Engineering Functional DNA Superstructures For Its Counting Antimicrobial Resistance Bacteria

Bacterial resistance is currently one of the most complex threats in the field of global public health.The increasingly prominent problem of Antimicrobial-Resistant Bacteria(ARB)infection can lead to prolonged hospitalization,increased treatment costs,increased mortality and so on.The detection of ARB is extremely important for the diagnosis of infectious diseases,medication guidance,efficacy monitoring and infection control.Therefore,it is urgent to find a simple,rapid,sensitive and specific diagnosis method.Fluorescence-based digital or counting assays have become a powerful tool in biological and biochemical research.Until now,various fluorescent biolabels coupled with microdroplets,epifluorescence microscopy,confocal microscopy and total internal reflection fluorescence microscopy have been employed for sensitive detection of biomolecules.This article mainly designed a universal bio-sensing platform to meet the requirements in high sensitivity,simple and rapid detection of ARB markers(proteins)and cancer markers(RNA).The following research were mainly carried out:1.Preparation two functional micrometer-sized DNA superstructure(3D DNA)biolabels.The circular DNA template was induced by phi29 DNA polymerase to synthesize 3D DNA(rolling circle amplification reaction).Subsequently,the β-lactamase capture antibody was bound to the surface of the 3D DNA to recognize the target molecule.2.A detection method(3DMCA)was builded through the double antibody sandwich reaction on a 384-well plate and counting the fluorescent spots.There is a linear relationship between the number of spots and β-lactamase,and the detection limit reaching to 100 a M.3.This method was used to detect the ARB.First,the ARB which can secrete β-lactamase was cultivated,and the cell lysate was detected with a detection limit of 10 CFU/m L of ARB.In addition,3DMCA realizes the specific identification of clinically β-lactam-Resistant bacteria.4.Then demonstrated the use of the detection method in the detection of cancer markers(miRNA).The ultra-micro detection of miR-21 can be achieved through simple steps.The capture DNA paired with miR-21 was immobilized in a 384-well plate,and the detect DNA paired with miR-21 formed a 3D DNA biolabel on the surface of the 3D DNA,the 3D DNA biolabel was combined with single miR-21 and stained with fluorescent dye to form a uniform fluorescence dot.There is a linear relationship between the number of fluorescent dots and miR-21,and the detection limit can reach 1 f M.In addition,3DMCA can be used to determine the content of endogenous mature miR-21 in various cell lines,and the results are comparable to q RT-PCR.This study first reported the functional micrometer-sized DNA superstructure biolabel,building a detection method to achieve ultra-micro detection of protein and RNA.The reaction is rapid,sensitive and selective.We envision that the approach described herein will find useful applications in chemical biology,medical diagnostics,bio-sensing.