Biomacromolecule Detection Based On Superlong Poly(thymine)-templated Fluorescent Copper Nanoparticles

The study of biomacromolecules such as enzymes and nucleic acids is of great significance for exploring life processes,revealing genetic mysteries and other life sciences.Traditional fluorescent analysis based on organic dyes faces the disadvantages of small Stokes shift,easy to photobleaching and high biological toxicity.In recent years,the rapid development of nanotechnology has contributed a wide variety of new fluorescent nanomaterials for biological analysis,such as semiconductor quantum dots,upconversion fluorescent nanomaterials and fluorescent metal nanomaterials.Among them,nucleic acid stabilized fluorescent copper nanoparticles(CuNPs)have the advantages of simple and rapid synthesis,low cost,nonpoisonous and large Stokes shift.In particular,the fluorescent copper nanoparticles templated by single poly T strand(poly T-CuNPs),because of their flexibility in the design of fluorescent probes,have emerged in the field of biological analysis and have attracted much research enthusiasm.However,in applications,poly T-CuNPs still exhibit the problems of relatively weak fluorescence emission intensity and poor fluorescence stability.In this thesis,we focus on this frontier research,and to improving the fluorescence properties of poly T-CuNPs.Based on the sequence dependence of CuNPs(the fluorescence intensity of poly T-CuNPs is proportional to the length of the template sequence),by using terminal transferase(TdT,a template free DNA polymerase),a biological analytical platform based on TdT polymerization"superlong" poly T mediated fluorescent copper nanoparticles was constructed.Biological enzymes and nucleic acid were selected as the object of analysis,combined with the nucleic acid tool enzyme and graphene oxide,a series of simple,convenient,low-cost,sensitive,specific and label-free biological macromolecules assay were developed.The details are as follows:1.Label-free and sensitive assay for deoxyribonuclease I activity based on enzymatically-polymerized "superlong" poly(thymine)-hosted fluorescent copper nanoparticlesDeoxyribonuclease I(DNase I),a common DNA enzyme,plays an important role in DNA repair,waste management and apoptosis.The change of DNase I activityis closely related to many diseases.Herein,by skillfully combined with degradation products with 3’ hydroxyl groups of DNase I and TdT can only identify and extend 3’ hydroxylated DNA substrates,fluorescence of poly T-CuNPs as signal,a simple,green,label-free and highly sensitive DNase I activity analysis based on"superlong" poly T templated fluorescent copper nanoparticles has been established for the first time.In this strategy,a 3’-phosphorylated DNA primer is designed to block TdT polymerization.After addition of DNase I,the primer could be digested to release 3’-hydroxylated fragments,which could further be tailed by TdT in dTTP pool with superlong poly T ssDNA for CuNPs formation.Fluorescence measurements and gel electrophoresis demonstrated its feasibility for DNase I analysis.The results indicated that with a size of 3-4 nm,the CuNPs templated by TdT-polymerized superlong poly T(>>500 mer)had several advantages such as short synthetic time(<5 min),large Stokes shift(～275 nm)and intense red fluorescence emission.Under the optimal conditions,quantitative detection of DNase I was realized,showing a good linear correlation between 0.02 and 2.0 U/mL(R2=0.9928)and a detection limit of 0.02 U/mL.By selecting six other nucleases(λ Exo,DpnI,DSN,KF-)or proteins(SSB,BSA)as controls,an excellent specificity was also verified.Then,the strategy was successfully applied to detect DNase I in diluted serum with a standard addition method,thus implying its reliability and practicability for biological samples.The proposed strategy might be promising as a sensing platform for related molecular biology and disease studies.2.Label-free and cyclic amplification assay for miRNA based on enzymatically-polymerized "superlong" poly(thymine)-hosted fluorescent copper nanoparticles combined with graphene oxideMicro RNA(miRNA)is a class of small noncoding ssRNA with an average of 22 nucleotides.Its abnormal expression is closely related to human cancer,diabetes,hepatitis and so on,therefore,it is an ideal biomarker for clinical diagnosis,treatment and prognosis.In this paper,graphene oxide(GO)was introduced into the"superlong" poly T templated copper nanoparticles signal amplification platform for the first time.Based on the property of GO in difference of adsorption capacity of ssDNA and dsDNA and anti-enzymatic protection of adsorbed nucleic acids,a new miRNA cycle amplification method with simple design,label-free,high sensitivity and good specificity has been developed by using DNase I.Herein,the let-7a was used as model target.First of all,on the basis of graphene oxide in single/double chain adsorption capacity differences,combined with the polymerization of TdT to synthesize fluorescent copper nanoparticles and the fluorescence of copper nanoparticles as signal output,a non-amplification method used for direct detection of miRNA are designed.The method showed a good linear correlation within the range of 5-100 nM(R2=0.9965),with a minimum concentration of 5 nM.Further,we use the DNase I as the tool enzyme,combined with the method established by graphene oxide to achieve dual-amplification of the signal:The first signal amplification is that DNase I degrading the DNA probe which is competed to leave from the surface of graphene oxide by target miRNA to produce 3’-OH DNA fragment and the free DNA fragment increasing the substrate for TdT polymerization,at last,achie-ving amplification by the TdT extension.The second amplification is the recycling of target miRNA.DNA probes were competed to leave from the surface of graphene oxide by hybridization with miRNA,and the DNA in DNA/RNA can be cleavage by DNase I.Due to this dual-amplification mechanism,the detection limit of miRNA is decreased to 50 pM,the sensitivity was improved by 100 times,and the linear detection is good in the range of 50 pM-10 nM(R2=0.9938).Furthermore,this strategy showed a good detection specificity for let-7a by utilized let-7e,let-7d,let-7i,miR-21 and miR-222 as controls.This strategy is expected to be a universal platform for RNA detection.