| In recent years,researchers have been developed highly sensitive biosensing technologies that can diagnose human diseases early or detect harmful substances in food and the environment.In the presence of disease markers or food contaminants with extremely low concentrations,traditional biosensor methods based on receptor-ligand interactions are usually not sensitive enough to meet the requirments of early detection.Therefore,the development of isothermal nucleic acid amplification technology is of great significance for constructing biosensors and applying that in the ultrasensitive detection of biosensing strategies in the fields of modern biology,biomedicine and food safety.As one of the most widely used isothermal enzyme amplification technologies,rolling circle amplification technology has the characteristics of high efficiency and rapid reaction kinetics,and can realize exponential signal amplification to improve the detection sensitivity.In view of different disease markers and food safety contaminants,this research paper has developed several simple,efficient,ultrasensitive and novel optical biosensors for the detection of different analyzes taking advantages of nucleic acid molecules as the bioreceptor element,combining rolling circle amplification technology and other signal amplification strategies with fluorescence assay.The main contents of the research paper are summarized as follows:Firstly,in this work,a fluorescence biosensor based on target-mediated endonuclease-assisted rolling circle amplification strategy for the ultrasensitive detection of uracil-DNA glycosylase activity.In the presence of target UDG,UDG can catalyze the removal of uracil on hairpin probe?HP?leaving an apurinic/apyrimidinic?AP site?which can be cleaved by Endo IV and generating a primer for triggering the RCA reaction.Subsequently,numerous AP site-embedded signal probes namely as fluorescent-quenched probes combine with the RCA products to perform signal transduction and the quadradic signal amplification through Endo IV-catalyzed cleavage reaction,thus significantly enhanced fluorescent signal can be achieved for UDG activity screening.Under optimal conditions,this biosensor exhibits improved sensitivity toward target UDG with a detection limits of as low as 9.3×10-5 U mL-1and a widened detection range of 5 orders of magnitude.Additionally,our biosensor demonstrates high selectivity toward UDG and owns the superiority of simplicity,rapidness,and low-cost detections.Furthermore,by redesigning the modification of HP and using of the suitable endonuclease enzymes,this RCA coupled with Endo IV-assisted signal amplification strategy might be applied for the detection of other various targets,such as thymine DNA glycosylase,8-oxoguanine DNA glycosylase,DNA methyltransferase,and so on.Hence,the proposed strategy indeed provides a useful and versatile biosensing platform for ultrasensitive detection of UDG activity and related fundamental biomedicine research and clinical diagnosis.Next,the development of isothermal nucleic acid amplification techniques has great significance for the highly sensitive biosensing in modern biology and biomedicine.A facile and robust exponential rolling circular amplification?RCA?strategy is proposed based on repair enzyme-assisted primer-remodeling amplification?REPRA?,and uracil-DNA glycosylase?UDG?is selected as a model analyte.Two kinds of complexes,Complex I and Complex II,are pre-prepared by hybridizing circular template?CT?with uracil-containing hairpin probe and tetrahydrofuran abasic site mimic?AP site?-embedded fluorescence-quenched probe?AFP?,respectively.Target UDG specifically binds to Complex I,resulting in the generation of AP site and the following cleavage via Endonuclease IV?Endo IV?and the successive trimming of unmatched 3'terminus via phi29 DNA polymerase,thus usable primer-CT complex is produced that actuates primary RCA.Then numerous Complex II anneal with the first-generation RCA product?RP?,generating Complex II-RP assembly containing AP sites within the DNA duplex.With the aid of Endo IV and phi29,AFP as pre-primer in Complex II is converted into mature primer to initiate additional rounds of RCA.So,countless AFP is cleaved,releasing remarkably strong fluorescent signals.The biosensor is demonstrated to enable rapid and accurate detection of UDG activity with improved detection limit as low as 4.7×10-5 U mL-1 compared with Scheme 1.Therefore,this proposed strategy may create a useful and practical platform for ultrasensitive detection of low levels of analytes in clinical diagnosis and fundamental biomedicine research.Finally,a label-free fluorescent signal-on biosensor based on the target-driven primer remodeling rolling circle amplification?RCA?-activated multisite-catalytic hairpin assembly?CHA?enabling concurrent formation of Y-shaped DNA nanotorches?Y-DNTs?for ultrasensitive detection of ochratoxin A?OTA?.Aptamer structure-switching concept can be implemented for RCA process through the facile design of a switchable DNA assembly namely as Complex I made of a circular template?CT?,OTA aptamer?S1?and a substrate probe?S2?as the pre-primer binding with both S1 and CT.Thus,OTA binding to the aptamer releases the aptamer-binding element triggering primer remodeling owing to phi29 DNA polymerase,featuring high amplification efficiency and excellent selectivity.The RCA products as the promoter of CHA can provide multisite that can combine with ingenious-designed Complex II achieving concurrent duplex-amplification reaction.Y-DNTs are obtained through CHA process and bring the two split G-quadruplex fragments into close-enough proximity resulting in the formation of the intact G-quadruplex structures which used as signal-transducing units to enhance the fluorescence signal output of the NMM expressly.Moreover,this approach has demonstrated to enable accurate determination of OTA with improved detection limit as low as 0.2×10-3 ng mL-1 and widened dynamic range over 4orders of magnitude,and this method is proved to be capable for analyzing actual samples. |
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