We describe a turn-on electrochemical biosensor for the detection of methyltransferases (MTases) causing DNA adenine methylation. This biosensor is based on insertion, methylation-resistant cleavage, signal enrichment caused by gold nanoparticles (AuNPs), and a signal probe-dragging strategy. A double-stranded DNA (dsDNA) containing identical MTase and methylation-resistant endonuclease (Mbo I) sites was immobilized on the surface of a gold electrode via Au-S covalent binding. The surface was subsequently treated with MTase and Mbo I and then washed. Results revealed that the surface of the electrode contains methylated dsDNA and 12-base nucleotides residual. Depending on biotin-streptavidin interactions that enabled signal probes and nucleotide residue hybridization and AuNP enrichment, a large number of signal probes labeled with ferrocene (Fc) are captured by the electrode. Under optimal conditions, the differential pulse voltammetry signals of Fc tags (at a working voltage of 0.24 V vs. Ag/AgCl) are linearly related to the log of the MTase activity in the 0.1 to 40 U·mL−1 range. The dynamic range extends from 0.05 to 50 U·mL−1, and the limit of detection is 0.024 U·mL−1 (at an S/N ratio of 3). The assay is well reproducible and highly selective. In our perception, this strategy provides a promising approach for simple, sensitive and selective detection of Dam MTase and may be extended to the determination of other MTase by exchanging the corresponding DNA.
相似文献Alloy nanoparticles of the type PtxFe (where x is 1, 2 or 3) were synthesized by coreduction with sodium borohydride in the presence of carbon acting as a chemical support. The resulting nanocomposites were characterized by scanning electron microscopy and X-ray diffraction. The nanocomposite was placed on a glassy carbon electrode, and electrochemical measurements indicated an excellent catalytic activity for the oxidation of glucose even a near-neutral pH values and at a working voltage as low as 50 mV (vs. SCE). Under optimized conditions, the sensor responds to glucose in the 10.0 μM to 18.9 mM concentration range and with a 3.0 μM detection limit (at an S/N ratio of 3). Interferences by ascorbic acid, uric acid, fructose, acetamidophenol and chloride ions are negligible.
相似文献A label-free and single-step method is reported for rapid and highly sensitive detection of bisphenol A (BPA) in aqueous samples. It utilizes an aptamer acting as a probe molecule immobilized on a commercially available array of interdigitated aluminum microelectrodes. BPA was quantified by measuring the interfacial capacitance change rate caused by the specific binding between bisphenol A and the immobilized aptamer. The AC signal also induces an AC electrokinetic effect to generate microfluidic motion for enhanced binding. The capacitive aptasensor achieves a limit of detection as low as 10 fM(2.8 fg ⋅ mL − 1) with a 20 s response time. The method is inexpensive, highly sensitive, rapid and therefore provides a promising technology for on-site detection of BPA in food and water samples.
B. The ACEK capacitive aptasensor has a limit of detection as low as 10 fM (2.8 fg ⋅ mL − 1) with a 20-s response time.
相似文献We report on a new amplification strategy for use in an immunoassay for influenza virus subtype H7N9. Graphene sheets were first placed on a glassy carbon electrode (GCE), and gold nanoparticles were then electrodeposited as a support for a layer of alcohol dehydrogenase (ADH) in a sol–gel containing thiol groups. Protein A was used to properly orientate immobilized antibody against H7N9 on the sol–gel, and this is shown to result in strongly improved specificity of the antigen-antibody binding. Thus, a sensitive and specific immunosensor was obtained in which a quadruple signal amplification strategy is employed, viz. (a) via the use of graphene sheets, (b) via a hybridization chain reaction, (c) the use of hemin/G-quadruplex DNAzyme concatamers, and (d) the use of ADH. The hemin/G-quadruplex is a typical DNAzyme, which simultaneously acts as NADH oxidase and HRP-mimicking DNAzyme. The hybridization chain reaction-based DNAzyme concatamers assembled on multi-walled carbon nanotubes (MWCNTs) and the ADH represent a triple electrocatalytic enzyme cascade system. Sandwich immunoreactions occurred between the capture antibody on the electrode and the secondary antibody labeled with MWCNTs. Positively charged Methylene Blue (MB) was then used as an intercalator to detect the DNAzyme concatamer formed. The differential pulse voltammetric signals for MB are related to the concentration of H7N9 in the range from 8 to 60 pg · mL−1, and the detection limit is 0.81 pg · mL−1 (at an S/N ratio of 3). This immunoassay is very sensitive, specific and robust.
相似文献This article describes an electrochemical sensor for the dye additive Sunset Yellow (SY). It consists of a carbon paste electrode modified with nanostructured resorcinol-formaldehyde (RF) resin. The RF resin warrants strong signal enhancement and a strongly increased oxidation peak currents of SY at 0.66 V (vs. SCE). The effects of pH value, amount of RF polymer, accumulation potential and time were optimized. The sensor has a linear response to SY in the 0.3 to 125 nM concentration range, and the limit of detection is 0.09 nM after a 2-min accumulation time. The electrode was applied to the analysis of samples of wastewater and drinks, and the results are consistent with those obtained by HPLC.
相似文献We have prepared a surface imprinted polymer (SIP) film for label-free recognition of immunoglobulin G (IgG). The IgG-SIPs were obtained by covalent immobilization of IgG via a cleavable covalent bond and a suitable spacer unit to a gold electrode, followed by electrodepostion of a nm-thin film of polydopamine (PDA). The IgG was then removed by destruction of the cleavable bond so that complementary binding sites were created on the surface of the film. IgG-SIPs with various thicknesses of the PDA films were compared with respect to their affinity to IgG using a quartz crystal microbalance combined with flow injection analysis. The films were also characterized by cyclic voltammetry and scanning electron microscopy. The IgG-SIPs with a film thickness of around 17 nm showed the most pronounced imprinting effect (IF 1.66) and a binding constant of 296 nM.
相似文献We report on an electrochemical method for the determination of the activity of trypsin. A multi-functional substrate peptide (HHHAKSSATGGC-HS) is designed and immobilized on a gold electrode. The three His residues in the N-terminal are able to recruit thionine-loaded graphene oxide (GO/thionine), a nanocover adopted for signal amplification. Once the peptide is cleaved under enzymatic catalysis by trypsin (cleavage site: Lys residue), the His residues leave the electrode, and the GO/thionine cannot cover the peptide-modified electrode anymore. Thus, the changes of the electrochemical signal of thionine, typically acquired at a voltage of -0.35 V, can be used to determine the activity of trypsin. A detection range of 1 × 10−4 to 1 U, with a detection limit of 3.3 × 10−5 U, can be achieved, which is better than some currently available methods. In addition, the method is highly specific, facile, and has the potential for the detection of trypsin-like proteases.
相似文献We describe a sensitive and selective biosensor for the environmental metabolite 2-hydroxyfluorene (2-HOFlu). It is based on electrochemical impedance spectroscopy and was obtained by assembling a thiolated single-stranded DNA on a gold electrode via S-Au covalent bonding. It is then transformed to a K+-stabilized G-quadruplex-hemin complex which exhibits peroxidase-like activity to catalyze the oxidation of 2-HOFlu by H2O2. This results in the formation of insoluble products that are precipitated on the gold electrode. As a result, the charge transfer resistance (R CT) between the solution and the electrode surface is strongly increased within 10 min as demonstrated by using the ferro/ferricyanide system as a redox probe. The difference in the charge transfer resistances (ΔR CT) before and after incubation of the DNA film with 2-HOFlu and H2O2 serves as the signal for the quantitation of 2-HOFlu with a 1.2. nM detection limit in water of pH 7.4. The assay is highly selective over other selected fluorene derivatives. It was exploited to determine 2-HOFlu in spiked lake water samples where it displayed a detection limit of 3.6 nM. Conceivably, this method has a wide scope in that it may be applied to other analytes for which respective G-quadruplexes are available.
相似文献We are presenting an electrochemical method for the determination of pyrophosphate ions (PPi) that is based on the competitive coordination of Cu(II) ion to a nanofilm of cysteine (Cys) and dissolved PPi. Cys was immobilized on the surface of a gold electrode by self-assembly. The Cys-modified gold electrode was loaded with Cu(II) ion which is released from the surface on addition of a sample containing PPi. The sensor shows an unprecedented electrochemical response to PPi, and the reduction peak currents is linearly related to the logarithm of the concentration of PPi in the 100 nM to 10 mM range (with an R2 or 0.982). The limit of detection is ~10 nM which is lower than the detection limits hitherto reported for PPi. Adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP) and common anions give a much weaker response. The method demonstrated here is simple, effective, highly sensitive, hardly interfered, and does not require the addition of a reagent. The method was applied to the determination of PPi in (spiked) serum samples.
相似文献We describe a simple and homogenous fluorimetric method for sensitive determination of DNA. It is based on target-triggered isothermal cycling and a cascade exponential amplification reaction that generates a large amount of a G-quadruplex. This results in strong fluorescence signal when using thioflavin T as a G-quadruplex-specific light-up fluorescent probe. Tedious handling after amplification is widely eliminated by the addition of thioflavin T. No other exogenous reagent is required. This detection platform is inexpensive and rapid, and displays high sensitivity for target DNA, with a detection limit as low as 91 pM.
相似文献We describe an electrochemical sensor for nitric oxide that was obtained by modifying the surface of a nanofiber carbon paste microelectrode with a film composed of hexadecyl trimethylammonium bromide and nafion. The modified microelectrode displays excellent catalytic activity in the electrochemical oxidation of nitric oxide. The mechanism was studied by scanning electron microscopy and cyclic voltammetry. Under optimal conditions, the oxidation peak current at a working voltage of 0.75 V (vs. SCE) is related to the concentration of nitric oxide in the 2 nM to 0.2 mM range, and the detection limit is as low as 2 nM (at an S/N ratio of 3). The sensor was successfully applied to the determination of nitric oxide released from mouse hepatocytes.
相似文献We describe the modification of a carbon paste electrode (CPE) with multiwalled carbon nanotubes (MWCNT) and an ionic liquid (IL). Electrochemical studies revealed an optimized composition of 60 % graphite, 20 % paraffin, 10 % MWCNT and 10 % IL. In a next step, the optimized CPE was modified with palladium nanoparticles (Pd-NPs) by applying a double-pulse electrochemical technique. The resulting electrode was characterized by scanning electron microscopy, energy dispersive X-ray spectroscopy, X-ray diffraction, cyclic voltammetry, and electrochemical impedance spectroscopy. It gives three sharp and well separated oxidation peaks for ascorbic acid (AA), dopamine (DA), and uric acid (UA), with peak separations of 180 and 200 mV for AA-DA and DA-UA, respectively. The sensor enables simultaneous determination of AA, DA and UA with linear responses from 0.6 to 112, 0.1 to 151, and 0.5 to 225 μM, respectively, and with 200, 30 and 150 nM detection limits (at an S/N of 3). The method was successfully applied to the determination of AA, DA, and UA in spiked samples of human serum and urine.
相似文献A molecularly imprinted polymer (MIP) for the specific retention of neopterin has been developed. A set of 6 polymers was prepared by radical polymerization under different experimental condition using methacrylic acid as functional monomer and ethylene glycol dimethacrylate as crosslinker, with the aim to understand their influence on the efficiency of the MIP. The performance of each MIP was tested in batch experiments via their binding capacity. The MIP prepared in the presence of nickel ions in dimethylsulfoxide-acetonitrile mixture (P4) exhibited the highest binding capacity for neopterin (260 μmol per gram of polymer). A selectivity study with two other pteridines demonstrated the polymer P4 also to possess the best selectivity.
相似文献Thin-layered molybdenum disulfide (MoS2) was intercalated, via ultrasonic exfoliation, into self-doped polyaniline (SPAN). This material, when placed on a glassy carbon electrode (GCE), exhibits excellent electrical conductivity and synergistic catalytic activity with respect to the detection of bisphenol A (BPA). The electrochemical response of the modified GCE to BPA was investigated by cyclic voltammetry and differential pulse voltammetry. Under optimal conditions, the oxidation peak current (measured best at 446 mV vs. SCE) is related to the concentration of BPA in the range from 1.0 nM to 1.0 μM, and the detection limit is 0.6 nM.
相似文献We have combined the molecular imprinting and the layer-by-layer assembly techniques to obtain molecularly imprint polymers (MIPs) for the electrochemical determination of p-nitrophenol (p-NPh). Silica microspheres functionalized with thiol groups and gold nanoparticles (Au-NPs) were assembled on a gold electrode surface layer by layer. The electrode was then immersed into a solution of pyrrole and p-NPh (the template), and electropolymerization led to the creation of a polymer-modified surface. After the removal of the silica spheres and the template, electrochemical impedance spectroscopy and differential pulse voltammetry (DPV) were employed to characterize the surface. The results demonstrated the successful fabrication of macroporous MIPs embedded with Au-NPs on the gold electrode. The effects of monomer concentration and scan rate on the performance of the electrode were optimized. Excellent recognition capacity is found for p-NPh over chemically similar species. The DPV peak current is linearly related to concentration of p-NPh in the 0.1 μM to 1.4 mM range, with a 0.1 μM limit of detection (at S/N = 3).
相似文献We have prepared a pyrolytic graphite electrode (PGE) whose surface is covered with a thin film of a nano-mixture of graphite/diamond (NGD). The electrode is shown to be capable of electrochemically sensing of tryptophan (TRP) and 5-hydroxytryptophan (HTRP). The presence of the NGD film resulted in a remarkable increase in the peak currents and sharpness of the waves so that submicromolar concentrations of TRP and HTRP become detectable. Potential scan rates, the pH of the solution, the accumulation conditions and the amount of the modifier were optimized via cyclic voltammetry. Linear sweep voltammetry, under optimized accumulation time and in open circuit operation, was applied to the determination of TRP and HTRP with detection limits (S/N = 3) of 30 nM (TRP) and 6 nM (HTRP). The electrode can be easily prepared, displays high sensitivity, sharp peaks, long-term stability, and remarkable voltammetric reproducibility and repeatability. These properties make the sensor suitable for the trace analysis of TRP and HTRP in pharmaceutical and clinical preparations.
相似文献We report on a sensitive aptamer-antibody interaction-based assay for cytochrome c (Cyt c) using electrochemical impedance. 4-Amino benzoic acid is used for the oriented immobilization of aminated aptamers onto multi-walled carbon nanotubes on the surface of a screen-printed electrode via electrochemical grafting. Impedance was measured in a solution containing the redox system ferro/ferricyanide. The change in interfacial charge transfer resistance (Rct) experienced by the redox marker was recorded to confirm the formation of a complex between aptamer and the target (Cyt c). A biotinylated antibody against cytochrome c was then used in a sandwich type of assay. The addition of streptavidin conjugated to gold nanoparticles and signal enhancement by treatment with silver led to a further increase in Rct. Under optimized conditions, a detection limit as low as 12 pM was obtained. Cross-reactivity against other serum proteins including fibrinogen, BSA and immunoglobulin G demonstrated improved selectivity.
相似文献Electrochemical immunodetection has attracted considerable attention due to its high sensitivity, low cost and simplicity. Large efforts have recently made in order to design ultrasensitive assays. Noble metal nanoparticles (NM-NPs) offer advantages such as high conductivity and large surface-to-volume ratio. NM-NPs therefore are excellent candidates for developing electrochemical platforms for immunodetection and as signal tags. The use of biofunctionalized NM-NPs often results in amplified recognition via stronger loading of signal tags, and also in enhanced signal. This review (with 87 references) gives an overview on the current state in the use of NM-NPs in Non-enzymatic electrochemical immunosensing. We discuss the application of NM-NPs as electrode matrices and as electroactive labels (either as a carrier or as electrocatalytic labels), and compare the materials (mainly nanoparticles of gold, platinum, or of bimetallic materials) in terms of performance (for example by increasing sensitivity via label amplification or via high densities of capture molecules). A conclusion covers current challenges and gives an outlook. Rather than being exhaustive, the review focuses on representative examples that illustrate novel concepts and promising applications. NM-NPs based immunosensing opens a series of concepts for basic research and offers new tools for determination of trace amounts of protein-related analytes in environment and clinical applications.
相似文献We describe an electrochemical sensor for simultaneous determination of hydroquinone (HQ) and catechol (CC). A glassy carbon electrode (GCE) was modified with gold nanoparticles, L-cysteine, and ZnS/NiS@ZnS quantum dots using a layer-by-layer technique. The materials were characterized by X-ray diffractometry, field emission scanning electron microscopy, and electrochemical impedance and Fourier transform infrared spectroscopy. Cyclic voltammetry and differential pulse voltammetry revealed this modified GCE to represent a highly sensitive sensor for the simultaneous determination of HQ and CC. The anodic peak current for HQ at a working voltage of 80 mV (vs. Ag/AgCl) is related to its concentration in the 0.1 to 300 μM range (even in the presence of 0.1 mM of CC). The anodic peak current for CC at a working voltage of 184 mV is related to its concentration in the 0.5 to 400 μM range (even in the presence of 0.1 mM of HQ). The detection limits (at an S/N ratio of 3) are 24 and 71 nM for HQ and CC, respectively. The modified GCE was successfully applied to the determination of HQ and CC in aqueous solutions and gave satisfactory results.
相似文献A novel strategy was devised for colorimetric analysis of the products of the polymerase chain reaction (PCR). The method takes advantage of simultaneous amplification of a horseradish peroxidase-mimicking DNAzyme (HRPzyme) during the PCR process. It is performed using a DNA specific forward primer and a universal reverse primer containing a complementary HRPzyme sequence. The double-strand PCR products, which include the HRPzyme sequence, are treated with a mixture of hemin and TMB (3,3′,5,5′–tetramethylbenzidine) in the presence of hydrogen peroxide. The resulting HRPzyme/hemin complex then promotes a peroxidase mimicking reaction, which produces the blue colored oxidized TMB. This colorimetric method can be more easily performed than previously developed gel based detection procedures and, as a result, can be conveniently applied to the specific and sensitive colorimetric analysis of DNA sequences arising from pathogenic bacteria. The potentially broad applicability of the new method has been demonstrated by its use in the identification of the 16s rDNA of Salmonella Typhimurium.
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