| Electrochemical biosensors are valuable analytical tools as the advantages of rapid response, high sensitivity, good selectivity, simple manipulation and low cost, which combined the merits of electrochemical and biological technology. Graphene and fullerene based on carbon nanomaterials is widely applied to the field of biosensors because of its high surface area, good electrical conductivity and biocompatible. Recently, amplified electrochemical biosensors have been received much attention, which achieved by employing nanomaterials, enzymes catalysis and biological amplification technique. Thus, this research focuses on the preparation of multi-functionalized carbon nanocomposites, the construction of sensitive interface and the development of novel signal amplificatory strategy to construct the electrochemical biosensors. The detail contents are mainly as follows:1. Novel electrochemical catalysis as signal amplified strategy for label-free detection of neuron-specific enolaseA label-free electrochemical immunoassay for neuron-specific enolase (NSE), a kind of lung cancer marker, was developed in this work via novel electrochemical catalysis for signal amplification. The new amplified strategy was based on the electrochemical catalysis of nickel hexacyanoferrates nanoparticles (NiHCFNPs) in the presence of dopamine (DA). NiHCFNPs, which were assembled on the porous gold nanocrystals (AuNCs) modified glassy carbon electrode (GCE), could exhibit a distinct pair of redox peaks corresponding to anodic and cathodic reactions of hexacyanoferrate (Ⅱ/Ⅲ). Subsequently, gold nanoparticles functionalized graphene nanosheets (Au-Gra) were coated on the surface of NiHCFNPs/AuNCs film. Then an enhanced amount of neuron-specific enolase antibody (anti-NSE) could be loaded to obtain a sensitive immunosensor of anti-NSE/Au-Gra/NiHCFNPs/AuNCs/GCE due to the strong adsorption capacity and large specific surface area of Au-Gra. More importantly, the oxidation peak current can be enormously enhanced towards the electrocatalytic oxidation of DA based on NiHCFNPs, resulting in the further improvement of the immunosensor sensitivity. Under optimal conditions, the electrochemical immunosensor exhibited a linear range of 0.001-100 ng mL-1 with a detection limit of 0.3 pg mL-1 (S/N =3). Thus, the proposed immunosensor provides a rapid, simple, and sensitive immunoassay protocol for NSE detection, which may hold a promise for clinical diagnosis.2. Simultaneous electrochemical detection of multiple tumor markers based on dual catalysis amplification of multi-functionalized onion-like mesoporous graphene sheetsIn this work, a sandwich-type electrochemical immunosensor for simultaneous sensitive detection of prostate specific antigen (PSA) and free prostate specific antigen (fPSA) is fabricated. Gold nanoparticles (AuNPs) modified Prussian blue and nickel hexacyanoferrates nanoparticles were firstly prepared, respectively, and then decorated onion-like mesoporous graphene sheets (denoted as Au@PBNPs/O-GS and Au@NiNPs/O-GS) as distinguishable signal tags to label different detection antibodies. Subsequently, streptavidin and biotinylated alkaline phosphatase (bio-AP) were employed to block the possible remaining active sites. With the employment of the as prepared nanohybrids, the dual catalysis amplification can be achieved by catalysis of the ascorbic acid 2-phosphate to in situ produce AA in the presence of bio-AP, and then AA was further catalyzed by Au@PBNPs/O-GS and Au@NiNPs/O-GS nanohybrids, respectively, to obtain the higher signal responses. The experiment results show that the linear range of the proposed immunosensor for simultaneous determination of fPSA is from 0.02 to 10 ng mL"1 with a detection limit of 6.7 pg mL-1 and PSA is from 0.01 to 50 ng mL-1 with a detection limit of 3.4 pg mL-1. Importantly, the proposed method offers promise for rapid, simple and cost-effective analysis of biological samples.3. Electrochemical immunoassay for thyroxine detection using cascade catalysis as signal amplified enhancer and multi-functionalized magnetic graphene sphere as signal tagThis paper constructed a reusable electrochemical immunosensor for the detection of thyroxine at an ultralow concentration using cascade catalysis of cytochrome c (Cyt c) and glucose oxidase (GOx) as signal amplified enhancer. It is worth pointing out that numerous Cyt c and GOx were firstly carried onto the double-stranded DNA polymers based on hybridization chain reaction (HCR), and then the amplified responses could be achieved by cascade catalysis of Cyt c and GOx recycling with the help of glucose. Moreover, multi-functionalized magnetic graphene sphere was synthesized and used as signal tag, which not only exhibited good mechanical properties, large surface area and an excellent electron transfer rate of graphene, but also possessed excellent redox activity and desirable magnetic property. With a sandwich type immunoreaction, the proposed cascade catalysis amplification strategy could greatly enhance the sensitivity for the detection of thyroxine. Under the optimal conditions, the immunosensor showed a wide linear ranged from 0.05 pg mL-1 to 5 ng mL-1 and a low detection limit down to 15 fg mL-1. Importantly, the proposed method offers promise for reproducible and cost-effective analysis of biological samples.4. Multi-labeled functionalized C60 nanohybrid as tracing tag for ultrasensitive electrochemical aptasensingThis work reports a new supramolecular method for the synthesis of the amino and thiol groups functionalized C60 nanoparticles (FC6oNPs) with the large surface active sites and good water solubility. First, Prussian blue carried gold nanoparticles were decorated onto the surface of the obtained FC6oNPs (abbreviated as Au@PB/FC6o). Subsequently, the Au@PB/FC6o was labeled by detection aptamers and alkaline phosphatase to act as tracer. On the other hand, onion-like mesoporous graphene sheets and gold nanoparticles were utilized as the biosensor platform to immobilize a large amount of capture aptamers, owing to theirs porous structure and high surface-to-volume ratio. Based on the sandwich format, a dual signal amplification strategy based on multi-labeled functionalized C60 nanohybrid as tracing tag has been successfully developed for platelet-derived growth factor B-chain electrochemical detection with a wide linear response in the range of 0.002-40 nmol mL-1 and a limit of detection of 0.6 pmol mL-1. The proposed aptasensor demonstrated good specificity and high sensitivity, implying potential applications in bioanalysis and biomedicine.5. New type of redox nanoprobe:C60-based nanomaterial and its application in electrochemical immunoassay for doping detectionCarbon nanomaterials were usually exploited as nanocarriers in an electrochemical immunosensor but rarely acted as redox nanoprobes. Herein, our motivation is to adequately utilize the inner redox activity of fullerene (C60) to obtain a new type of redox nanoprobe based on a hydrophilic C60 nanomaterial. First, C6o nanoparticles (C60NPs) were prepared by phase-transfer method and functionalized with amino-terminated polyamidoamine (PAMAM) to obtain the PAMAM decorated C6oNPs (PAMAM-C6oNPs) which have better hydrophilicity compared to that of unmodified C60NPs and possesses abundant amine groups for further modification. Following that, gold nanoparticles (nano-Au) were absorbed on the PAMAM-C6oNPs surface, and the resultant Au-PAMAM-C6oNPs were employed as a new type of redox nanoprobe and nanocarrier to label detection antibodies (Ab2). Doping control has become the biggest problem facing international sport. Erythropoietin (EPO) as a blood doping agent has been a hotspot in doping control. After sandwich-type immunoreaction between EPO (as a model) and Ab2-labeled Au-PAMAM-C6oNPs, the resultant immunosensor was further incubated with a drop of tetraoctylammonium bromide (TOAB) which acts as booster to arouse the inner redox activity of AuPAMAM-C6oNPs, thus a pair of reversible redox peaks is observed. As a result, the proposed immunosensor shows a wide linear range and a relatively low detection limit for EPO. This strategy paves a new avenue for exploring the redox nanoprobe based on carbon nanomaterials in the electrochemical biosensor field. |
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