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Study And Application Of Biosensors Based On The Nucleic Acid

Posted on:2015-10-22Degree:DoctorType:Dissertation
Country:ChinaCandidate:X Y GaoFull Text:PDF
GTID:1108330461969582Subject:Food safety and pharmaceutical chemistry
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Based on the diversity of structure, nucleic acid plays an important role in the life processes, and it has high stability, ease synthesis and modification. This thesis study some new nucleic acid biosensors and their application.The thesis has been divided into six chapters, and main contents are listed as follows:In chapter 1, the concept and characteristics of nucleic acid and one of the functional nucleic acid (aptamer) was described in detail, and their applications in biosensor fields were introduced in brief also. Meanwhile, several types of biosensors and their applications were summarized. In the end, the purpose and signification of this thesis were described.In chapter 2, the intermolecular i-switch based on i-motif structure was fabricated. In the study, two C-rich strands were employed, and one of the strand with thiol at 5’-terminus was immobilized on the gold electrode through the thiol-Au interaction, and ferrocene (Fc)-labelled strand hybridized with the above strand to form i-switch nanostructure. In neutral or basic aqueous solution, the i-switch was present in the form of an extended duplex that made Fc far away from the electrode a weak faradaic current was obtained, so-called "OFF " state. While in the slight acidic condition, an obvious signal was detected from Fc which approached the electrode due to the formation of i-motif, that was " ON " state. And the faradaic current had a linear relationship with the pH value in the range of 5.8-8.0. This proposed switch had lower background; moreover, based on it, a possible platform for pH measurement had been established.In chapter 3, a sensitive fluorescence sensor was developed for sequence-specific recognition of dsDNA in vitro using metal-organic framework (MOF, H2dtoaCu). This material could strongly chemisorb the dye-labeled probe triplex-forming oligonucleotide (TFO), and quench fluorescence from the dye. While in the presence of the target dsDNA, the TFO could interact with the major groove in dsDNA (via Hoogsteen hydrogen bonding) to form a rigid triplex structure, resulting in the fluorescent recovery. The enhanced fluorescence signal had a relationship with the dsDNA concentration, the detection limit is as low as 1.3 nmol/L(S/N=3) with well selectivity, which was lower than that based on the graphene oxide platform and the electrochemical-DNA sensor.In chapter 4, a simple and label-free DNA electrochemical impedance biosensor had been developed for the sequence-specific recognition of dsDNA. The probe triplex-forming oligonucleotide (TFO) was immobilized on the gold electrode through Au-S interaction. In the presence of the target dsDNA, the probe TFO interacted with the bases in the major groove of the dsDNA to form a rigid triplex structure leading to an increase of the electron-transfer resistance. It was found that the change of impedance had a linear relationship with the concentration of the target in the range of 0.1~40 nmol/L, and the detection limit as low as 0.04 nmol/L(S/N=3).In chapter 5, a label-free hyperbranched rolling circle amplification (HRCA) based fluorescence method had been developed for Hg2+ detection with high sensitivity and specificity. In the presence of Hg2+, two termini of the padlock probe were perfectly hybridized with the complementary trigger probe through the stable T-Hg2+-T biomimetic structure, which induced the 5’ends and 3’ ends of the padlock probe brought into proximity and generated a circular padlock probe with the assistant of E. coli DNA ligase. Subsequently, the circularized padlock probe was served as a template for the initiation of HRCA reaction. The HRCA product contained large amounts of various lengths dsDNA, which could be detected using SYBR Green I to achieve a strong fluorescence. It was found that the fluorescent intensity has a linear relationship with the concentration of target in the range of 4.25×10-13~4.25×10-8 mol/L, and the detection limit was as low as 1.4×10-13mol/L (S/N=3).In chapter 6, an electrochemical biosensor based on the GCE-APTES-rGO electrode had been fabricated for thrombin detection. Firstly, the complementary strand of thrombin aptamer was immobilized on the GCE-APTES-rGO electrode surface via covalent bonding, and Fc-labeled thrombin aptamer hybridized with the immobilized complementary sequence to fabricate DNA sensor. In the absence of thrombin, Fc approached the electrode surface, and an obvious faradaic current could be obtained. While in the presence of thrombin, Fc-labeled strand would combine with thrombin, resulting in the reduction of the faradaic current. By this means, the concentration of thrombin in solution can be obtained indirectly, and the detection was 0.03 nmol/L (S/N=3). Moreover, this sensor had good stability.
Keywords/Search Tags:nucleic acid, functional nucleic acid, sensor, aptamer, fluorescence, electrochemistry, MOF
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