Studies On The New Methodology Of DNA Electrochemical Biosensors

With improved understanding of human gene structure and function, and the development of the Human Genome Project, DNA separation and analysis has taken a more and more important role in the areas of clinical diagnosis, medicine, epidemic prevention, environmental protection and bioengineering. Wide-scale genetic testing requires the development of easy-to-use, fast, inexpensive, miniaturized devices. Many new biological technologies emerged and found their applications in this field. Among them, DNA biosensors are rapidly developed and have received considerable attentions. DNA electrochemical biosensor is a novel and developing technique that combining biochemical, electrochemical, medical and electronic techniques with the advantages of being simple, reliable, cheap, sensitive and selective for genetic detection, and has been a hot topic in the field of biochemistry and medicine.Nanotechnology is opening new horizons for the application of nanoparticles in analytical chemistry. Owing to unique physical and chemical properties, nanoparticles receive considerable interest and have been used in the fields of catalysis, optical absorption, medicine, magnetic medium, new materials synthesis. Such properties offer excellent prospects for chemical and biological sensing. The power and scope of such nanoparticles can be greatly enhanced by coupling them with biological recognition reactions and electrical processes (i.e. nanobioelectronics). Nanoparticle-biopolymer conjugates 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 the material science, biological science, information technology and nanometer technology, it has been the supramolecular science and has been employed as a vital method to design, and 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. Cyclodextrin (CD), as the most important host, have received considerable attention because of its particular characterization, and the studies on the host-guest interaction based on CD 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 analysis, medicine, environment protection and sensors.The goal of the present study is to design novel DNA hybridization detection techniques with high sensitivity and selectivity. This dissertation focuses on fabricating novel electrochemical DNA biosensors based on host-guest recognition technology, combining electrochemical analysis technique and nano-materials, thus developing a sensitive, sequence-specific and quantifiable gene detection method, and establishing the bases, especially one base mutation, then for application of electrochemical DNA biosensor to clinic diagnose. And based on protein-induced strand displacement, we constructed a novel protein electrochemical biosensor.Firstly, we introduce the DNA biosensor, including its principle (probe identification principle and immobilization method of ssDNA on solid support) and its electrochemical label. Among these, we emphatically review the principle, progress, the application and development trends of electrochemical DNA biosensors. Second, we introduce the principle of aptamer biosensor, including selex technique and application. The application of carbon nano-tube on biosensors was introduced. At last, we pointed out the purpose and significance of the 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 bind, and a DNA labeled CdS nanoparticles (DP-CdS) was used as a detection probe. When the so 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 was released one single strand and returned to IP strand which consequently hybridized with DP-CdS. After dissolving the captured CdS particles 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 was 4.3×10-13 mol/L of thrombin. 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 report on a new electrochemical method to detect the hybridization specificity by using host-guest recognition technique. A hairpin DNA with dabcyl-labeled at its 3' and NH2 group at 5' terminal was combined with CdS nanoparticle to construct a double-labeled probe (DLP), which could selectively hybridize with its target DNA in homogenous solution. Aβ-CD modified Poly(N-acetylaniline) glassy carbon electrode was used for capturing the dabcyl label in DLP. When without binding with target DNA, the DLP kept its stem-loop structure which shielded the dabcyl molecule due to the loop of the hairpin DNA and CdS nanoparticle blocking dabcyl enter into the cavity of theseβ-CD molecules on the electrode. However, in present of complementary sequence, the target-binding DLP was incorporated into double stranded DNA, causing the DLP's loop-stem structure opened and then the dabcyl was easily captured by theβ-CD modified electrode. During electrochemical measurement, the signal from the dissolved Cd2+ was used for target DNA quantitative analysis.We report a new strategy for electrochemical DNA detection in homogeneous solution based on the host-guest molecule recognition technique. In this sensing protocol, a novel dually-labeled DNA probe (DLP) in a stem-loop structure was employed, which was designed with dabcyl labeled at one end as a guest molecule, and with Au nanoparticle labeled at the other end as electrochemical tag to indicate the hybridization occurrence. Oneα-CD/MCNTs/GCE was used for capturing the DNA hybridization and electrochemical signal transduction. Before the hybridization, the DLP remained in the stem-loop structure, which forced the dabcyl molecular to be closed to the Au nanoparticle. Due to the steric effect of the Au nanoparticle, the dabcyl was prevented from conjugating with theα-CD on the electrode and resulting in that the DLP could not be captured by the electrode. After hybridized with the target DNA, the target-binding DLP caused the DLP's loop-stem structure opened and then the dabcyl molecule was easily entering the cavity of the a-CD modified electrode and resulting in that the DLP could be captured by the a-CD modified electrode and the capture efficiency was proportion with the concentration of the target DNA. Therefore, the target hybridization event can be sensitively transduced via detecting the electrochemical reduction current signal of AuCl4- of Au nanoparticles labeled at the DLP. By using this strategy, as low as 2.6×10-10 M DNA target could be detected with excellent differentiation ability for even single mismatch.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.