Application Of DNA Signal Amplification Technology And Novel Nanomaterials In Biosensors

Biosensors have broad application prospects in analytical chemistry and life science due to the advantages of low cost,fast analysis speed,good selectivity,high sensitivity and on-line continuous monitoring in complex systems.The extensive application and achievements of DNA signal amplification technology and nanomaterials in the field of biosensing provide researchers with more ideas for sensing design.In this paper,we developed a series of quantitative analysis methods for sensitive,simple and low-cost detection of disease markers based on DNA amplification technology and new nano materials such as fluorescence polydopamine nanoparticles（PDANPs）,terbium ion metal organic framework（Tb MOF）,bovine serum albumin coated gold nano clusters（BSA@Au NCs）and so on to,the detail contents were as follows:Uracil DNA glycosylase（UDG）is a typical DNA glycosylase that plays a fundamental role in base excision and repair.The occurrence of human neurological diseases,human immunodeficiency diseases,lymphoma and Bloom’s syndrome are closely related to the abnormal expression of UDG in human body.In Chapter 2,a unique 3’-5’exonuclease activity of endonucleaseⅣ（EndoⅣ）at the 3’terminus of DNA has been demonstrated,which enabled the development of a novel 3’-5’exonuclease activity-based EndoⅣcyclic signal amplification strategy（EAECSA）for a highly sensitive uracil DNA glycosylase（UDG）activity assay.In this strategy,a hairpin probe with one uracil nucleotide is efficiently excised by UDG,yielding an apyrimidinic site（AP site）.The AP site is cleaved by EndoⅣand a shorter DNA probe is released to trigger the EAECSA strategy via the cyclic formation of a three-way junction,leading to the repeated digestion of the detection probe and the liberation of FAM-labeled single bases.Then,a strong fluorescence signal is observed due to the extremely weak interaction between the FAM-labeled single bases and GO.In contrast,when UDG is absent,the cleavage of the hairpin probe fails,the three-way junction structure is not formed,and no cleavage of the detection probe occurs,and thus,a negligible fluorescence signal is detected because the fluorophore of the detection probe is efficiently quenched by GO.The designed EAECSA technology demonstrated a detection limit of 7.0×10^-4 U m L^-1 and showed a superb performance in screening UDG inhibitors,as well as cancer cell analysis.The proposed strategy provides a new way for isothermal nucleic acid amplification and may be promising for UDG profiling applications and related disease diagnosis.Alkaline phosphatase（ALP）has a high content in kidney,bone,liver,placenta and other organs of human body,and the concentration of ALP in organs is different from the normal level of human body,which is closely related to diabetes,hepatitis,cancer and other related diseases.In recent years,fluorescent PDA NPs has gradually become a new type of biopolymer nanoprobes,and has been successfully applied in the fields of biosensing and bioimaging.In chapter 3,we simply synthesized intrinsic fluorescent polydopamine nanoparticles（PDA NPs）in sodium hydroxide solution（Na OH,p H=11）,and constructed a new fluorescence nanoplatform for the detection of alkaline phosphatase（ALP）using PDA NPs as an effective signal reporter.The nanoplatform was constructed by the combination of enzymatic hydrolysis of ALP to the substrate L-ascorbic acid-2-phosphate（AA2P）and the chemical redox reaction between L-ascorbic acid（AA）and mercury ion(Hg²⁺).The fluorescence of PDA NPs could be effectively quenched by Hg²⁺through the coordination effect between Hg²⁺and the functional groups on the surface of PDA NPs.However,the quenching effect was greatly inhibited by the addition of AA into the solution.Based on this point,the activity of ALP could be monitored by hydrolysis of the substrate AA2P to AA and the fluorescence output of PDA NPs.The nanoplatform exhibited high sensitivity and desirable selectivity for ALP detection.With a wide linear range of 0 to 18 U L^-1,a detection limit of 0.4 U L^-1 was achieved using the developed nanosensor.The proposed method could not only be used to screen the inhibitor of ALP but also be used to detect ALP activity in human serum samples successfully.Moreover,the strategy can easily be expanded to determining other kinds of enzymes participating in AA-generation reactions.Heparin（Hep）plays an important role in physiological processes such as venous thromboembolism,inflammation and blood coagulation,while sulfate（CS）is an important component of vertebrate tissues such as cartilage,bone and ligament.They play an important role in many biological systems and are widely used in clinical practice.In chapter 4,we designed a novel dual-emission reverse change ratio fluorescence nanoplatform for fluorimetric and colorimetric sensing of heparin（Hep）and chondroitin sulfate（CS）based on green emissive terbium metalorganic framework（Tb MOF）and red emissive bovine serum albumin capped gold nanoclusters（BSA@Au NCs）.When excess Tb MOF was mixed with BSA@Au NCs,the nanoaggregates of Tb MOF and BSA@Au NCs could be precipitated by centrifugation due to the electrostatic interactions between them,and the supernatant displayed bright green color of Tb MOF under 254 nm UV lamp.However,the introduced Hep or CS caused the decomposition of Tb MOF since Tb³⁺has stronger affinity with Hep and CS,which resulted in the release of BSA@Au NCs into the supernatant.As a result,the red fluorescence of BSA@Au NCs turned on while the green fluorescence of Tb MOF turned off under the 254 nm UV illumination.The dual-emission reverse change ratio fluorescence nanoplatform possessed the variations of the emission intensity ratio at two different wavelength and the corresponding green-to-red fluorescent color changes,endowing a novel mechanism for fluorimetric and colorimetric analysis of Hep and CS using fluorescence spectrophotometer and smartphone,respectively.With the ratiometric fluorescence nanoprobe,simple and rapid sensing of Hep and CS with high sensitivity,selectivity and accuracy could be realized.Moreover,successful recoveries of the analytes in human serum samples were also validated,indicating promising application for Hep and CS analysis in biomedical diagnosis.