Research Of Electrochemical Biosensing In Detection Of Single Base Mutation And Proteins

It's of great significance to establish rapid and efficient detection techniques of nucleic acids and proteins in environmental monitoring, food engineering, biomedical researches and clinical diagnosis. Electrochemical biosensing technologies, because of high sensitivity, simple implementation, and low cost, is currently widely used in scientific researches. In nucleic acids and proteins detection, the use of nucleic acid hybridization, immunoreaction and small molecule-protein interaction offers desirable selectivity for the assays. Meanwhile, the applications of electroactive materials and nanomaterials as signal reporters has provided enormous possibilities of sensitivity enhancement. Electrochemical biosensors for nucleic acids and proteins have been developed in the present thesis:(1) Based on S1 nuclease'cleavage, an electrochemical sensor for the single base mutation detection is constructed with ferrocene as an electroactive lable in Chapter 2. Detection probe with ferrocene (Fc) tag at the 3'end hybrids with wild-type and mutant target, forming noncomplementary and complementary sequence respectively at the 3'end. S1 nuclease cuts the Fc tag at noncomplementary hybrids, but holds it at complementary hybrids. After denaturation, only Fc-reserved detection probe captured by gold electrode can response electrochemical signal, leading to the recognition of single base mutation.(2) With the unique interaction (affinity) between small organic molecules and their protein receptors, a single-stranded DNA (ssDNA) terminally tethered to a small molecule is protected from the degradation by exonuclease I (Exoâ… ) in the presence of specific protein. Such an Exoâ… applied ssDNA terminal protection assay is established in Chapter 3. A ssDNA (detection probe) is labled by Fc at the 5'end and biotin at the 3'end, the new-coming streptavidin connects with biotin, resulting large steric, avoiding the degradation of Exoâ… . This ssDNA is then captured by the fixed probe on the gold electrode and electrochemical response will be obtained.(3) An electrochemical immunosensing technology is developed based on phospholipid-modified MWNTs (PL/MWNTs) as a signal regulator in Chapter 4. MWNTs is modified with phospholipid which is cross-linked to the monoclonal antibody(MAb) of tumor marker prostate specific antigen (PSA). After the sandwich reaction, MWNTs/MAb is collected on the eletrode modified with octadecyl mercaptan by magnetic separation. The deposited conductive MWNTs alter the electrical situation on the electrode, and then produce electrochemical signal to the dynamic range of 5 pg/mL to 100 ng/mL of PSA with low detection limit (3 pg/mL). This technology also is ideally responsed to the real sample.