The HCR-based Fluorescent Biosensor For Detection Of Tumor Markers

Tumor markers are substances that can be distinguished from normal cells with the development of cancerous cells.They are applied to clinical diagnosis,clinical treatment and prognostic evaluation and can provide a diagnostic indicator for the early detection and treatment of tumors.Thus they have potential clinical application value.Nowadays,traditional detection methods toward tumor markers mainly include enzyme-linked immunoassay,electrochemical immunoassay,chemiluminescence immunoassay and colorimetric immunoassay.These methods have some problems such as high cost,long time-consuming,poor selectivity and low sensitivity,which make them difficult to practical application.However,tumor markers are usually low abundance and cannot be early detected by conventional methods.Therefore,constructing a simple,fast and efficient method for detecting these tumor markers has become the focus of current analysis field.In this thesis,Hybridization Chain Reaction(HCR)was combined with a fluorescent biosensor for the detection of Carcinoembryonic Antigen(CEA)and circulating tumor DNA(ct DNA),and the analysis ability of the constructed sensors was evaluated in real samples.The specific details are as follows:Chapter 1: It was introduced that the clinical value and classification of tumor markers and the research progress of CEA and ct DNA.The definition,characteristics and application in detecting tumor markers of HCR were described.In addition,the main research contents and innovation of this thesis were briefly summarized.Chapter 2: A fluorescent aptasensor based on HCR and G-quadruplex DNAzyme for the detection of CEA was constructed.In this system,Aptamer Probe(AP)composed of the CEA aptamer and an initiator was rationally designed.In the presence of CEA,the aptamer blocked in AP specifically recognized the target CEA and made the AP hairpin structure open.The AP underwent a conformation change and released the initiator.Furthermore,the initiator triggered a hybrid event between H1 and H2,and formed a catalytically active G-quadruplex DNAzyme in the presence of hemin.On the other hand,the G-quadruplex DNAzyme further catalyzed the oxidation of thiamine to emit fluorescence upon the addition of H2O2,and thus the quantitative detection of CEA is based on this fluorescence intensity changes.A good linear relationship between(F-F0)/F0 and CEA concentration in the range of 0.25-1.5 n M with a detection limit of 0.2 n M was obtained,so the constructed fluorescent aptasensor had a high sensitivity to CEA.In addition,the sensor has a good selectivity for CEA without affected by other proteins such as Ig G,AFP and PSA.Furthermore,the recovery in the real samples was in the range of 102.47%-115.33%.Chapter 3: A fluorescent DNA biosensor based on HCR was constructed to detect ct DNA.In this system,ct DNA served as an initiator of HCR and then two dual-labeled auxiliary hairpin probes(H1L and H2L)were ingeniously designed.In the absence of ct DNA,H1 L and H2 L were in a metastable hairpin structures and coexisted in the solution.Thus FAM and BHQ1 were close to each other and fluorescence was quenched due to fluorescence resonance energy transfer(FRET).In the presence of ct DNA,H1 L and H2 L hybridized via a chain reaction and the generated double-stranded DNA can emit fluorescence owing to the opening of hairpin structures and the separation of fluorophores.Therefore,ct DNA is quantitatively determined according to the change in fluorescence intensity.(F-F0)/F0 was linear with ct DNA concentration ranging from 5 to 100 n M with a detection limit of 2 n M.Thus,the developed fluorescence DNA sensor has a good analytical performance.At the same time,the sensor exhibited good selectivity without affected by the interference of other base mismatch DNA sequences.The recovery ranged from 95.52% to 109.90%.