for immediate on-site detection of food safety and even environmental substances.