Study On The Construction Of Nanosensor Based On Resonance Light Scattering And Fluorescence Technology

Thrombin,a protein involved in the blood coagulation cascade,was the first biological macromolecule exploited for aptamer selection.Thrombin is a kind of serine proteases in blood clotting system with functions of coagulation and anticoagulation.Thrombin generated by prothrombin activation,and catalysed soluble fibrinogen to chain fibrin,thus promotes blood clotting.Activation of thrombin is crucial in physiological and pathological coagulation,which plays an important role in tissue repair and tumor biology.Thrombin can be seen as a biological marker related disease diagnosis,such as pulmonary metastasis and cartilage.Due to the clinical importance of thrombin,it would be tremendously valuable to establish a fast and reliable way for the detection of thrombin.Micro RNA(mi RN A)is a new class of non-protein-coding RNA,contains approximately 22 nucleotides,which is capable of controlling gene expression by directly interacting with target messenger RN A(m RNA).A lot of research works showed that many diseases including human cancer are directly related to the expression of mi RN As.Therefore,it is significantly important to find an effective method to detect mi RNAs in clinical early diagnosis and disease prevention.Pyrophosphate(PPi)is the basic substance of life processes in biological systems.PPi is produced by many biochemical reactions,such as ATP hydrolysis,the formation of DNA and RNA polymerase ring adenosine phosphate enzyme adenylate cyclase aggregation,and enzymatic activation.Many pathological diseases,such as familial cartilage calcification,are related to inorganic phosphate ions transport and regulation.Therefore,mi RN A,thrombin,and pyrophosphate as the most popular analytes attracted many interests.The focus of this thesis is to construct biosensors of mi RNAs,thrombin and pyrophosphate based on resonance light scattering technology,using magnetic nanoparticles and carbon dots.There are three parts in this thesis.1.A reusable aptasensor of thrombin based on DNA machine employing resonance light scattering techniqueThe design of molecular nanodevices attracted great interest in these years.Herein,a reusable,sensitive and specific aptasensor was constructed based on an extension-contraction movement of DNA interconversion for the application of human thrombin detection.The present biosensor was based on resonance light scattering(RLS)using magnetic nanoparticles(MNPs)as the RLS probe.MNPs were coated with streptavidin which can combine with biotin labeled thrombin aptamers.The combined nanoparticles composite is monodispersed in aqueous medium.After thrombin was added to the system a sandwich structure was formed on the surface of MNPs,which induced MNPs aggregation.RLS signal was therefore enhanced,and there was a linear relationship between RLS increment and thrombin concentration in the range of 60 p M-6.0 n M with a limit of detection at 3.5 p M(3.29 SB/m,according to the recent recommendation of IUPAC).The present aptasensor can be repeatedly used for at least 6 cycling times by heat to transfer G-quadruplex conformation to single strand of DNA sequence and release thrombin,and applying the external magnetic field to capture MNPs.Furthermore,the proposed biosensor was successfully applied to detect thrombin in human plasma.2.Detection of mi RNAs based on magnetic nanoparticles using the resonance light scattering techniqueIn this work,we developed a label-free biosensor for the detection of mi RNAs through the adoption of magnetic nanoparticles(MNPs)modified with streptavidin.O n the basis of the specific affinity between biotin and streptavidin,the probes can be attached to the surface of MNPs.Due to the hybridization between probes and target mi RNA,MNPs aggregated and induced the increment of RLS signal.In addition,the RLS signal increased with increasing concentrations of the target mi RNA.This method exhibits ultrahigh sensitivity toward mi RNA with a dynamic range of 3.0-300 p M.The present biosensor can discriminate the target sequence from even single-base mismatched sequence or other mi RN A sequences.The result indicates that the propsed biosensor could become a promising mi RNA detection method in early clinical diagnostics and biomedical research.3.Construction of an off-on fluorescence system based on carbon dots for trace pyrophosphate sensingA novel and simple fluorescence O ff-On system is proposed for selective pyrophosphate(PPi)sensing in an aqueous solution.The method is constructed based on the strong blue emission of carbon dots(C Ds)owing to its outstanding photoluminescence and easy synthesis.The fluorescence of C Ds can be remarkably quenched bysome transition metal ions such as C u2+,N i2+,Mn2+ and Co2+ due to the coordination reaction between metal ions and the carboxylic groups on the surface of C Ds.When PPi was introduced to CDs-metal ion system the fluorescence of CDs was recovered regularly.The increment of fluorescence intensity was proportional with the concentration of PPi in the range of 1–200 ?M and correspondingly the limit of detection was calculated as 0.32 ?M according to the recommendation of IUPAC as 3.29 SB/m.Furthermore,the proposed system was successfully used to monitor the content of PPi in water samples from artificial wetland.