Fabrication Of Biosensors Based On Nicking Endonuclease And Deoxyribozyme

A biosensor is an analytical device using biological components （e.g., enzymes,antibodies, nucleic acids, cells, microorganisms, etc.） as sensing elements for thedetection of an analyte. Because of its high sensitivity and specificity, it is widelyapplied in clinical diagnosis, bioengineering, environmental protection and foodscience. Nicking endonuclease and deoxyribozyme, due to their unique properties,can be employed as signal-amplifying elements to develop novel kinds of biosensors,thus, fabrication of biosensors based on nicking endonuclease and deoxyribozyme hasbeen received more and more research interests.1. A novel aptasensor forATP detectionAptamers are oligonucleic acid molecules that may bind to target molecules （e.g.,inorganic ions, organic molecules, proteins, cells, etc.） with high affinity andspecificity. In this work, we have fabricated a novel aptasensor for adenosinetriphosphate （ATP） detection. Firstly, an oligodeoxynucleotide containing ATPaptamer sequence is employed to bind ATP specifically. Then it leaves a binding sitefor deoxyribozyme strand to form a double-stranded DNA （dsDNA） which containsthe recognition site of nicking endonuclease. The nicking endonuclease can onlycleave the deoxyribozyme strand in the dsDNA, which transfers and amplifies thequantitative information of ATP to that of deoxyribozymes. Taking advantage of thedeoxyribozyme-catalyzed colorimetric reaction, we are able to quantify the amountsof ATP simply by UV-Vis spectroscopic analysis or even with naked eyes.Experimental results show the detection limit can be as low as100fmol/L, and ATPcan be easily distinguished from other nucleoside triphosphates such as CTP and GTP.2. Sensitive DNA assay based on cascade enzyme-linked signal amplification strategyDetection of specific sequences in target DNA is critical to clinical diagnosissuch as early detection of cancers, identification of pathogens and confirmation ofgenetic disorders. In order to detect DNA molecules with high sensitivity andaccuracy, several signal amplification reactions can be combined as cascade reactions,so we have proposed a sensitive DNA assay method which integrates threeconsecutive steps of signal amplification. Firstly, each target DNA molecule starts anicking endonuclease-assisted signal amplification reaction and results in a largemolar excess of primers which can trigger polymerase-mediated rolling circleamplification （RCA）. Then, the RCA reaction enriches the deoxyribozymes byproducing a long chain with repetitive deoxyribozyme moieties. Thesedeoxyribozymes have the peroxidase-mimicking activities, so they can catalyze aH₂O₂-mediated ABTS oxidation and finally generate a colorimetric output. Byemploying this cascade enzyme-linked signal amplification strategy, this DNA assaymay have processed a high sensitivity and accuracy with a great ability to distinguishone-base mismatched oligonucleotide, which might have promising potential forclinical applications in the future.