New Methods Based On The SERS Nano-probe And Electrochemical Nano-interface For Biological Diagnosis

Early diagnosis of disease is one of the great challenges in clinical medicine.The development of high sensitivity,simple,fast and portable methods of biological diagnosis is particularly important.In recent years,with the rapid development of DNA nanotechnology and inorganic nanomaterials,new ideas have been emerged for the development of new biological diagnostic methods.SERS spectroscopy is widely used in bioanalysis and imaging because of its narrow peak,high sensitivity,anti-photobleaching,non-biological sample spontaneous fluorescence interference and rapid real-time in situ detection.Electrochemical detection has the characteristics of high sensitivity,fast,cheap and easy to integrate,which has alao been popular in the biosensors.In this paper,combined with biological nano-materials and the means of SERS and electrochemical detection,we have done the following four aspects,specifically: 1.Ready-to-Use SERS Probes for Ultrasensitive and Multiplex DNA/mi RNA DetectionUniform silver-containing metal nanostructures with strong and stable surface-enhanced Raman scattering(SERS)signals hold great promise for developing ultrasensitive probes for biodetection.Nevertheless,the direct synthesis of ready-to-use such nanoprobes remain extremely challenging.Herein we report a DNA-mediated gold-silver nano-mushroom with interior nanogaps directly synthesized and used for multiplex and simultaneous SERS detection of various DNA and RNA targets.The DNA involved in the nanostructures can act as not only gap DNA(mediated DNA)but also probe DNA(hybridized DNA),and allow it have the inherent ability to recognize DNA and RNA targets.Importantly,we were the first establishing a new method for the generation of multicolor SERS probes using two different strategies.First Raman-labeled alkanethiol probe DNA was assembled on gold nanoparticles and second thiol-containing Raman reporters were co-assembled with the probe DNA.The ready-to-use probes also give great potential to develop ultrasensitive detection methods for various biological molecules.2.A Multiplexing Methylation Specific Enzyme-linked Oligonucleotide Assays(MS-ELONA)for DNA Methylation AnalysisAlterations in the methylation of promoters of cancer-related genes play an important role in carcinogenesis and the level of methylation has a potential for the early cancer diagnosis.Here we report a multiplexing and ultrasensitive assay,MS-ELONA for detection and quantification of DNA methylation.Bisulfite treatment of DNA is amplified with primers specific for methylated DNA,then the DNA double strands was immobilized on the screen-printed carbon electrode(SPCE).We could detect as little as 2 pg of methylated DNA in the presence of a 100000-fold excess of unmethylated alleles.We also demonstrate the reversibility of DNA methylation modification by treatment with demethylation drugs.The platform can be used for the screening of antitumor drug and DNA methylation of tumor markers With 16-channel electrochemical work station our research reveals a simple,inexpensive and multiplex method to quantify the methylation level of specially appointed genes,and have the potential to be applied in the clinical research.3.Smartphone based Point of Care Test for Directly Detecting Serum-derived Exosomes and Monitoring the Secretion of ExosomesExosomes are small membrane-bound phospholipid nanovesicles actively secreted by mammalian cells and carry the parental information,which play an important role in the intercellular communication.Exosomes could also serve as the potential biomarkers and could be used for cancer diagnosis and prognosis prediction.Herein we developed an ultrasensitive and portable electrochemical approach in combination with smartphone for quantitative analysis of exosomes.With the poly-HRP based signal amplification,we can detect as little as 7.23 ng exosomes in 5 ?L volume within 2 h.We could also directly capture the exosome in 5 ?L serum and the cell culture supernatant on the working electrode.With the CD63 positive exosome detection system,CD63 was less expressed on the exosome in the prostate patient serum,which hold great potential for diagnose of prostate cancer.Our platform could also be used to monitor the secretion of exosomes with the drug therapy.Our work provides a novel way toward point of care test for the diagnosis and prognosis prediction of diseases.4.A Carbon-based DNA Tetrahedron Nano-bio Interface for Biosensing with a High Signal-to-Noise Ratio of Various Bioactive MoleculesIn recent years,with the development of nano-science and technology,the nano-materials modified screen-printed carbon electrode has been widely used as electrochemical biosensor platform in the field of environment,food,diagnostics and other fields,because of its advantages such as simple preparation,low cost and easy integration.It is very important to construct a simple and universal electrochemical detection platform based on screen printed carbon electrode.However,the screen printed carbon electrode has a rough surface,easy to generate non-specific adsorption,and the direction of the probe on the surface of carbon is not controllable,the nucleic acid detection platform research based on screen printing carbon electrode cannot always meets the requirement,which greatly limits its application in nucleic acid detection.In this paper,we first reported that the use of a DNA tetrahedron-modified screen printing carbon electrode which can solve or partially solve the defects of the carbon interface,and can be applied for the detection of a variety of bioactive molecules.With simple and fast covalent coupling,the DNA tetrahedron can be immobilized on the carbon interface.By changing the probe sequence extending from DNA tetrahedron,it can be used in different target analysis.Here we designed gap hybridization,aptamer “sandwich” and aptamer competition reduction strategy for the detection of mi RNA141,thrombin and ATP,respectively,and high signal-to-noise ratio,sensitivity and specificity were obtained.In addition,the DNA-tetrahedron modified screen-printed carbon electrode as a general-purpose detection platform can also be successfully applied to serum samples.Therefore,the high-signal-to-noise ratio of DNA tetrahedral universal carbon interface based nano-biosensor greatly expands its application in nucleic acid research and has potential application in clinical disease diagnosis.