The Study Of Fluorescent Biosensor Based On Nucleic Acid Amplification Strategy And Novel Nanomaterials

Fluorescent biosensors can selectively identify target molecules with different concentrations and transform the physicochemical information generated in the process into fluorescent signals that are easy to detect by analytical instruments.The concentration of target and the fluorescent signal intensity exist in a significant dose-response relationship,which was the basis of quantitative analysis and detection.Owing to the homogenous detection environment,fluorescent biosensors have played an increasingly important role in medical diagnosis and drug efficacy monitoring and evaluation with the preponderances of simple instrument,easy operation,fast signal response and real-time monitoring.Unfortunately,conventional fluorescent biosensors can hardly meet the trouble of accurate and sensitive analysis of trace targets.Therefore,it is of great significance and imperative need to engineer a new fluorescent biosensor with high sensitivity and selectivity via direct or indirect methods coupled nucleic acid amplification strategy for the detection of trace targets.Because of this,we construct a series of highly sensitive and selective fluorescent biosensors based on novel nanomaterials as carrier and a lot of nucleic acid signal amplification strategies for detection of tumor-associated biomarker miRNAs.The specific works are as follows:1.A novel fluorescent assay for the ultrasensitive detection of miRNA-21 with the use of G-quadruplex structures as an immobilization material for a signal indicatorConventional fluorescent detection methods used a sequence containing G-base to directly immobilize signal indicator protoporphyrin IX(PPIX)with limited length.Due to the limited number of G-quadruplex structures,the immobilizing capacity of indicator PPIX is greatly limited.Herein,a novel fluorescent assay was proposed for ultrasensitive detection of miRNA-21 based on the efficient immobilization of PPIX as signal indicators in massive G-quadruplex structures obtained by target recycling,three-dimensional DNA walker and rolling circle amplification(RCA)-coupled cascade nucleic acid amplification strategy.The sequence containing G-base could further fold into a great number of G-quadruplex structures with the participation of K~+to efficiently immobilize substantial PPIX as signal probe,which significantly increased the efficiency and amount of PPIX immobilization,realizing a prominent amplification efficiency and detection sensitivity.As expected,the present fluorescent method demonstrated excellent performances for detection of target miRNA-21 with a detection limit of 0.35 fmol/L in a linear range from1 fmol/L to 1 pmol/L.Moreover,this proposed fluorescent method could be employed for effectively distinguishing miRNA-21 expression levels change in cancer-cell lysates and possessed great prospects to be applied for ultrasensitive detection of various biomolecules,opening up a novel paradigm for accurately clinical diagnosis.2.A Janus 3D DNA nanomachine for simultaneous and fluorescence detection and imaging of dual miRNAs in cancer cellsSome three-dimensional(3D)DNA nanostructures have been employed in simultaneous detection and imaging of dual miRNA targets via stochastically immobilizing two different signal probes on the surface of the homogeneous nanoparticles to realize relatively precise co-location in the same position of a cell.However,the fluorescence signal interference could be still caused by the proximity of the two different signal probes immobilized on the homogeneous nanoparticle-based 3D DNA nanostructure,resulting in low sensitivity for the simultaneous detection and fluorescence imaging of miRNAs in cells.Moreover,it also greatly reduced the walking efficiency of the 3D DNA nanomachine.Fortunately,Janus nanoparticles have two sides,and this asymmetric structure has opposite or complementary properties,providing the possibility of multicolor imaging at the same location in cells.Herein,a Janus 3D DNA nanomachine was constructed for the simultaneous and sensitive fluorescence detection and imaging of dual miRNAs in cancer cells,which could effectively eliminate signal interference in a homogeneous nanoparticle-based 3D DNA nanostructure caused by the proximity of the two different signal probes to achieve accurate co-location in the same position of living cancer cells.Impressively,in comparison with current miRNA imaging methods based on nanoparticle assemblies,the proposed strategy could efficiently eliminate“false positive”results obtained in single type miRNA detection.Meanwhile,the proposed fluorescence imaging technology makes it possible to visualize low concentrations of miRNAs with tiny change associated with some cancers,which could significantly improve the accuracy and precision compared to those of the conventional fluorescence in situ hybridization(FISH)approach.Therefore,it could serve as persuasive evidence for supplying accurate information to better understand biological processes and investigate mechanisms of various biomolecules and subcellular organelles.This strategy provided an innovative approach to design new generations of nanomachines with ultimate applications in bioanalysis and clinical diagnoses.3.An enzyme-free fluorescent biosensor with the use of perovskite quantum dots as a signal indicator for the detection and imaging of miRNA-21Commercial organic dye has suffered from large absorption and scattering in the visible region and are easy to be bleached by light in the process of fluorescent imaging,which could not monitor the long-term dynamic expression changes of biomarkers owing to the low penetration depth and imaging resolution.With high quantum yield and narrow band gap,CPB QDs have been widely used in photovoltaic cells and LED lamps.However,CPB QDs are known to have poor stability when exposed to moisture,light,or heat due to dynamic ligand binding to the crystals'surface,and these properties have severely limited their practical bioapplications in analysis and detection.In this work,the CPB QDs were wraped by polyester,which could significantly improve the stability of CPB QDs in queous solution and make it possible to decorate different DNA substrates.The dendrimer-like polymerization nanostructure was constructed based on the self-assembly and wraped on the surface of CPB@PMMA nanospheres,which could significantly reduce the energy transfer distance between the donor and receptor to increase the efficiency of fluorescence resonance energy transfer,realizing sensitive detection and imaging of miRNA-21 targets in cancer cells.Compared with traditional CdTe QDs as signal indicator,CPB QDs with narrow band gap significantly reduced background noise and enhanced imaging contrast,improving the accuracy of imaging and detection.As expected,the proposed strategy exhibited well excellent performance for miRNA-21 imaging in living cells.In addition,this work may provide a new avenue for the construction of DNA nanostructures,and a novel technology for biosensors,drug delivery.