| The biosensor is a cross-field involving biology, chemistry, materials and nano, micro-electronics, information technology and other subjects, and have some significant characteristics, such as, rapid, accurate, high selectivity. Whether it’s mechanism biological electron system research, or practical applications, investigators who at home or abroad show great interesting in the biosensors research. With further research and the completion of gene structure and function of the human genome project, marking the development of life sciences has officially entered the post-genome era. The main object of study in the life sciences is functional genomics, including genomic and proteomic research studies and DNA and proteins are basic research unit in genomics and proteomics is. DNA aptamer can specifically bind a variety of targets to form flexible molecular recognition and signal conversion mechanism, so some excellent performance new biosensors based on DNA aptamer probes were developed and widely applied in gene analysis, disease diagnosis, environmental monitoring, food safety and other fields. In this paper, We use DNA probes to construct some simple biosensors for thrombin, PDGF-BB and pesticides detection.This Paper is divided into four parts:Chapter Ⅰ:PrefaceThe biosensor basic structure, including principle, classification and application fields and prospects are introduced. Biosensors are divided into two classes, one is aptamer biosensor, which is highlighting the principle of configuration and DNA conformational changes in DNA and related case; and the other is acetylcholinesterase activity inhibited biosensor which include two types biosensors.Chapter Ⅱ:A signal-on electrochemical biosensor based on triple-helix molecular switch for thrombin detectionIn the present study, a versatile "signal-on" electrochemical aptasensor based on a triple-helix molecular switch has been developed. An aptamer probe is designed to hybridize with the methylene blue (MB)-modified DNA capture probe immobilized on the gold electrode to form rigid triple-helix DNA, impeding the efficient electron transfer of MB to the electrode and resulting in the decreased oxidation peak current of MB. However, upon introduction of the perfectly matched target, for example, human a-thrombin (Tmb), the interaction between Tmb and the aptamer probe leads to the dissociation of the triple-helix DNA structure and thereby liberates the MB-modified end of the capture probe, allowing the MB to collide with the electrode surface and resulting in an increase of the oxidation peak currents of MB. Therefore, the sensitive signal-on detection of Tmb is realized, linear range:0.5 nM～10μM and the detection limit of Tmb is 0.12 nM. The proposed approach also demonstrates excellent regenerability, reproducibility and stability. Additionally, it also has the advantages of simplicity in design and easy operation.Chapter Ⅲ:A free enzyme and free labeled strategy based on DNA conformational changes for sensing platelet-derived growth factorIn this charpter, a novel fluorescent aptasensing strategy for protein assay has been developed based on target-triggered hybridization chain reaction (HCR) and graphene oxide (GO)-based selective fluorescence quenching. Three DNA probes, a helper DNA probe (HP), hairpin probe 1 (H1) and hairpin probe 2 (H2) are ingeniously designed. In the presence of the target, the aptamer sequences in HP recognize the target to form a target-aptamer complex, which causes the HP conformation change, and then triggers the chain-like assembly of H1 and H2 through the hybridization chain reaction, generating a long chain of HP leading complex of H1 and H2. At last the fluorescence indicator SYBR Green I (SG) binds with the long double strands of the HCR product through both intercalation and minor groove binding. When GO was added into the solutions after HCR, the free H1, H2 and SG would be closely adsorbed onto GO surface via π-π:stacking. However, the HCR product cannot be adsorbed on GO surface, thereby the SG bound to HCR product gives a strong fluorescence signal dependent on the concentration of the target. With the use of platelet-derived growth factor BB (PDGF-BB) as the model analyte, this newly designed protocol provides a highly sensitive fluorescence detection of PDGF-BB with a limit of detection down to 1.25 pM, and also exhibit wide linear range:5 pM-5 nM. Therefore, the proposed aptasensing strategy based on target-triggered hybridization chain reaction amplification should have wide applications in the diagnosis of genetic diseases due to its simplicity, low cost, and high sensitivity at extremely low target concentrations.Chapter Ⅳ:Fluorescence biosensrs based on DNA conformational changes and cyclic amplification strategy for pesticide detectionThe sensor is based on metal-mediated DNA conformation changes and the enzyme triggered amplification strategy for pesticide residues detection. Two kinds of DNA, single-stranded DNA1 with T-T mismatch were designed which can form a hairpin DNA with T-Hg2+-T base pair in the presence of mercury ions. The other one is a short-chain DNA BHQ probe in which the fluorescence of the fluorophore (FAM) at one end was quenched by the quencher (BHQ1) at the other end due to the fluorescence resonance energy transfer (FRET) effect, could not only fully completely hybridize with the part of DNA1 to form a DNA duplex in case of the hairpin structure of the DNA1 was destroyed, but also include the recognition sequence as well as cleavage site for Nb.BtsI. AChE can catalyze the hydrolysis reaction of Ach to generate thiocholine containing the chemically reactive thiol group (-SH) which plays an essential role in this protocol. Due to the stronger S-Hg interaction, Hg2+ ions would release from the hairpin structure of the DNA1. As a result, the configuration of the DNA1 is destroyed and changed from the hairpin to linear strand structure. Subsequently, linear strand of the DNA1 in turn can easily hybridize with the BHQl probe to form complementary double strands structure. Once the DNA duplex formed, the nicking enzyme Nb.BtsI quickly binds to and cleaves the BHQ1 probe. So the fluorophore in BHQI probe was separated from the quencher to restore its fluorescence signal, and at the same time the Helper probe was released to hybridize with another BHQl probe to initiate the subsequent cycling cleavage process. As a consequence, significantly amplified fluorescence signals were obtained. In the presence of aldicarb, the activity of the AChE can be inhibited. The number of the thiol group by the hydrolysis reaction of Ach would be reduced, resulting in release a low concentration of Hg2+. The number of the hairpin structure of the DNA1 can be selectively opened would be reduced and the subsequent nicking enzyme-assisted cleavage processes could be affected, resulting in decreased fluorescence signals. The decreased fluorescence intensities increase with the increase of aldicarb concentration. The fluorescence intensity has a linear relationship with aldicarb pesticide concentration. The linear range is from 10 ug L-1 to 10 mg L-1 and the detection limit is 3.3μg L-1. |
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