Studies On The Employment Of Nanotechnique And Biotechnique To Fabricate Ultrasensitive Electrochemical Aptasensor

Electrochemical aptasensor, a kind of modern biosensor combines aptamer-target recognition technology and electrochemical analysis, which contains signal generation, signal detection, the specific recognition reaction. It holds advantages of the two methods above, such as simple operation, high sensitivity, low detection limit, good selectivity and reproducibility. In recent years, in order to achieve high sensitivity for disease markersin early detection of disease, the new nanomaterials and signal amplification strategy are widely used to prepare aptasensor. In this paper, different nanomaterials serve as the immobilization matrix of biomolecular. Moreover, the electrochemical sensing technology, nanotechnique based on amplification and enzymatic amplification strategies are combined to construct several new systems for the ultrasensitive detection of target protein. The research contents are mainly as follows:1. Aptamer-based highly sensitive electrochemical detection of thrombin via the amplification of grapheneExcept the fact that nanomaterials have been extensively employed to work as a promoter to increase the surface area and improve the electron transfer at the electrode interface, they could also be adopted as efficient carriers to load a large quantity of biomolecules and other elements, which could afford substantial elctrochemical signal amplification and the enhancement of performances of the biosensors. For example, biocompatible graphene sheets act as sensor platform, which can bring many advantages attributing to the unique properties of Gra:Firstly, abundant electrochemically active surface areas are provided for biomolecular immobilization. Secondly, a perfect microenvironment is afforded to retain the biological activity of biomolecules. Finally, Gra can also play a role of fast electron-transfer kinetics to effectively accelerate the electron transfer between electrode and detection molecules, leading to a more rapid and sensitive current response. Herein, we successfully fabricated a sensitive electrochemical aptasensor for the detection of thrombin with high specificity, stability and a relative low detection limit. Firstly, Nf-Gra-NiHCFNPs nanocomposites were coated onto the pretreated electrode surface and dried in air. Following that, gold nanoparticle (AuNPs) was introduced to the modified electrode by electrodeposition. The obtained AuNPs monolayer could not only be used to immobilize the thrombin aptamer but also enhance the stability of the composite film. After that, thrombin aptamer (TBA) could be immobilized on the electrode surface by Au-S covalent bond. Its advantages are as follows:on the one hand, the loading amount of TBA was increased attributing to the large specific surface of graphene (Gra). On the other hand, the detection sensitivity of proposed aptasensor was remarkably enhanced owing to the employment of Gra and AuNPs, which can facilitate the electron transfer between electroactive substance and electrode surface.2. An ultrasensitive electrochemical aptasensor for thrombin based on the triplex-amplification of hemin/G-quadruplex horseradish peroxidasemimicking DNAzyme and horseradish peroxidase decorated FeTe nanorodsIn recent years, researchers are enthusing about amplify elements linking to replace the original single amplification technology, which can amplify the signal response, and improves the detection sensitivity. Herein, we report here an aptamer based, triplex-amplified novel biorecognition system for thrombin (TB), where horseradish peroxidase mimicking DNAzyme (HRP-DNAzyme), iron telluride nanorods (FeTe NRs) and horseradish peroxidase (HRP) could amplify the signal response simultaneously. Attributing to the aforementioned amplification factors, this proposed aptasensor showed outstanding analytical performance for thrombin detection. Besides that, this sensing strategy could also provide a new model for a sandwich-type assay in aptamer-based sensing systems. AuNPs was electrodeposited onto the glass carbon electrode (GCE) surface for the following immobilization of thrombin aptamer1(TBA1). An electroactive species thionine (Thi), as the electron mediator was firstly immobilized onto the FeTe NRs decorated by gold nanoparticles (AuNPs@FeTe NRs). Thereafter, NH2-labeled secondary thrombin aptamer (TBA2), a single-stranded guanine-rich nucleic acid that was employed to form a hemin/G-quadruplex biocatalytic structure and act as a bio-recognition element, was introduced via the conjugate between AuNPs@-FeTe NRs and the amido of the aptamers. Subsequently, HRP served as a blocking reagent to block possible remaining active sites. It is worthwhile to note that FeTe NRs here not only served as a novel carrier but also a kind of horseradish peroxidasemimicking enzyme. The as-prepared aptasensor dramatically amplified the reduction peak current of Thi in the presence of H2O2.3. An aptasensing platform for simultaneous detection of multiple analytes based on the amplification of exonuclease-catalyzed target recycling and DNA concatemers Compared with the traditional single-analyte assay, the simultaneous multiplex assay could offer several potential advantages, such as shorter analytical times and procedures, smaller sample volumes required, higher test efficiencies and lower cost. Therefore, designing an electrochemical aptasensor for the simultaneous detection of multiple targets is necessary and of great importance. Until now, enzyme-catalyzed target recycling and DNA concatemers have been extensively adopted for signal amplication individually; nevertheless, research that couples the amplication capability of the two favorable methods has never been reported. Therefore, it is promising to use united amplication protocols to further enhance the sensitivity of the proposed aptasensor. In the present study, an ultrasensitive electrochemical aptasensor for the simultaneous detection of thrombin (TB) and ochratoxin A (OTA) was fabricated by using exonuclease-catalyzed target recycling and DNA concatemers for signal amplification. The previously hybridized double-stranded DNAs (SH-cTBA/TBA and SH-cOBA/OBA) were self-assembled on a gold electrode. In the presence of targets, the formation of aptamer-target complexes would lead to not only the dissociation of aptamers (TBA and OBA) from the double-stranded DNAs but also the transformation of the complementary DNAs (SH-cTBA and SH-cOBA) into hairpin structures. Subsequently, owing to employment of RecJf exonuclease, which is a single-stranded DNA-specific exonuclease to selectively digest the appointed DNAs (TBA and OBA), the targets could be liberated from the aptamer-target complexes for recycling of the analytes. Thereafter, probe DNAs (Tl and T2) were employed to hybridize with SH-cTBA and SH-cOBA respectively to provide primers for the concatemer reaction. After that, when four auxiliary DNA strands S1, anthraquinone-2-carboxylic acid (AQ)-labeled S2, S3, S4, as well as hemin were introduced, extended dsDNA polymers with lots of AQ moieties and hemin-G-quadruplex complexes could form on the electrode surface. Then, based on the signal of the AQ and hemin-G-quadruplex complex, an electrochemical aptasensor for the simultaneous detection of TB and OTA was successfully fabricated.