Construction Of Organic Dye-based Label-Free Fluorescent Biosensors

Biomarkers are used for risk assessment,diagnosis,and prognosis of diseases,as well as for predicting treatment efficacy,toxicity,and relapse.The development of low-cost,accurate,highly sensitive,and stable biosensing strategies for detection of biomarkers contributes to environmental monitoring,biomedical research and monitoring of tumorigenesis,progression,and prognosis.Currently,despite the great success of label-based strategies,they usually suffer from the limitations（e.g.,the need of sophisticated,laborious,and time-consuming label processes,low reproducibility and low stability of labeled bioconjugates,poor photostability,p H-sensitivity,the incomplete fluorescence quenching-induced false positive,and the interference on the biomolecules function after label processes）.However,label-free strategies may avoid the above-mentioned limitations,and it has several significant features including no need of fluorophore/radioactive isotopes labels,no side-effects caused by fluorophore modifications,and genetically encoded fluorophore/aptamer in vivo,low cost,and easiness.Traditional nucleic acid-based fluorescent probes are labeled with fluorescent dyes（e.g.,Cy5,Cy3,and FAM）based on covalent binding,but there are various drawbacks,such as the need of purification,not easy to store,poor stability,and relatively high cost.Alternatively,some organic dyes can bind to specific nucleic acid structure/aptamers for generation of fluorescence signal,and they have some advantages of no labeling,high stability,high quantum yield,good specificity,reproducibility,and strong signals.Therefore,the development of organic dye-based label-free fluorescent biosensors has great potential for biomarker detection.In this thesis,we combined the nucleic acid amplification,organic dyes,and functional nucleic acids,and selected the histone acetyltransferase（HAT）activity,micro-ribonucleic acids（mi RNAs）,and long noncoding RNA（lnc RNA）as research models for developing various label-free fluorescent biosensing strategies with some advantages of low cost,programmability,autonomy,good stability,simple operation,high specificity,and high sensitivity.This thesis achieves the detection of enzymes/nucleic acids in human cells or human tissues.The main aspects of this thesis are as follows:1.An aptamer-mediated rolling circle amplification（RCA）was developed for label-free detection of histone acetyltransferase（Tip60）activity.In the existence of Tip60,it catalyzes the transfer of acetyl groups from the acetyl-coenzyme A（Ac Co A）to lysine 16（K16）residue of H4peptide substrate,generating an H4K16ac site.Then,the aptamer in DNA probes can bind to H4K16ac with the high binding affinity.The resulting acetylated peptide-DNA probes can be immobilized on the surface of streptavidin-coated magnetic bead through specific biotin-streptavidin interaction.After the magnetic separation,the H4K16ac-bound DNA probes are dissociated by heating at 95°C.The released DNA probe can bind the RCA template and is subsequently elongated,generating abundant single-stranded RCA products.SYBR Gold are inserted into products for a significant fluorescent signal.Nevertheless,in the non-existence of Tip60,the H4 peptide substrate remains unmodified,and no H14K16ac site is produced and no RCA can be triggered for generation of an amplified DNA signal.This assay utilizes aptamer for recognition and SYBR Gold（note:SYBR Gold is an organic fluorescent dye that can bind to DNA）for signal detection without the use of any antibodies and fluorescent/radioactive labels,and the introduction of histone modification site-specific RCA significantly improves the assay sensitivity（LOD of 220 f M）.It can be further applied for histone modifying enzyme（HME）inhibitor screening,enzyme kinetic analysis,and endogenous HME measurement in cancer cells,providing a powerful tool for HME-related biological studies and clinical diagnosis.2.Target-triggered assembly of G-quadruplex DNAzyme nanowires was developed for sensitive mi RNA assay in lung tissues.Target mi RNA is complementary to the circular templates to trigger primer generation-mediated rolling circle amplification,generating numerous primers.The resultant primers then are complementary to catalytic hairpin templates to initiate primer exchange reaction（PER）,followed by the production of long G-quadruplex nanowires.Finally,G-quadruplex DNAzyme selectively bind to Thioflavin T（Th T）（note:Th T is an organic fluorescent dye that can bind to G-quadruplex）as a fluorescence indicator to generate an amplified signal.In contrast,the absence of mi R-486-5p cannot induce the PG-RCA-PER,and no enhanced signal is detected.Notably,the combination of PG-RCA with PER endows this assay with high amplification efficiency,and the generation of Th T/G-quadruplex DNAzyme nanowires can induce 2100-fold fluorescence enhancement,facilitating sensitive detection of mi RNA without the involvement of any fluorescence-labeled nucleic acid probes.This strategy can detect mi RNA over a wide linear range（up to 6 orders of magnitude）,and it exhibits high sensitivity with a LOD of 61.34 a M.It can highly selectively distinguish even a single-base mutation in mi R-486-5p.Moreover,it may measure the mi R-486-5p concentrations in various cells types,and it achieves the identification of differentially expressed mi R-486-5p in lung tissue of nonsmall-cell lung carcinoma（NSCLC）patients and healthy individuals,with promising applications for cancer prognosis and biomedical analysis.3.On-bead functional nucleic acid nanowires was demonstrated on the basis of cyclic ligation reaction-driven rolling circle amplification for label-free measurement of long noncoding RNAs in lung and breast tissues.The presence of lnc RNA initiates the cyclic ligation reaction,converting low-abundance targets to numerous biotinylated ligation products by repetitive denaturation-hybridization-ligation.The resulting ligation products can be immobilized on the surface of magnetic bead（MB）to initiate the downstream rolling circle amplification（RCA）,facilitating the synthesis of long G-quadruplex nanowires.The resultant nanowires can light up Th T to generate a high fluorescence signal.This method possesses significant advantages:（1）the improved ligation efficiency of T4 RNA ligase 2 by altering DNA bases to RNA bases at conjunction site;（2）the enhanced sensitivity resulting from repetitive denaturation-hybridization-ligation and RCA-mediated formation of G-quadruplex nanowires,and elimination of background interference by magnetic separation.This method displays ultrahigh sensitivity with a LOD of 9.97 a M,and it possesses excellent single-base mismatch discrimination capability.Moreover,it can accurately quantify cellular Homeobox（HOX）gene antisense intergenic RNA（HOTAIR）at single-cell level and differentiate the HOTAIR level in normal individuals and lung/breast cancer patients tissues,holding significant promise in clinical diagnosis.4.A sensitive and simple fluorescent biosensor was developed for label-free and rapid measurement of lnc RNAs based on Rec J_f-assistant recombinase polymerase amplification（RPA）without the use of thermal cycling and reverse transcription.Target lnc RNAs can bind with the5′-end of DNA template to create a DNA-lnc RNA hybrid,protecting the DNA template from Rec J_f exonuclease-mediated degradation.Subsequently,the primers hybridize with the intact DNA templates and are extended to generate the ds DNA products with the assistance of polymerase.The resulting ds DNA products may be amplified through exponential RPA to produce abundant ds DNAs,producing a distinct fluorescence signal within 10 min with SYBR Green I as indicator（note:SYBR Green I is an organic fluorescent dye that can bind to ds DNA）.This biosensor achieves a wide dynamic range from 10^-17 to 10^-9 M and high sensitivity with a detection limit of 1.23 a M.In addition,it can distinguish the expressions of lnc RNA HOTAIR in tissues of breast cancer patients and healthy individuals,with broad application prospects in lnc RNA-related research and early diagnosis of cancers.