| DNA analysis has been more and more important in the areas of medicine; clinical diagnosis;environmental protection;epidemic prevention and bioengineering with improved the development of the Human Genome Project and understanding of human gene structure and function.Development of easy-to-use, fast, inexpensive, miniaturized devices are required for wide-scale genetic testing. In this field, various new biological technologies emerged and found their applications, and among them, DNA biosensors have received considerable attention due to their advantage in many aspects.As a novel and developing technique, electrochemical DNA detection combines electrochemical, medical,biochemical and electronic techniques with the advantages of being simple, reliable, cheap, sensitive and selective for genetic detection, and thus has been one of hot topics in the fields of biochemistry and medicine.Nanotechnology has opened new horizons for the application of nanoparticles in analytical chemistry. Owing to their unique physical and chemical properties, nanoparticles receive considerable interest and have been widely used in the fields of catalysis, medicine, optical absorption, magnetic medium, new materials synthesis an d biological sensing. By coupling them with biological recognition reactions and electrical processes (i.e. nanobioelectronics), the power and scope of such nanoparticles can be greatly enhanced, e.g. nanoparticle-biopolymer conjugates could offer great potential for DNA diagnostics.The molecular recognition technology, defined as the supramolecular noncovalent interaction between the "host" and "guest" molecules, has played an important role in the chemical sensing field. Combing with material science, information technology, nanometer technology and biological science, supramolecular science has been employed as a key method to prepare new materials and obtain novel properties.Therefore,supramolecular chemistry is believed to be the base of new concept and technology in the 21st century. Cyclodextrins (CDs),as the most important host molecule, have received considerable attention due to its particular characterization. Till now, the studies focusing on the host-guest interaction had been transferred from the processes and mechanism of inclusion complex between a pair of host and guest to the application in the fields such as medicine, analysis, environment protection and sensors.The objective of present study is to develop novel DNA hybridization and protein detection techniques with high sensitivity and selectivity. This dissertation centers on fabricating novel electrochemical DNA and protein biosensors that are based on displacement mechanism and host-guest recognition technology, combining electrochemical analysis method and nanoparticle technique, and thus developing sensitive, sequence-specific and quantifiable gene detection methods.Firstly, we introduced the development of DNA biosensor, including its principle (probe identification principle and immobilization procedure of ssDNA on solid support) and classification. Among those, we emphatically reviewed the principle, development, application and trends of electrochemical DNA biosensors. Secondly, the application of host-guest recognition technique on biosensors was introduced. At last, we pointed out the purpose and significance of this dissertation.A sensitive electrochemical aptasensor for detection of thrombin based on target protein-induced strand displacement is presented. For this proposed aptasensor, dsDNA which was prepared by the hybridization reaction of the immobilized probe ssDNA (IP) containing thiol group and thrombin aptamer base sequence was initially immobilized on the Au electrode by self-assemble via Au-S bond,and a DNA labeled CdS nanoparticles (DP-CdS) was used as the detection probe. When such prepared dsDNA modified Au electrode was immersed into a solution containing target protein and DP-CdS,the aptamer in the dsDNA preferred to form G-quarter structure with the present target protein and the dsDNA sequence released one single strand and returned to IP strand which consequently hybridized with DP-CdS.After dissolving the captured CdS nanoparticles from the electrode, a mercury-film electrode was used for electrochemical detection of these Cd2+ ions which offered sensitive electrochemical signal transduction. The peak current of Cd2+ ions had a good linear relationship with the thrombin concentration in the range of 2.3×10-9-2.3×10-12 mol/L and the detection limit of thrombin was 4.3×10-13 mol/L.The detection was also specific for thrombin without being affected by the coexistence of other proteins, such as BSA and lysozyme.A competitor-switching electrochemical sensor based on a generic displacement strategy was designed for DNA detection. In this strategy, an unmodified single-stranded DNA (cDNA) completely complementary to the target DNA served as the molecular recognition element, while a hairpin DNA (hDNA) labeled with a ferrocene (Fc) and a thiol group at its terminals served as both the competitor element and the probe. This electrochemical sensor was fabricated by self-assembling a dsDNA onto a gold electrode surface.The dsDNA was pre-formed through the hybridization of Fc-labeled hDNA and cDNA with their part complementary sequences.Initially, the labeled ferrocene in the dsDNA was far from surface of the electrode,the electrochemical sensor exhibited a "switch-off" mode due to unfavorable electron transfer of Fc label. However, in the presence of target DNA, cDNA was displaced from hDNA by target DNA, the hairpin-open hDNA restored its original hairpin structure and the ferrocene approached onto the electrode surface, thus the electrochemical sensor exhibited a "switch-on" mode accompanying with a change in the current response.The experimental results showed that as low as 4.4×10-10 mol/L target DNA could be distinguishingly detected, and this method had obvious advantages such as facile operation, low cost and reagentless procedure.We herein constructed a sensor that converts target DNA hybridization-induced conformational transformation of the probe DNA to electrochemical response based on host-guest recognition and nanoparticle label.In the sensor, the hairpin DNA terminal-labeled with 4-((4-(Dimethylamino)phenyl)azo)benzoic acid(dabcyl)and thiol group was immobilized on Au electrode surface as the probe DNA by Au-S bond, and the CdS nanoparticles surface-modified withβ-cyclodextrins (CdS-CDs) were employed as electrochemical signal provider and host-guest recognition element. Initially, the probe DNA immobilized on electrode kept the stem-loop configuration, which shielded dabcyl from docking with the CdS-CDs in solution due to the steric effect. After target hybridization, the probe DNA underwent a significant conformational change, which forced dabcyl away from the electrode. As a result, formerly-shielded dabcyl became accessible to host-guest recognition betweenβ-cyclodextrin (β-CD) and dabcyl, thus the target hybridization event could be sensitively transduced to electrochemical signal provided by CdS-CDs. This host-guest recognition-based electrochemical sensor has been able to detect as low as picomolar DNA target with excellent differentiation ability for even single mismatch. |
PDF Full Text Request