Construction Of Enzyme Assisted Signal Amplification-Based Fluorescence Biosensors For Biomedical Applications

Selective detection of biomolecules with high sensitivity is highly desired in diverse research areas including disease diagnosis,food safety control,forensic analysis,and environmental monitoring.Fluorescence is a useful technique for the detection of biomolecules such as nucleic acids and proteins,and it is widely used in biological and biomedical applications due to its high spatial and temporal resolution.However,the content of certain biomolecules related to disease are in very low abundance(such as circulating cell-free micro RNA and other tumor biomarkers)and beyond the limits of detection of conventional fluorescent technologies.Therefore,designing biosensors with higher selectivity and sensitivity is of great importance.The nucleic acid-based isothermal signal amplification is involved in the construction of fluorescence biosensors to achieve sensitive detection of low abundance biomolecules at a constant temperature without the requirement of thermocycling.Among them,the enzyme assisted signal amplification-based fluorescence biosensors exhibit high detection selectivity,high signal amplification efficiency and high detection sensitivity,holding great potential in biomedical research and clinical diagnosis.In this thesis,we develop a variety of biosensors based on enzyme assisted signal amplification such as helicase-dependent amplification(HDA),strand displacement amplification(SDA),and nuclease-assisted recycling signal amplification for the quantification of various biomolecules including nucleic acids and enzymes with high sensitivity and selectivity.The detailed contents are as follows:1.A simple,rapid,and sensitive helicase-dependent amplification(HDA)-based fluorescent biosensor was constructed for label-free quantification of low-abundance micro RNAs(mi RNAs).Dysregulation of mi RNA expression levels is closely associated with a variety of human diseases,and their rapid and sensitive quantification is essential to clinical diagnosis and therapy.Because of their poor sensitivity,conventional quantification methods are unable to detect low-abundance mi RNAs.Alternatively,nucleic acid amplification approaches have been introduced to improve the detection sensitivity,but most of them involve complicated probe design and time-consuming procedures.In this assay,the target mi RNA may specifically hybridize with the 3'-terminus of the linear probe to form a DNA-mi RNA heteroduplex,protecting the probes from exonuclease?(Exo?)digestion.The remaining probes may be subsequently amplified by HDA,generating an ultrahigh fluorescence signal within 30 min.This biosensor is very sensitive with a low detection limit of 12.8 f M and exhibits a large dynamical range from 100 f M to 10 n M.Moreover,the biosensor can discriminate different mi RNA family members,and it can be used to absolutely quantify endogenous mi RNA of total RNA samples extracted from cancer cells,providing a powerful tool for biomedical research and clinical diagnostics.2.A new fluorescent biosensor with zero background signal was built for sensitive detection of?-glucosyltransferase(?-GT)activity based on 5-hydroxymethylcytosine(5-hm C)glucosylation-triggered HDA.?-GT catalyzes the glucosylation of 5-hm C to enable the survival of bacteriophage and parasite in host cells,and it is a critical tool enzyme for 5-hm C assay.However,few methods are available for?-GT assay,and they usually have the drawbacks of radioactive contamination,high background,laborious procedures,and unsatisfactory sensitivity.In this biosensor,the designed detection probe may act as both a probe for?-GT recognition and a template for HDA.The?-GT-catalyzed 5-hm C glucosylation can protect the detection probes from both the cleavage by Mfe I restrictive enzyme and the digestion by Exo?and exonucleases?(Exo?).The remaining detection probes can subsequently act as the templates for exponential HDA to generate numerous double-stranded DNA products,which can be easily detected by SYBR Green?in a label-free manner.The zero background can be achieved by efficient elimination of primer–dimer nonspecific amplification and complete digestion of non-glucosylated detection probes.This biosensor exhibits high sensitivity and good specificity,and it can be further used to analyze?-GT kinetic parameters and screen the inhibitors,providing a powerful platform for deeper understanding of?-GT biological functions and promoting?-GT-related epigenetic studies.Furthermore,this biosensor can be extended to detect various DNA-modifying enzymes by simply replacing the recognition sequence and restriction enzyme.3.A simple and label-free fluorescent biosensor was developed for the polynucleotide kinase(PNK)assay based on phosphorylation-triggered two-stage strand displacement amplification(SDA).The PNK-catalyzed phosphorylation plays crucial roles in a variety of fundamental cellular processes,especially in DNA recombination,replication and damage repair.PNK is now regarded as a potential target for the treatment of cancers,and it is of great importance to develop sensitive methods for the PNK assay.Among the current PNK assays,the fluorescence methods attract more attention due to their distinct advantages of high sensitivity,the capability of homogeneous assay and being easy to operate.In spite of their good performance,most of them require expensive fluorescent labeling,and poor sensitivity due to the lack of an efficient signal amplification strategy.In this biosensor,PNK catalyzes the phosphorylation of the 5'-hydroxyl terminus to generate a 5'-phosphorylated probe-S.With the addition of lambda exonuclease(?exo),the 5'-phosphorylated probe-S in the probe-S/probe-A duplex is digested,releasing the free probe-A.The released probe-A can spontaneously fold into a hairpin structure with a 5'protruding terminus,with the 3'-end of the hairpin serving as a primer to initiate the two-stage SDA,producing abundant double-stranded DNAs(ds DNAs),which can be simply monitored using SYBR Green I in real time.This biosensor exhibits ultrahigh sensitivity with a detection limit of as low as 0.0002 U/m L,which is superior to most existing PNK assays.Importantly,it enables sensitive detection of endogenous PNK activity at the single-cell level,holding great potential in clinical diagnosis and biomedical research.4.A simple fluorescent nanosensor was constructed for sensitive simultaneous measurement of glycosylases based on excision repair-initiated enzyme-assisted bicyclic cascade signal amplification.Human single-stranded selective monofunctional uracil DNA glycosylase(h SMUG1)is a member of the uracil DNA glycosylases(UDG)superfamily.In addition to uracil,h SMUG1 has been shown to remove some oxidized pyrimidines,indicating it plays a role in the repair of DNA oxidation damage.Human alkyladenine DNA glycosylase(h AAG)exhibits broad substrate specificity and is responsible for the recognition and excision of a diverse group of alkylated purine bases and the removal of hypoxanthine from deoxyinosine-containing DNA.Abnormal DNA glycosylase activities are associated with a variety of diseases,suggesting great importance of the simultaneous measurement of DNA glycosylases.The nanosensor involved a bifunctional dumbbell probe and two signal probe-conjugated gold nanoparticles(signal probes@Au NPs).There are three regions including a stem region and two loop regions in the bifunctional dumbbell probe.The stem of the dumbbell probe is modified with one uracil base(U)and one hypoxanthine base(I)near the two loops,respectively,with the U for h SMUG1 sensing and I for h AAG sensing.The presence of DNA glycosylase enables the removal of damaged base in the stem of dumbbell probe and generates an apurinic/apyrimidinic(AP)site.The APE1-catalyzed cleavage of the AP site leaded to the break of dumbbell probe.The cleaved dumbbell probe functions as both a primer and a template to initiate the SDA,producing abundant triggers.The resultant triggers may selectively hybridize with the Cy5/Alexa Fluor 488 signal probes which are assembled on the surface of Au NPs.Each ds DNA with an AP site can act as the substrate for APE1,inducing recycling cleavage of signal probes to liberate numerous of Cy5 or Alexa Fluor488 molecules from the Au NPs,which can be simply measured by total internal reflection fluorescence(TIRF)-based single molecule detection,with Cy5 indicating the presence of h SMUG1 and Alexa Fluor 488 indicating the presence of h AAG.Due to the high amplification efficiency of the enzyme-assisted bicyclic cascade signal amplification and the high signal-to-ratio of single molecule detection,this nanosensor can achieve a detection limit of 8.14×10^-10 U/?L for h SMUG1 and 4.50×10^-9 U/?L for h AAG.It can be further applied for simultaneous detection of multiple DNA glycosylases in cancer cells at the single-cell level and can distinguish between normal cells and cancer cells.