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Fabrication Of Highly Sensitive Electrochemical Nucleic Acid-Based Biosensor And Detection Performance

Posted on:2016-06-08Degree:DoctorType:Dissertation
Country:ChinaCandidate:L WangFull Text:PDF
GTID:1108330461993477Subject:Applied Chemistry
Abstract/Summary:PDF Full Text Request
It would be of significant importance for the early diagnosis and clinical treatment toward some major diseases by fabricating the highly sensitive nucleic acid-based biosensing platform. It further constituted the key issues to achieve the highly sensitive and selective detection toward some important disease-relevant biomolecules including DNA, protein and small biomolecules, etc. In the current dissertation, several new electrochemical biosensors were developed based on the proposed nucleic acid-based sensing and signal amplification strategies. It was further used for the achievement of highly sensitive and selective detection toward DNA、protein and small molecules. The obtained main experimental results could be concluded as below:1. The dendritic gold nanospheres were firstly prepared with the reduction of HAuCl4 by using 3-(4-aminophenyl) propionic acid as the reductant at a room temperature. Then, the electrochemical DNA biosensor was fabricated with the modification of the dendritic gold nanospheres on the electrode surface. The current dendritic gold nanospheres modified electrode possessed a large surface area. Thus it could evidently increase the immobilization amount for probe DNA and further hybridization efficiency toward target DNA. The detection limit toward target DNA could be obtained as 1 pM, which was about three orders of magnitudes better than that of unmodified electrode. Also, the obtained dendritic gold nanospheres by a one-step method showed the better stability. Furthermore, the regeneration ability of the fabricated biosensor was investigated. It could be found that the biosensor still possessed the good recognition performance after its regeneration for three times. The detection performance toward target DNA was further upgraded with the combination of enzymatic amplification strategy of alkaline phosphatase.2. In this section, a new strategy for the highly sensitive electrochemical detection of DNA was proposed by combining hybridization chain reaction (HCR):metallization of DNA template and solid-state electrochemical detection techniques. The probe DNA was firstly assembled onto the electrode surface and then hybridized with target DNA. The linked target DNA could further propagate the alternative hybridization of two hairpin DNAs to form the DNA concatamers by hybridization chain reaction technique on the electrode. Then the DNA concatamers could be used as the DNA templates for the electrostatic adsorption of silver ions. The reduced silver ions were then reduced to be silver nanoclusters or particles. The electrochemical signal could be directly recorded by the solid-state transformation reaction of Ag and AgCl, which corresponded to the recognition information of target DNA. The detection limit toward target DNA was obtained as 10 fM, which was about upgraded for four orders of magnitude compared with that in the absence of HCR amplification. Also it demonstrated a high selectivity toward target DNA and could effectively discrimate the complementary, non-complementary and even single-base mismatched target DNA.3. The electrochemical aptasensor for the detection toward the model analyte of ATP was fabricated by using a split aptamer strategy. The dendritic DNA concatamers were autonomously formed by the hybridization chain reaction of the designed three auxiliary DNA probes. It was then used as the signal carrier by electrostatic adsorption of RuHex and played the signal amplification role toward target DNA detection. It could achieve the sensitive detection toward the ATP with a low detection limit of 20 fM and a linear detection range of 6 orders of magnitudes. It also demonstrated the selective detection toward ATP compared with the ATP analogues. The comparable responses were obtained for the detection of ATP in both buffer and the diluted fetal bovine serum, indicating the potential for real analytical application.4. The levels of thrombin concentration have been well-known to be closely associated with some diseases, including thromboembolic disease and Alzheimer’s disease. Herein, two unique hairpin DNAs including recognition probe and signal probe were designed. The recognition probe contains the aptamer segment for thrombin. In the presence of thrombin, it could be recognized by the aptamer and the recognition probe was opened. Then the liberated T-DNA segment could easily hybridize with the signal probe to trigger the successive cleavage process by Exo Ⅲ toward the signal probe, accompanied with the release of the ferrocene-labeled mononucleotide for the amplified electrochemical signal toward thrombin detection. Furthermore, following with the Exo III cleavage process, the T-DNA segment could be recycled and the new secondary DNA segment with the same role of T-DNA segment could be generated for the cascade cleavage toward signal probe. The strategy could be denoted as an Exo Ⅲ-assisted autocatalytic sensing strategy. It demonstrated the high sensitivity and selectivity toward thrombin detection. The low detection limit toward thrombin was as low as 5 pM. It should be also pointed out that the developed electrochemical method was homogenous operation and avoided the immobilization procedure of DNA on the electrode surface.5. The label-free and ultrasensitive electrochemical assay for T4 polynucleotide kinase phosphatase (PNK) activity was developed, which took full advantage of three enzymes including polymerase, nicking endonuclease and Lambda exonuclease to realize the signal amplification. The assembled hairpin DNA probe with a 3’-phosphoryl end on the electrode surface could resist the polymerase elongation reaction. Also the electron transfer of ferricyanide toward the electrode surface was largely inhibited. In the presence of PNK, the hairpin DNA could be dephosphorylated into a 3’-hydroxyl group, which then could serve as the preferred substrate for DNA polymerase. Following with the polymerase elongation reaction, a long double-helix hairpin product was produced, which could be recognized and cleaved by nicking endonuclease. The duplex hairpin DNA was removed from the electrode, resulting in the enhancement of the diffusion of ferryanide towards the electrode surface. The further digestion of the dissociated duplex hairpin DNA by lambda exonoclease lead to the release of the corresponding complementary strands, which could then hybridize with another hairpin DNA to form the partially complementary DNA duplexes. It then triggered a new cleavage and digestion recycle. Following this principle, the PNK activity could be achieved for amplified detection. The detection limit toward DNA PNK could be obtained as 1 mU/mL.
Keywords/Search Tags:electrochemical biosensor, DNA, target recycling, aptamer, signal amplification
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