Study On Electrochemical Biosensors Using Nanomaterial As Signal Amplification

Electrochemical biosensors have widely attracted the interests of researchers due to the advantages of high sensitivity, simple operation, avoid separation, good selectivity, and no need of sample preparation. The current applications for its research and development is very fast, which has been applied to industrial processes control, clinical testing, environmental monitoring, chemical safety assessment food, pharmaceutical and other areas. In addition, the nanomaterial is widely used in electrochemical sensing technology because of its excellent chemical and physical properties, high surface area, the sensitivity of molecular adsorption capacity, and the capacity of improving the speed of biochemical reactions. In this paper, we carried out the research work focused on the electrochemical biosensors studying using nanomaterial as signal amplification, the main work are listed as follows:1. Electrochemical detection of thrombin based on aptamer and ferrocenylhexanethiol loaded silica nanocapsulesA sensitive and specific electrochemical assay for detection of thrombin based on aptamer and ferrocenylhexanethiol loaded silica nanocapsules (FcSH/SiNCs) amplification is described. In the protocol, a double aptamer sandwichs tructure was formed in the presence of thrombin, in which an aptamer-labeled FcSH/SiNCs for electrochemical detection and a streptavidin-coated magnetic bead immobilized aptamer. For rapid and specific separation of target protein.After separated from the sample mixture under a magnetic field, the sandwich complex was treated with NaOH to release the loaded ferrocenylhexanethiol (FcSH) from the silica nanocapsules (SiNCs). Differential pulse voltammetry (DPV) was employed to detect the released FcSH, which was related to the concentration of the thrombin. The method took advantage of sandwich binding of two affinity aptamers for increased specificity, high payload of FcSH in SiNCs for signal amplification, magnetic beads for fast magnetic separation. The peak current of released FcSH had a good linear relationship with the thrombin concentration in the range of0.1-5.0nmol/L, and the detectionlimitof thrombin in the method was0.06nmol/L.The detection was also specific for thrombin without being affected by other proteins, such as immunoglobulin G, bovine serum albumin, lysozyme and human serum albumin.The method has been used to detect thrombin in human serum albumin with minimum background interference. 2. Single Wall Carbon Nanotubes-Based Electrochemical Sensor for Detection of the Methyltransferase ActivityA sensitive and simple electrochemical approach for the assay of methyltransferases (MTase) activity based on the single wall carbon nanotubes (SWCNTs) signal amplification is developed. To realize this purpose, the duplex strand DNA (dsDNA), contained specific recognition sequence of Dam MTase and methylation-sensitive restriction endonuclease Dpn I, was modified on electrode at first. In the presence of Dam MTase and Dpn I, the recognition sequence was methylated and cleaved to single-stranded DNA (ssDNA). Then, the SWCNTs were controllably adsorbed on the isolating ssDNA modified electrode. The SWCNTs assembly can mediate efficient electron transfer between the electrode and electroactive species, generating a high faradic current due to the signal amplification, which was related to the concentration of the Dam MTase. Through detecting the currents mediated by SWCNTs, a linear response to concentration of Dam MTase range from0.1U/mL to1.0U/mL and a detection limit of0.04U/mL were obtained. Such a SWCNTs based biosensor opens a simple, sensitive, nonradioactive route for the detection of MTase activity. In addition, the screening of the inhibitors of MTase can be achieved based on the developed method.3. Ferrocene-functionalized SWCNT for Electrochemical Detection of T4polynucleotide kinase ActivityA novel label-free electrochemical strategy for monitoring the activity and inhibition of T4polynucleotide kinase (PNK) is developed by use of titanium ion (Ti4+) mediated signal transition coupled with signal amplification of single wall carbon nanotubes (SWCNTs). In this method, a DNA containing5’-hydroxyl group is self-assembled onto the gold electrode and used as substrate for PNK. The biofunctionalized SWCNTs with anchor DNA and ferrocene are chosen as the signal indicator by virtue of the intrinsic5’-phosphate end of anchor DNA and the high loading of ferrocene for electrochemical signal generation and amplification. The5’-hydroxyl group of the substrate DNA on the electrode is phosphorylated by T4PNK in the presence of ATP, and the resulting5’-phosphoryl end product can be linked with the signal indicator by Ti4+. The redox ferrocene group on the SWCNTs is grafted to the electrode and generates the electrochemical signal, the intensity of which is proportional to the activity of T4PNK. This assay can measure activity of T4PNK down to0.01UmL-1. The developed method is a potentially useful tool in researching the interactions between proteins and nucleic acids and provides a diversified platform for a kinase activity assay.4. Barbated Skullcup herb extract-mediated biosynthesis of gold nanoparticles and its primary application in electrochemistryThe design, synthesis, characterization and application of biologically synthesized nanomaterials have become an important branch of nanotechnology. In this paper, we report the extracellular synthesis of gold nanoparticles using Barbated Skullcup (BS) herb (a dried whole plant of Scutellaria barbata D. Don) as the reducing agent. After exposing the gold ions to BS herb extract, rapid reduction of gold ions is observed leading to the formation of gold nanoparticles in solution. UV-vis spectrum of the aqueous medium containing gold nanoparticles showed a peak at around540nm. Transmission electron microscopy (TEM) micrograph analysis of the gold nanoparticles indicated they were well-dispersed and ranged in size5-30nm. When the gold nanoparticles were modified on the glassy carbon electrode (GCE), it could enhance electronic transmission rate between the electrode and the p-nitrophenol.5. Electrogenerated chemiluminescence biosensor based on Ru(bpy)32+doped mesoporous silica nanoparticlesA novel electrogenerated chemiluminescence (ECL) sensor based on Ru(bpy)32+-doped MCM-41mesoporous silica nanoparticles (MSN) was developed. The Ru(bpy)32+was first loaded into the sulfhydryl modified MSN through the electrostatic absorption. Then the Ru(bpy)32+-doped MSN was immobilized on the surface of Au electrode by Au-S interaction. The behavior of electrochemistry and ECL were also investigated. Because the amino structure of melamine is similar to tripropylamine, we attempted to branch out the application of MCM-41into the field of melamine detection, the ECL sensor provided a new assay for the detection of melamine. Furthermore, the research provided a new approach for the immobilization of Ru(bpy)32+on the electrode surface.6. A sensitive ligase-based ATP electrochemical assay using molecular beacon-like DNA.A sensitive and selective ligase-based signal-on electrochemical sensing method for adenosine-5-triphosphate (ATP) detection had been developed using molecular beacon (MB)-likeDNA.In this method, the biotin-tagged MB-like DNA was self-assembled onto gold electrode to form a stem-loop structure by means of facile gold-thiol chemistry, which resulted in blockage of electronic transmission. It was eT OFF state. In the presence of ATP, two nucleotide fragments which were complementary to the loop of the MB-like DNA could be ligated by the ATP-dependent T4DNA ligase. Hybridization of the ligated DNA with the MB-like DNA induced a significant conformational change in this surface-confined DNA structure, which in turn released the biotin from the surface allowing free exchange of electrons with the electrode generating a measurable electrochemical signal (eT ON). The resulting change in electron transfer efficiency was readily measured by differential pulse voltammetry at target ATP concentrations as low as0.05nM and with linear response range from0.1to1000nM. Moreover, it was also able to discriminate ATP from its analogues. The proposed method had been successfully applied to the determination of ATP in the Escherichia coli O157:H7extracts of water samples, and the linear response was found between the concentrations of103and10’cfu/mL.