| The latest decade has witnessed increasing interest in composite membrane for electrode fabrication of biosensors, because it has many advantages:increase of both the mechanical strength in the dry state and the dimensional stability in the wet state; high flux and high selectivity; ease of handling; availability of very thin membranes. Nafion, a perfluorosulfonated cation-exchange polymer, is a permselective polymer known for its capability of incorporating cations and rejecting anions, which makes it a good candidate for successful incorporation of various cationic electrocatalysts into Nafion film at the surface of the modified electrodes. In addition, Nafion film has high chemical and thermal stability, good biocompatibility and the ability to resist interferents from anions and biological macromolecules. Thus, it is promising to fabricate functional composite membrane based on Nafion to construct kinds of sensors.Electrochemical immunosensor is a kind of sensing device combined by electrochemical analysis and immunoassay. It has many advantages, such as simple operation, rapid response, high sensitivity and good selectivity. Furthermore, electrochemical methods are free from color and turbid interferences in vivo monitoring. Electrochemiluminescent aptasensor is one kind of biosensors which is tight combination of electrochemiluminescence and aptamer-ligand specific recognition. Thus, it presents both of their merits, such as high sensitivity, low detection limit, good repeatability and selectivity. Therefore, it is of great significance to construct highly sensitive, rapid and effective immunosensors and aptasensors by employing composite membrane based on Nafion.Part 1 Study on an Amperometric Immunosensor Based on Nafion-Cys Composite Membrane for Detection of Carcinoembryonic AntigenIn this work, protonated L-cysteine was entrapped in Nafion membrane by cation-exchange function, forming Nafion-Cys composite membrane, which was more stable, compact, biocompatible and favorable for mass and electron transfer compared with Nafion film solely. Then gold nanoparticles were adsorbed onto the electrode surface by thiol groups on the composite membrane. After that, nano-Au monolayer was formed, onto which carcinoembryonic antibody (anti-CEA) was loaded to prepare carcinoembryonic antigen (CEA) immunosensor. The results indicated that the immunosensor had good current response for CEA, using potassium ferricyanide as the redox probe. A linear concentration range of 0.01 to 100 ng/mL with a detection limit of 3.3 pg/mL (S/N=3) was observed. Moreover, the morphology of the modified gold substrates were investigated with atomic force microscopy (AFM), the electrochemical properties and performance of modified electrodes were investigated by cyclic voltammograms (CV) and electrochemical impendence spectroscopy (EIS). The results exhibited that the immusensor had advantages of simple preparation, high sensitivity, good stability and long life-expandency. Thus, the method can be used for CEA analysis.Part 2 Electrochemiluminescence Quenching via Capture of Ferrocene-labeled Ligand-bound Aptamer Molecular Beacon for Ultrasensitive Detection of ThrombinHerein, we present an ultrasensitive protein-detection protocol based on electrochemiluminescence (ECL) quenching through capture of ferrocene-labeled ligand-bound aptamer molecular beacon (MB). Our device included two main parts:a solid-state ECL sensing platform and an ECL switch. The sensing platform was constructed by modifying the composite of tris(2,2’-bipyridyl) ruthenium(II) and platinum nanoparticles (Ru-PtNPs) and followed by immobilization of capture DNA (CaDNA). MB worked as the ECL intensity switch. In the presence of thrombin (TB), the hairpin-shaped MB binded to TB, inducing the exposure of a partial single-strand (MBs at this state was ligand-bound MBs). The exposed single stand was complementary to CaDNA and hybridized with CaDNA, leading to the capture of ligand-bound MB onto ECL sensing platform and finally resulting in ECL quenching by ferrocene that linked to MB. Otherwise, the ligand-unbound MB couldn’t be captured and ECL quenching wouldn’t happen. Accordingly, the change of ECL intensity indirectly reflected the concentration of TB in the samples. The results indicated that our protocol realized recognition of TB directly in the solution and exhibited much higher sensitivity with a detection limit of 1.7 pmol/L. Thus, this approach would be a promising protein-detection procedure directly performed in the solution.Part 3 In-situ Produced Ascorbic Acid as Coreactant for an Ultrasensitive Solid-state Tris(2,2’-bipyridyl) Ruthenium(Ⅱ) Electrochemiluminescence AptasensorHerein, an ultrasensitive solid-state tris(2,2’-bipyridyl) ruthenium(II) (Ru(bpy)32+) electrochemiluminescence (ECL) aptasensor using in-situ produced ascorbic acid as coreactant was successfully constructed for detection of thrombin. Firstly, the composite of Ru(bpy)32+and platinum nanoparticles (Ru-PtNPs) were immobilized onto Nafion coated glass carbon electrode, followed by successive adsorption of streptavidin-alkaine phosphatase conjugate (SA-ALP) and biotinylated anti-thrombin aptamer to successfully construct an ECL aptasensor for thrombin determination. In our design, Pt nanoparticles in Ru(bpy)32+-Nafion film successfully inhibited the migration of Ru(bpy)32+into the electrochemically hydrophobic region of Nafion and facilitated the election transfer between Ru(bpy)32+and electrode surface. Furthermore, ALP on the electrode surface could catalyze hydrolysis of ascorbic acid 2-phosphate to in-situ produce ascorbic acid, which co-reacted with Ru(bpy)32+ to obtain quite fast, stable and greatly-amplified ECL signal. The experimental results indicated that the aptasensor exhibited good response for thrombin with excellent sensitivity, selectivity and stability. A linear range of 1×10-15-1×10-8 mol/L with an ultralow detection limit of 0.33 fmol/L (S/N=3) was obtained. Thus, this procedure has great promise for detection of thrombin present at ultra-trace levels during early stage of diseases. |
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