Study On The Fabrication Of DNA Electrochemical Biosensor And Its Applications In Biochemical Sensing

The sensitive, selective, and simple detection of trace amount of DNA fragments is of vitally importance in the research field of application in the precision medicine, clinic detection, forensic analysis, and pharmacy. DNA electrochemical biosensors have attracted great interests due to lots of merits such as low cost, high sensitivity, simple to operate, and ease to be miniaturized. In vitro nucleic acid amplification techniques are commonly employed in molecular biology research. DNA electrochemical biosensor combining nucleic acid amplification technologies with high sensitive characteristic is of great value to realize the determination of trace target DNA. In this dissertation, valuable explorations have been conducted on how to rapid construct a DNA biosensor, design a reliable and regenerated DNA biosensor, and amplify the electrochemical signal for biomolecules detection. The main contents of this dissertation are summarized as follows: 1. Rapid assembly of ssDNA on gold electrode surfaces at low pH and high salt concentration conditionsThis chapter reports a simple method to immediately functionalize ssDNA onto gold electrodes using a low pH-induced and high salt concentration solution route. Electrochemical impedance spectroscopy, cyclic voltammetry, and chronocoulometry were used to characterize the ssDNA self-assembled monolayer(SAM). The effects of pH, ionic strength, and ssDNA sequences for DNA adsorption were investigated. It was found that thiolated ssDNA can be attached to gold electrodes using a low pH-induced route in a high salt concentration solution. A synergistic effect between pH and the salt has been suggested by studying the fundamental kinetics. The surface coverage of 3.054 × 1013 molecules/cm2 in pH 3.4 buffer was higher than that of 4.574 × 1012 molecules/cm2 in pH 7.4 buffer. The ssDNA on the gold surface was functional and was able to recognize complementary DNA strands. 2. Detection of single-nucleotide polymorphisms using an ON-OFF switching of regenerated biosensor based on a locked nucleic acid- integrated and toehold-mediated strand displacement reactionAlthough various strategies have been reported for single-nucleotide polymorphisms(SNPs) detection, development of a time-saving, specific, and regenerated electrochemical sensing platform still remains a realistic goal. In this chapter, an ON-OFF switching of a regenerated biosensor based on a locked nucleic acid(LNA)-integrated and toehold-mediated strand displacement reaction technique is constructed for detection of SNPs. The LNA-integrated and methylene blue-labeled capture probe with an external toehold is designed to switch on the sensing system. The mutant-type DNA probe completes complementary with the capture probe to trigger the strand displacement reaction, which switches off the sensing system. However, when the single-base mismatched wild-type DNA probe is presented, the strand displacement reaction cannot be achieved; therefore, the sensing system still keeps the ON state. This DNA sensor is stable over five reuses. We further testify that the LNA-integrated sequence has better recognition ability for SNPs detection compared to the DNA-integrated sequence. Moreover, this DNA senor exhibits a remarkable is crimination capability of SNPs among abundant wild-type targets and 6000-fold(m/m) excess of genomic DNA. In addition, it is selective enough in complex and contaminant-ridden samples, such as human urine, soil, saliva, and beer. Overall, these results demonstrate that this reliable DNA sensor is easy to be fabricated, simple to operate, and stable enough to be readily regenerated. 3. A regenerated electrochemical biosensor for label-free detection of glucose and urea based on conformational switch of i-motif oligonucleotide probeImproving the reproducibility of electrochemical signal remains a great challenge over the past decades. In this chapter, i-motif oligonucleotide probe-based electrochemical DNA(E-DNA) sensor is introduced as a regenerated sensing platform, which enhances the reproducibility of electrochemical signal, for label-free detection of glucose and urea. The addition of glucose or urea is able to activate glucose oxidase-catalyzed or urease-catalyzed reaction, inducing or destroying the formation of i-motif oligonucleotide probe. The conformational switch of oligonucleotide probe can be recorded by electrochemical impedance spectroscopy. Thus, the difference of electron transfer resistance is utilized for the quantitative determination of glucose and urea. We further demonstrate that the E-DNA sensor exhibits high selectivity, excellent stability, and remarkable regenerated ability. The human serum analysis indicates that this simple and regenerated strategy holds promising potential in future biosensing applications 4. Guanine nanowire based amplification strategy: Enzyme-free biosensing of nucleic acidsSensitive and specific detection of nucleic acids plays a vital role in food, forensic screening, clinical and environmental monitoring. There remains a great challenge in the development of signal amplification method for biomolecules detection. Herein, we describe a novel signal amplification strategy based on the formation of guanine nanowire for quantitative detection of nucleic acids at room temperature. In the presence of analytes and magnesium ions, the guanine nanowire could be formed within 10 min. Compared to the widely used single G-quadruplex biocatalytic label unit, the detection limits are improved by two orders of magnitude in our assay. The proposed enzyme-free method avoids fussy chemical label-ling process, complex programming task, and sophisticated equipment, which might provide an ideal candidate for the fabrication of selective and sensitive biosensing platform 5. Sensitive detection thrombin based on guanine nanowire amplification strategyCreating a simple, rapid, high selectivity and high sensitivity strategy for detection of thrombin is extremely important due to the concentration of thrombin reaches to nmol L-1 in blood. In this chapter, we described a label-free and economy-free electrochemical DNA biosensor for the detection of thrombin based on guanine nanowire amplification strategy. The guanine nanowire is formed on the electrode surface in the presence of thrombin. In the absence of thrombin, guanine nanowire cannot be formed and no catalytic DNA nanochain existed on the electrode surface. This amplification strategy avoids the fuzzy labeling process and leads to a great increase of catalytic DNA\G- quadruplex amount on electrode surface. This method possesses some advantages, such as fast response, simple operation, and high specificity. At the same time, the constructing strategy of this DNA biosensor is applicable to many other aptamers and might provide a broad application prospects in the field of biomarker detection and disease diagnosis. 6. Polyepinephrine-based fluorescent organic dots and its application in intracellular metal ions sensingIn this chapter, we present a class of bio-dots, polyepinephrine(PEP)-based fluorescent organic dots(PEP-FODs) for selective and sensitive detection of Fe2+, Fe3+ and Cu2+. The PEP-FODs were derived from epinephrine via self-polymerization at relatively low temperature down to 60 ℃ with low cytotoxicity and relative long lifetime(7.24 ns). The surface morphology and optical properties of the synthesized PEP-FODs were characterized. We found that the diameters of PEP-FODs were mainly distributed in the narrow range of 2-4 nm with an average diameter of 2.9 nm. An optimal emission peak located at 490 nm was observed when the green light-emitting PEP-FODs were excited at 400 nm. It is discovered that Fe2+, Fe3+, and Cu2+ can strongly quench the fluorescence of PEP-FODs through the nonradiative electron-transfer. The detection limit of 0.16, 0.67, and 0.15 μM was obtained for Fe2+, Fe3+, and Cu2+, respectively. The independent sensing platform of Fe2+, Fe3+, and Cu2+ could be established by using NaF as a chelating agent and by regulating the reaction time between NaF and metal ions. Cell viability studies reveal that the as-prepared PEP-FODs possess good solubility and biocompatibility, making it as excellent imaging nanoprobes for intracellular Fe2+, Fe3+, and Cu2+ sensing. The developed PEP-FODs might hold great promise to broaden applications in nanotechnology and bioanalysis.