| Electrochemical aptasensors are valuable analytical tools for monitoring target analytes through the changes of electrochemical signals before and after the binding between aptamers and target analytes, which hold the advantages of high sensitivity, good selectivity, rapid response and simple operation. Using a variety of amplification technology such as chemical, material and biological combination, could effectively enhance the detection sensitivity. Therefore, this research focuses on the preparation of functionalized nanocomposite, the construction of sensitive interface, the application of novel signal amplification strategies and the enhancement of detection efficiency to construct the electrochemical aptasensors. The research contents are mainly as follows:1. Bi-enzyme functionalized hollow PtCo nanochains as labels for an electrochemical aptasensorHollow PtCo nanochains (HPtCoNCs) were synthesized by employing Co nanoparticles produced in situ as templates, which were used for the immobilization of ferrocene-labeled secondary thrombin aptamer (Fc-TBA2) and further functionalized by bi-enzyme—glucose oxidase (GOD) and horseradish peroxidase mimicking DNAzyme (HRP-DNAzyme). This work successfully constructed a sandwich-type electrochemical aptasensor for thrombin detection using the as-prepared multi-functionalized nanomaterials as tracer labels. With the surface area enlarged by HPtCoNCs, the amount of immobilized Fc-TBA2, hemin and GOD can be enhanced. Under the enzyme catalysis of GOD, glucose is rapidly oxidized into gluconic acid accompanying with the generation of H2O2, which is further electrocatalyzed by Pt nanoparticles and HPR-DNAzyme to improve the electrochemical signal of Fc. The present work demonstrates that using HPtCoNCs as labels is a promising way to amplify the analysis signal and improve the sensitivity of aptasensors.2. Direct electrochemistry and electrocatalysis of a glucose oxidase-functionalized bioconjugate as a trace label for ultrasensitive detection of thrombinPoly(amino-amine) dendrimers-encapsulated carbon nanotubes (PAMAM-CNTs) with high surface area was modified on the surface of glassy carbon electrode as the sensor platform to enhance effective area of the modified electrode and increase the immobilization of primary thrombin aptamers (TBA1). In addition, reduced graphene oxide (rGO) was employed to support platinum nanoparticles (PtNPs), forming PtNPs@rGO nanocomposite with good biocompatibility for the decoration of glucose oxidase (GOD) and secondary thrombin aptamers (TBA2) with high loading amount and good biological activity. With the excellent direct electron transfer of GOD and the synergistic electrocatalysis of GOD and PtNPs, a new sandwich-type electrochemical aptasensor was constructed for ultrasensitive detection of thrombin. The preparation and characterization of nanocomposites, as well as the construction and performance of the aptasensor, were also studied. The results showed that this method is feasible with low detection limit and good selectivity.3. Amperometric aptasensor for thrombin detection using enzyme-mediated direct electrochemistry and DNA-based signal amplification strategyThis work developed an electrochemical aptasensor for highly sensitive detection of thrombin based on direct electron transfer and electrocatalysis of horseradish peroxidase (HRP) using two typical biotechnology amplification—exonuclease-catalyzed target recycling and hybridization chain reaction (HCR) for signal amplification. To construct the aptasensor, double-stranded DNA (dsDNA) of the thiolated capture probe and thrombin binding aptamer was immobilized on gold nanoparticles (AuNPs) modified electrode through Au-S bond. In the presence of thrombin, the formation of aptamer-thrombin complex could result in the dissociation of aptamer from the dsDNA. Subsequently, with the employment of exonuclease, aptamer was selectively digested and thrombin could be released for analyte recycling. The resulted single stranded capture probe was used as the initiator to trigger the HCR of two biotin-labeled hairpin helper DNAs and lead to the formation of extended dsDNA polymers on the electrode surface. Then the biotin-labeled dsDNA polymers could introduce numerous avidin-labeled HRP, resulting in significantly amplified electrochemical detection signal through the direct electrochemistry and electrocatalysis of HRP. The proposed strategy combined the amplification of exonuclease-catalyzed analyte recycling and HCR, as well as the inherent electroactivity and electrocatalytic activity of HRP, which exhibited high sensitivity for thrombin determination.4. A signal-on electrochemical aptasensor for ultrasensitive detection of endotoxin using three-way DNA junction-aided enzymatic recycling and graphene nanohybrid for amplificationTo date, using lipopolysaccharide (LPS) binding aptamer as molecular recognition element of aptasensors for detection LPS have been rarely reported, and the sensitivity of these aptasensors are dissatisfactory. Thus, this work described a new signal-on electrochemical aptasensor for ultrasensitive detection of LPS by combining the three-way DNA junction acided enzymatic target recycling and nanotechnology for amplification. Toluidine blue (Tb), a kind of aromatic molecules with electrochemical activity, can decorate graphene (Gra) to obtain the Tb-Gra nanocomposite through π-π stacking, which not only improved the solubility and self-assembly properties of Gra, but also increased the immobilization of Tb and enhanced the electrochemical signal. Moreover, the application of three-way DNA junction makes the recognition site of restrictive endonuclease unconstrained on the specified sequence of target DNA. Thus, the high sensitivity and specificity make this method versatile for the detection of other biomolecules by changing the corresponding sequences of capture probe and assistant probe.5. Simultaneous electrochemical detection of multiple analytes based on dual signal amplification of carbon nanotubes and multi-labeled graphene sheetsTo improve the detection efficiency of aptasensor, this work fabricated a sandwich-type electrochemical aptasensor for one-spot simultaneous sensitive detection of platelet-derived growth factor (PDGF) and thrombin using graphene-nanocomposites as tracer labels. Reduced graphene oxide sheets (rGO) were used as matrices to immobilize the different redox probes, which were subsequently coated with the platinum nanoparticles (PtNPs) to form the PtNPs-redox probes-rGO nanocomposites. With the employment of the as prepared nanocomposites, a signal amplification strategy was described based on bi-enzyme (glucose oxidase and horseradish peroxidase) modified PtNPs-redox probes-rGO nanocomposites as tracer labels for secondary aptamers (Apt II) through sandwiched assay. Gold nanoparticles functionalized carbon nanotubes (AuNPs@CNTs) as the sensor platform could enhance the surface area of electrode to capture a large amount of primary aptamers (Apt I), thus amplifying the detection response. The experiment results showed that multi-labeled PtNPs-redox probes-rGO nanocomposites display satisfactory electrochemical redox activity and highly electrocatalytic activity of PtNPs and bi-enzyme, which exhibited high sensitivity and specificity for detection of proteins. |
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