| Biosensors for highly sensitive,selective,and rapid quantification of specific biomolecules make great contributions to biomedical research,especially molecular diagnosis.However,the conventional methods for biomolecular assays often suffer from unsatisfactory sensitivity and poor specificity.In some case?e.g.early state of disease?,the concentrations of some target biomolecules are too low to be detected by these routine approaches,and cumbersome procedures are needed to improve the detection sensitivity.Therefore,there is an urgent need for the development of rapid and ultrasensitive analytical tools.Single-molecule detection technology may well satisfy the requirement and hold promising potential for the development of ultrasensitive biosensors.By in vitro/in vivo labeling of target biomolecules with proper fluorescent tags,the quantification of biomolecule at the single-molecule level can be achieved.In comparison with the conventional ensemble measurements,single-molecule detection technology possesses the advantages of ultrahigh sensitivity,good selectivity,rapid analysis,and low sample consumption.Consequently,single-molecule detection can be used as an ideal analytical approach to quantify the low-abundant biomolecules with rapidity and simplicity.In this theresis,we develop a variety of biosensors based on single-molecule detection for the quantiffcation of various biomolecules including enzymes,nucleic acids,and cells with high sensitivity and selectivity.?1?We constructed a molecular beacon-functionalized quantum dot-based single-molecule fluorescent biosensor for DNA methyltransferase detection.Aberrant DNA methyltransferases?MTases?level is closely associated with disease states,and the accurate and sensitive detection of DNA MTases activity is of great importance for disease diagnosis and drug development.However,most current DNA MTase assays suffer from the significant disadvantages of time-consuming procedures,multiple complicated probe design and unsatisfactory sensitivity.Herein,we demonstrate a single quantum dot?QD?-based FRET biosensor for sensitive monitoring of DNA MTase activity and screening of its inhibitor.As far as we know,this is first application of QD-based FRET technique for probing DNA MTase activity.In our assay,the QD605 is functionalized with BHQ/Cy5 double-labeled detection probe to form QD605/probe/BHQ2/Cy5 complex,in which the Cy5 emission originating from the fluorescence resonance energy transfer?FRET?between QD605and Cy5 is quenched by BHQ2,thus no Cy5 signal can be detected.In the presence of target DNA adenine methyltransferase?Dam?,the detection probe is specifically methylated and subsequently cleaved by a methylation-sensitive restriction endonuclease DpnI,resulting in the release of BHQ2 moiety from the detection probe.As a result,the Cy5 emission originating from FRET is recovered,which can be easily detected by total internal reflection fluorescence?TIRF?imaging-based single molecule counting.The proposed assay involves only a single detection probe,avoiding complicated assay design and laborious signal amplification steps.The assay possesses significant advantages of high selectivity and high sensitivity with a detection limit of 0.002 U/mL.Moreover,it can be further applied for inhibitor screening,holding great potential for disease diagnosis and drug development.?2?We developed a ligase amplification reaction-assisted assembly of single quantum dot-based fluorescent biosesnor for alkaline phosphatase detection.Alkaline phosphatase?ALP?plays important roles in a variety of physiological and pathological processes,and the sensitive detection of ALP activity is essential for both biological research and clinical diagnosis.Fluorescet assay is most widely used for ALP detection,however,it often requires tendious and complex procedures for the preparation of inorginic nanomateries and chemical synthesis of orginic probes,and its sensitivity is quite limited because it only relies on the inherent fluorescent property of nanomaterial and fluorescent groups and lacks the appropriate signal amplification technique.To solve these issues,we introduce the ligase amplification reaction approach to quantitatively detect ALP activity.In this assay,ALP catalyzesthe dephosphorylation of 3'phosphorylated detection probe,enabling the further ligation between the detection probe and the assistant probe with the assistence of template and ligase to form the intact secondary template.The secondary template is able to catalyze the cyclical ligation of Cy5/biotin dual-labeled signal probe.The resultant siganl probes can be assembled on the surface of 605QD to form the 605QD/Cy5 complex,inducing efficient FRETfrom the 605QD to Cy5,which can be simply detected by single-molecule imaging.This assay is highly sensitive with a detection limit of as low as 5.63×10-8 U/mL,and it can be further applied for the detection of endogenous ALP in cancer cells and the screening of ALP inhibitors,providing a new approach for ALP-related biomedical researches and disease diagnosis.?3?We built a enzyme-free catalytic assembly of single quantum dot-based fluorescent biosesnor for circulating microRNA detection.MicroRNAs are key regulators of gene expression and have been regarded as potential biomarker for disease diagnosis.However,the current microRNA assays are often laborious and not sensitive for detection of low-abundant target such as circulating microRNA.Enzyme-assisted signal amplification strategies have been widely introduced to improve the assay sensitivity,but they always involve expensive and unstable protein enzymes,and precise thermal cycling,and complex assay design.Herein,we reported a simple,enzyme-free catalytic assembly of a single-QD-based biosensors and its application for highly sensitive detection of microRNA.The presence of target microRNA catalyzes the cyclical hybridization of two hairpin DNA probes,including biotin-labeled capture probes and Cy5-labeled detection probe,to form biotin/Cy5dual-labeled DNA duplexes,which can then be assembled on the surface of streptavidin-conjugated 605QDs.As a result,an efficient fluorescence resonance energy transfer?FRET?occurs from 605QD to Cy5,and the FRET signal from each single-QD/DNA/Cy5 assembly can be detected by total internal reflection fluorescence imaging-based single-molecule detection.The single-QD-based biosensor is highly selective and sensitive with a low detection limit of 8.16×10-16 M,and can be applied for accurate detection of endogenous microRNA in cells and circulating microRNA in human blood samples,without the requirement of anyprotein enzymes and thermal cycling process.As far as we know,this is the first report for construction of enzyme-free catalytic assembly of QD-based biosensors,opening a new way for simple,reliable,and highly sensitive biosensing applications.?4?We reported a DNA nanomachine-based single-molecule fluorescent biosensor for circulating tumor cell detection.Accurate and sensitive detection of rare cancer cells such as circulating tumor cells?CTCs?has attracted great interest in the fields of clinical diagnosis and cancer biomedical researches.However,the conventional methods for cancer cell detection often involve the complicated platform and laborious procedures with a limited sensitivity.Herein,we construct a new entropy-driven DNA nanomachine with the integration of single-molecule detection for rare cancer cell detection.The DNA nanomachine is constructed by three DNA components including a detection probe,a signal probe,and a fuel.Upon binding to the target cancer cell,the detection probe undergoes a conformational switch to expose the initiator which can hybridize with the signal probe to initiate the cascade toehold-mediated strand displacement,thus activating the DNA nanomachine and releasing abundant Cy5-labeled reporters which can be simply quantified by total internal reflection fluorescence?TIRF?imaging-based single-molecule detection.This assay employs the entropy-driven DNA nanomachine for efficient cancer cell recognition and signal amplification without the involvement of expensive and unstable antibodies and enzymes,and it enables one-step detection of living cancer cells with high sensitivity and good specificity.Moreover,it can be further applied for rare CTCs detection in patient sample. |
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