Improving the fluorometric determination of the cancer biomarker 8-hydroxy-2′-deoxyguanosine by using a 3D DNA nanomachine

The authors describe a fluorometric method for improving the determination of the cancer biomarker 8-hydroxy-2′-deoxyguanosine (8-OHdG). A nicking endonuclease (NEase)-powered 3-D DNA nanomachine was constructed by assembling hundreds of carboxyfluorescein-labeled single strand oligonucleotides (acting as signal reporter) and tens of swing arms (acting as single-foot DNA walkers) on a gold nanoparticle (AuNP). The activity of this DNA nanomachine was controlled by introducing the protecting oligonucleotides. In the presence of aptamer against 8-OHdG, the protecting oligonucleotides are removed from the swing arms by toehold-mediated strand displacement reaction. In the next step, detached DNA walker hybridizes to the labelled DNA so that the DNA nanomachine becomes activated. Special sequences of signal reporter in the formed duplex can be recognized and cleaved by NEase. As a result, the DNA walker autonomously and progressively moves along the surface of the AuNP, thereby releasing hundreds of signal reporters and causing a rapid increase in green fluorescence. This 3-D nanomachine is highly efficient because one aptamer can release hundreds of signal reporters. These unique properties allowed for the construction of a DNA nanomachine-based method for sensitively detecting 8-OHdG in concentrations as low as 4 pM. This is three orders of magnitude lower compared to previously reported methods.

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Graphical abstract Schematic of a fluorometric method for determination of the cancer biomarker 8-hydroxy-2′-deoxyguanosine. A nicking endonuclease powered 3D-DNA nanomachine was used to improve the sensitivity. Limit of detection is three orders of magnitude lower than reported methods.

     

Electrochemical immunoassay for α-fetoprotein through a phenylboronic acid monolayer on gold

A novel reusable electrochemical immunosensor for α-fetoprotein (AFP) based on phenylboronic acid monolayer on gold was proposed. The sensor was fabricated by immobilizing of 3-aminophenylboronic acid (APBA) conjugated thiol-mixed monolayer on gold through 2-(5-norbornene-2,3-dicarboximido)-1,1,3,3-tetramethyluronium tetrafluoroborate (TNTU) as linkage. AFP and enzyme-conjugated antibody were further trapped to the modified electrode surface through sugar-boronic acid and immunoaffinity interactions, respectively. The attached enzyme-conjugated antibody on the electrode surface could catalyze the reduction of hydrogen peroxide in the presence of thionine, which can be used to detect AFP in human serum by a competitive mechanism. Cyclic voltammetric, electrochemical impedance studies and photometric activity assays were used to probe the assembly and regeneration process of the immunosensor. The influences of the competitive ratio of antigen and antibody, pH value of the measuring solution, incubation temperature and time were explored for optimizing the analytical performance. The whole assay process including incubation, detection and regeneration of the electrode could be completed in 35 min. The detection of AFP in five serum samples provided from clinically diagnosed patients with liver cancer showed acceptable accuracy. The proposed immunosensor enabled fast, low-cost and would be valuable for clinical diagnosis.     

Insight into Iron Leaching from an Ascorbate-Oxidase-like Fe−N−C Nanozyme and Oxygen Reduction Selectivity**

Ascorbate (H₂A) is a well-known antioxidant to protect cellular components from free radical damage and has also emerged as a pro-oxidant in cancer therapies. However, such “contradictory” mechanisms underlying H₂A oxidation are not well understood. Herein, we report Fe leaching during catalytic H₂A oxidation using an Fe−N−C nanozyme as a ferritin mimic and its influence on the selectivity of the oxygen reduction reaction (ORR). Owing to the heterogeneity, the Fe-N_x sites in Fe−N−C primarily catalyzed H₂A oxidation and 4 e⁻ ORR via an iron-oxo intermediate. Nonetheless, trace O₂⋅⁻ produced by marginal N−C sites through 2 e⁻ ORR accumulated and attacked Fe-N_x sites, leading to the linear leakage of unstable Fe ions up to 420 ppb when the H₂A concentration increased to 2 mM. As a result, a substantial fraction (ca. 40 %) of the N−C sites on Fe−N−C were activated, and a new 2+2 e⁻ ORR path was finally enabled, along with Fenton-type H₂A oxidation. Consequently, after Fe ions diffused into the bulk solution, the ORR at the N−C sites stopped at H₂O₂ production, which was the origin of the pro-oxidant effect of H₂A.     

Carbon-nanotube-enhanced direct electron-transfer reactivity of hemoglobin immobilized on polyurethane elastomer film

In this study, we investigate the direct electron-transfer reactivity of immobilized hemoglobin (Hb) on a polyurethane elastomer (PUE) film for biosensor designs. The PUE film synthesized by an additional polymerization possesses good biocompatibility, uniformity, and conformability and is ready for protein immobilization. Electrochemical and spectroscopic measurements show that the presence of multiwalled carbon nanotubes (MWNTs) increased the protein-PUE interaction, varied polymer morphology, improved the permeability and the conductivity of the PUE film, and thus facilitated the direct electron transfer between the immobilized Hb and the conductivity surface through the conducting tunnels of MWNTs. The immobilized Hb maintains its bioactivities and displays an excellent electrochemical behavior with a formal potential of -(334 +/- 7) mV. The addition of NaNO2 leads to an increase of the electrocatalytic reduction current of nitrite at -0.7 V. This allows us to develop a nitrite sensor with a linear response range from 0.08 to 3.6 mM. The proposed method opens a way to develop biosensors by using nanostructured materials mixed with low electrical conductivity matrixes.     

Nitrogen‐Doped Carbon Hollow Spheres for Immobilization,Direct Electrochemistry,and Biosensing of Protein

Nitrogen‐doped carbon hollow spheres (NCHS) were designed for the immobilization and biosensing of proteins. Chitosan was first functionalized with glutaraldehyde to form cross‐linked chitosan with free ? CHO groups (GCS). The as‐prepared GCS was used for dispersion of nitrogen‐doped carbon hollow spheres. Using glucose oxidase (GOD) as a model, the NCHS was tested for immobilization of redox proteins and the design of electrochemical biosensors. GOD molecules immobilized in the nanocomposites showed direct electrochemistry with a formal potential of ?0.448 V and well electrochemical performance. The proposed biosensor exhibited a linear response to glucose concentrations ranging from 3.7 µM to 18.0 mM with a detection limit of 1.2 µM and a sensitivity of 11.85 µA mM^?1. This biosensor was also applied to detect glucose in human serum samples, accomplishing good recovery in the range of 92–105 %. The nanocomposites provided a good matrix for protein immobilization and biosensor fabrication.     

通用小型汽油机催化器应用研究

简要介绍了中国通用小型汽油机行业的现状及美国EPA对于通用小型汽油机的排放要求,并结合国内通用小型汽油机的排放统计现状,指出了催化器在通用小型汽油机行业应用的技术可能性,并针对通用小型汽油机的排放特点,指出了催化器应用于通用小型汽油机需注意的问题.     

Coadsorption of horseradish peroxidase with thionine on TiO2 nanotubes for biosensing

In this study, we investigate the coadsorption of protein with thionine on TiO(2) nanotubes for biosensor design. The TiO(2) nanotube arrays fabricated by anodic oxidation of titanium substrate possess large surface areas and good uniformity and conformability and are ready for enzyme immobilization. Electrochemical and spectroscopic measurements show that the TiO(2) nanotube arrays provide excellent matrixes for the coadsorption of horseradish peroxidase (HRP) and thionine and that the adsorbed HRP on these TiO(2) nanotube arrays effectively retains its bioactivity. The immobilized thionine can be electrochemically reduced but cannot be reoxidized in the electrode potential range between -0.7 and 0.0 V. The addition of H(2)O(2) leads to the biocatalytic oxidation of the reduced thionine in the presence of HRP, resulting in developing a novel H(2)O(2) sensor with good stability and reproducibility. The fabricated TiO(2) nanotubes offer a stage for further study of immobilization and electrochemistry of proteins. The proposed method opens a way to develop biosensors using nanostructured materials with low electrical conductivity.     

Thirty years of haemoglobin electrochemistry

Electrochemical investigations of the blood oxygen carrier protein include both mediated and direct electron transfer. The reaction of haemoglobin (Hb) with typical mediators, e.g., ferricyanide, can be quantified by measuring the produced ferrocyanide which is equivalent to the Hb concentration. Immobilization of the mediator within the electrode body allows reagentless electrochemical measuring of Hb. On the other hand, entrapment of the protein within layers of polyelectrolytes, lipids, nanoparticles of clay or gold leads to a fast heterogeneous electron exchange of the partially denatured Hb.     

Preparation of carbon nitride nanoparticles by nanoprecipitation method with high yield and enhanced photocatalytic activity

As an emerging 2D conjugated material,graphitic carbon nitride(CN) has attracted great research attention as important catalytic medium for transforming solar energy.Nanostructure modulation of CN is an effective way to improve catalytic activities and has been extensively investigated,but remains challenging due to complex processes,time consuming or low yield.Here,taking advantage of recent discovered good solvents for CN,a nanoprecipitation approach using poor solvents is proposed for preparation of CN nanoparticles(CN NPs).With simple processes of CN dissolution and precipitation,we can quickly synthesize CN NPs(^40 nm) with a yield of up to 50%,the highest one to the best of our knowledge.As an example of potential applications,the as-prepared CN NPs were applied to photocatalytic degradation of dyes with an evident boosted performance up to 2.5 times.This work would open a new way for batch preparation of nanostructured CN and pave its large-scale industrial applications.     

Application of capillary electrophoresis coupling with electrochemiluminescence detection to estimate activity of leucine aminopeptidas

A new method to estimate the leucine aminopeptidase (LAP, EC 3.4.11.1) activity using capillary electrophoresis coupled with electrochemiluminescence (ECL) is described. The liberated proline produced by LAP catalyzing the hydrolysis reaction of leucin–proline was used as an ECL coreagent to enhance Ru(bpy)₃²⁺ ECL signals efficiently. The detection limit for proline was 2.88 × 10^?6 m (signal‐to‐noise ratio 3), which was equal to 9.60 × 10^?8 units of LAP being used to catalyze leucin–proline for 1 min. The Michaelis constant K_m (2.07 × 10^?2 mol/L) and the maximum reaction velocity V_max (1.06 × 10^?5 mol/L/min) of LAP for leucin–proline are reported. The reaction conditions including the concentration of metal ions, incubation temperature and pH were optimized. This method was successfully applied to detect LAP activity in plasma and the results were in good agreement with that obtained by the clinical method. Copyright © 2013 John Wiley & Sons, Ltd.