Study On The Detection Of Sialic Acid-related Biomarkers Based On Aptamers And Quantum Dots

Sialic acid is a group of acidic monosaccharides with a 9-carbon skeleton.More than 50sialic acids with diverse structures have been found in nature,among which N-acetylneuraminic acid（Neu5Ac）,N-hydroxyacetylneuraminic acid,Ketodeoxynonulosonic acid and Neuraminic acid are the most common sialic acids,which are widely found in the glycan of mammalian cells and microorganisms.Many biological processes,such as intercellular communication,intercellular interaction,immune response and microbial infection,are closely related to the structure and expression level of sialic acid and sialicoglycan,so the structural identification and quantitative analysis of sialic acid and sialoglycan are very important.However,traditional detection methods such as high performance anion exchange chromatography,mass spectrometry,high performance liquid chromatography and flow cytometry have certain limitations.In this paper,a variety of biosensors based on aptamers and quantum dots（QDs）were developed to detect sialic acid related biomarkers with high sensitivity by taking advantage of the excellent characteristics of aptamers and QDs.The research contents are as follows:1.Based on the advantages of magnetic beads SELEX（MB-SELEX）for screening small molecular target aptamers,a ds DNA library immobilized MB-SELEX platform was constructed for screening the aptamers of polysialic acid（PSA）,sialyzed Lewis acid x（SLe^x）and 6’-sialyllactose（6’-SL）.A large number of candidate ss DNA aptamers were obtained after several rounds of screening and high-throughput sequencing.DNAMAN software was used to conduct homology comparison of candidate aptamers,and representative sequences were selected for affinity and specificity characterization according to Gibbs free energy and the number of repeated sequences.The 79-nucleotide length sequence PSA-Apt3(K_d=114.0nmol·L^-1),the 79-nucleotide length sequence SL-Apt9(K_d=152.3 nmol·L^-1)and the80-nucleotide length sequence SLe^x-Apt2(K_d=23.01 nmol·L^-1)(Kd=23.01 nmol·L^-1)were the best candidate for PSA,6’-SL and SLe^x,respectively,which were determined by affinity and specificity verification.2.Based on the structure prediction,the truncation optimization of the aptamer sequences was carried out systematically.Molecular docking and truncation optimization experiments were conducted for PSA and 6’-SL aptamers obtained by screening,and 3D models of target and aptamer were established.The binding sites of target and aptamer were predicted by molecular simulation docking,and then the retention area after truncation of aptamer was designed according to the binding sites.Finally,the affinity and specificity of truncated sequences were determined by GO adsorption method.The 30-nucleotide length sequence PSA-Apt3-1(K_d=282.7 nmol·L^-1),the 12-nucleotide length sequence PSA-Apt3-3(K_d=277.3 nmol·L^-1),and the 32-nucleotide length of SL-Apt9-1(K_d=91.75 nmol L^-1)were obtained.In addition,aptamer dimer and trimer were designed and constructed for the truncated PSA aptamer sequence.The affinity and specificity of homologous dimer（PSA-Apt3-4 and PSA-Apt3-5）,heterodimer（PSA-Apt3-6,PSA-Apt3-7 and PSA-Apt3-8）and trimer（PSA-Apt3-9）were evaluated.PSA-Apt3-7(K_d=80.58 nmol·L^-1)was the best dimer aptamer.3.Sialic acid related biomarker detection methods based on Cd Se/Zn S QDs and aptamer were constructed.Neu5Ac specific RNA aptamer reported in literature and PSA and 6’-SL specific ss DNA aptamers screened in this study were used to construct three fluorescence resonance energy transfer（FRET）based fluorescent biosensors for the detection of Neu5Ac,PSA and 6’-SL.The sensor combines the excellent optical properties of Cd Se/Zn S QDs,Exo III assisted cyclic amplification and catalytic hairpin self-assembly（CHA）signal amplification technology to amplify the detection signal of the target.The results showed that the linear range of Neu5Ac detection by FRET system based on Cd Se/Zn S QDs and Exo III assisted cyclic amplification was 0.2-12.5 pmol·L^-1 and 12.5-1000 pmol·L^-1,and the detection limit was as low as 24 fmol·L^-1.The method was applied to milk and serum samples with high recovery rates（98.8%-104.4%）.Meanwhile,a fluorescent biosensor based on FRET between fluorescein Cy5 and Cd Se/Zn S QDs and CHA signal amplification was constructed by using the screened PSA aptamer PSA-Apt3 for PSA detection.The linear range of the fluorescent biosensor was 10 pmol·L^-1-1μmol·L^-1,the detection limit was 0.63 pmol·L^-1,and the recovery rates in serum samples were 98.53%-102.9%.The biosensor has the characteristics of wide detection range,low detection limit and good anti-interference performance.A novel fluorescent biosensor based on FRET between fluorescein Cy5 and Cd Se/Zn S QDs and CHA signal amplification was constructed by using the screened DNA aptamer SL-Apt9 for ultra-sensitive detection of 6’-SL.The linear range of the biosensor was10-75 nmol·L^-1,the detection limit was 0.3 nmol·L^-1,and the recovery rates of milk samples is 92.0%-103.2%.The biosensor has the characteristics of high Signal-to-noise ratio,short time consuming,high efficiency,simple operation and low detection limit.The biosensors constructed above have great application potential in the fields of medical diagnosis and food detection.4.Sialic acid related biomarker detection methods based on aptamer bio-dots were constructed.The SLe^x ss DNA aptamer screened in this paper and the PSA and 6’-SL ss DNA aptamers obtained by truncation optimization were used to construct three biosensors based on aptamer bio-dots.Firstly,a 6’-SL fluorescent biosensor based on aptamer bio-dots and aminobenoborate functionalized carbon quantum dots was developed.The linear range of the biosensor was 10μmol·L^-1 to 5 mmol·L^-1,and the detection limit was 0.9μmol·L^-1.The method was applied to detect 6’-SL in milk samples,and the recovery rates were94.8%-105.4%under the best experimental conditions.Secondly,a SLe^x fluorescent biosensor was constructed by aptamer bio-dots and aminobenzenoborate modified magnetic bead.The linear range of the biosensor was 100μmol·L^-1-2 mmol·L^-1,and the detection limit was 32μmol·L^-1.The recovery rates of SLe^x were 96.62%-103.75%under the optimum experimental conditions.Finally,a dimer aptamer bio-dots was constructed to study PSA imaging on cell surface.PSA-Apt3-7 bio-dots was applied to detect human embryonic kidney cell 293,human breast cancer cell 468,human breast cancer cell 231 and colorectal adenocarcinoma cell CACO-2.It has good imaging effect and can clearly distinguish cancer cells from non-cancer cells.In this paper,the ss DNA aptamers of PSA,SLe^x and 6’-SL were screened by MB-SELEX,and the truncation and optimization of the aptamers were systematically studied based on structural prediction.Meanwhile,a series of sialic acid related biomarker detection methods were constructed by QDs and aptamers.The effects of inorganic quantum dots,bio-dots and signal amplification techniques on signal detection were studied to achieve high sensitivity detection of sialic acid related biomarkers.Finally,the constructed biosensors were applied to the detection of sialic acid related biomarkers in milk,serum samples and cells.The results show that the constructed biosensors have broad application potential in food field and medical imaging research.