Visual Point-of-care Assays For Telomerase Activity And Nucleic Acid Studies

Point-of-care testing(POCT)is a kind of rapid detection technology,which can detect and analyze samples on-site with many advantages such as being small,fast,simple,and instant.Visual detection is a method whose results can be read directly by naked eyes without large instruments,and thus has been applied in POCT.Yet traditional visual methods still require professional operation and there has great challenge to achieve POCT.The invention of test strips provides a potable and reliable tool for the development of visual method,and has been widely utilized in disease diagnosis,environmental monitoring,food safety and other aspects.By combining nucleic acid probe and test strips,the biosensor would offer a lower-cost,more portable and stable tool for disease marker detection.In this paper,we construct visual POCT method to detect disease markers with test strips as signal readouts.The main contents of this paper can be divided into the following two parts.1.Research on point-of-care testing for visual detection of telomerase activityThe point of care testing(POCT)of telomerase activity is critical for early diagnosis of cancer.Herein,a visual method was developed for detection of telomerase activity via hydrogen peroxide test strip.It is based on the telomerase-controlled in-site formation of hydrogen peroxide.Firstly,biotinylated telomerase substrate(TS)primer was attached on the surface of magnetic beads(MBs)via the streptavidin-biotin reaction to form MB-TS complex.Then,TS primers were elongated by telomerase to form long telomere elongated products(TEP)which contains TTAGGG repeat units.The in-situ formed MB-TEP complex specifically hybridized with glucose oxidase modified cDNA(GOD-cDNA).After magnetic separation and washing,the MB-TEP/GOD-cDNA complex incubated with glucose solution to in-situ produce hydrogen peroxide(H2O2)which was detected by hydrogen peroxide test strip.One long TEP hybridized with multiple GOD-cDNAs,which enriched GOD to highly efficiently catalyze glucose for generating hydrogen peroxide.Thus,the visual assay achieved sensitive detection of telomerase activity,and the limit of detection(LOD)reached as low as 10 HeLa cells/?L by naked eyes and 4.5 HeLa cells/?L by absorbance measurements.Therefore,it offers a sensitive and low-cost method for visual detection of telomerase activity,which also,widens the application of commercial hydrogen peroxide test strip in the development of non-H2O2 biosensors.2.Development of colloidal gold nucleic acid lateral flow test stripLateral flow test strip has the advantages of being portable,fast,and inexpensive,thus has been widely used in many fields.Colloidal gold is often used as a signal molecule in lateral flow assay due to its advantages of obvious color,low price,easy synthesis,good stability,and easy modification.In this work,colloidal gold lateral flow assay was prepared using nucleic acid as a recognition probe.Several factors were optimized to improve the performance of the test strips.Nucleic acid hybridization and the molecular recognition between aptamer and small molecule were designed for preliminary verification of the assembled test strips.The experimental results indicated that the assembled lateral flow test strip can visually detect nucleic acid and pesticide.However,the limited was limited and the performance need to be improved in the future study.