A sensitive fluorescent assay for thiamine based on metal-organic frameworks with intrinsic peroxidase-like activity

Metal-organic frameworks (MOFs) with tunable structures and properties have recently been emerged as very interesting functional materials. However, the catalytic properties of MOFs as enzymatic mimics remain to be further investigated. In this work, we for the first time demonstrated the peroxidase-like activity of copper-based MOFs (HKUST-1) by employing thiamine (TH) as a peroxidase substrate. In the presence of H₂O₂, HKUST-1 can catalyze efficiently the conversion of non-fluorescent TH to strong fluorescent thiochrome. The catalytic activity of HKUST-1 is highly dependent on the temperature, pH and H₂O₂ concentrations. As a peroxidase mimic, HKUST-1 not only has the features of low cost, high stability and easy preparation, but also follows Michaelis–Menten behaviors and shows stronger affinity to TH than horseradish peroxidase (HRP). Based on the peroxidase-like activity of HKUST-1, a simple and sensitive fluorescent method for TH detection has been developed. As low as 1 μM TH can be detected with a linear range from 4 to 700 μM. The detection limit for TH is about 50 fold lower than that of HRP-based fluorescent assay. The proposed method was successfully applied to detect TH in tablets and urine samples and showed a satisfactory result. We believed that the present work could improve the understanding of catalytic behaviors of MOFs as enzymatic mimics and find out a wider application in bioanalysis.     

Fluorescence assay of catecholamines based on the inhibition of peroxidase-like activity of magnetite nanoparticles

We report a fluorescence approach for the highly selective and sensitive detection of catecholamines using magnetite nanoparticles (Fe₃O₄ NPs) in the presence of Amplex UltraRed (AUR) and H₂O₂. Fe₃O₄ NPs catalyze H₂O₂-mediated oxidation of AUR. The resulting product fluoresces (excitation/emission maxima, ca. 568/587 nm) more strongly, relative to AUR. When catecholamines bind to Fe₃O₄, the complexes that are formed induce decreased activity of Fe₃O₄ NPs, mediated through the coordination between Fe³⁺ on the NP surface and the catechol moiety of catecholamines. As a result, Fe₃O₄ NPs-catalyzed H₂O₂-mediated oxidation of AUR is inhibited by catecholamines. The limits of detection for dopamine (DA), l-DOPA, norepinephrine, and epinephrine were 3 nM, 3 nM, 3 nM, and 6 nM, respectively. The Fe₃O₄ NPs-H₂O₂-AUR probe exhibited high selectivity (>1000-fold) toward catecholamines over other tested biomolecules that commonly exist in urine. Four catecholamines had similar sensitivity because the inhibition of the Fe₃O₄ NPs activity relies on the presence of the catechol moiety. This approach also allowed the determination of tyrosinase activity because tyrosinase catalyzes the conversion of l-tyrosine to l-DOPA. We validated the practicality of the use of the Fe₃O₄ NPs-H₂O₂-AUR probe for the determination of the concentrations of DA in urine samples.     

A new colorimetric assay for amylase based on starch-supported Cu/Au nanocluster peroxidase-like activity

In this paper, we present a new colorimetric technique as a novel assay for the easy and direct detection of α-amylase activity. This detection system utilizes the interaction of α-amylase with starch that is supporting copper/gold (Cu/Au) nanoclusters. The Cu/Au nanoclusters are synthesized using starch as a stabilizing agent at room temperature. These nanoclusters show robust peroxidase-like activity and are able to catalyze the oxidation of TMB (3,3,5,5-tetramethylbenzidine) in the presence of hydrogen peroxide (H₂O₂), leading to the generation of a blue-colored solution. The α-amylase detection mechanism is based on the digestion of the starch by α-amylase, which results in nanocluster aggregation, leading to increased nanoparticle size and thus decreased peroxidase-like activity of the Cu/Au NCs. Experiments showed that the gradual addition of α-amylase causes the peroxidase activity to decrease step by step in a linear fashion. Using this method, colorimetric sensing of α-amylase was achieved with a detection limit (LOD) of 0.04 U/mL and a linear range of 0.1–10 U/mL. This method is significantly selective for α-amylase and could be affordably and conveniently applied to the detection of α-amylase in blood serum.

Graphical Abstract

     

A highly selective colorimetric sulfide assay based on the inhibition of the peroxidase-like activity of copper nanoclusters

The authors report that sulfide ions are capable of inhibiting the peroxidase-like activity of copper nanoclusters (CuNCs). The catalytic activity of CuNCs toward the oxidation of the chromogenic substrate 3,3′,5,5′-tetramethylbenzidine by H₂O₂ is remarkably decreased in the presence of sulfide. Based on this finding, a colorimetric assay was developed for the rapid determination of sulfide. Best operated at a wavelength of 652 nm, it has a 0.5 μM detection limit. The method is highly selective and has been successfully applied to the quantification of sulfide in environmental water samples.

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Graphical abstract The catalytic activity of CuNCs toward the oxidation of 3,3′,5,5′-tetramethylbenzidine by H₂O₂ is remarkably decreased in the presence of sulfide ions. This finding has been applied to design a method for colorimetric quantification of sulfide ions in environmental samples.

     

Naked-eye sensitive detection of nuclease activity using positively-charged gold nanoparticles as colorimetric probes

Positively-charged gold nanoparticles can effectively differentiate long DNA and fragmented DNA, thus providing a simple and visual approach to colorimetric detection of nuclease activity.     

Colorimetric glutathione assay based on the peroxidase-like activity of a nanocomposite consisting of platinum nanoparticles and graphene oxide

Microchimica Acta - This work describes a colorimetric glutathione (GSH) assay that is taking advantage of (a) the peroxidase-like activity of a nanocomposite prepared from platinum nanoparticles...     

Aptamer based photometric assay for the antibiotic sulfadimethoxine based on the inhibition and reactivation of the peroxidase-like activity of gold nanoparticles

It is known that gold nanoparticles (AuNPs) possess peroxidase-like activity. They can catalyze the oxidation of 3,3,5,5-tetramethylbenzidine by H₂O₂ which leads to a color change from red to blue. It is shown here that the peroxidase-like activity of AuNPs can be inhibited by passivating its surface passivation with a ssDNA aptamer against sulfadimethoxine. If, however, the target molecule (sulfadimethoxine) is present, the aptamer is desorbed from the AuNPs surface, and this results in the reactivation of the catalytic property of the AuNPs. The color change of the solution (from purple to blue) is related to the analyte concentration, and this can be judged visually or by UV-visible absorptiometry at 650 nm. The assay, under optimized conditions, has a detection limit of 10 ng·mL^?1 of sulfadimethoxine, and the calibration plot is linear over a rather wide concentration range (0.01–1000 μg·mL^?1). The assay can be performed within <15 min, is sensitive, and therefore is well suited for fast screening in food analysis. Conceivably, it can be extended to many other small analytes for which aptamers are available.

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Graphical abstract Aptamer based photometric assay for sulfadimethoxine(SDM) based on the inhibition and reactivation of the peroxidase-like activity of gold nanoparticles (AuNPs) was performed with a rather wide linear range (0.01–1000 μg?mL^?1) and low detection limit of 10 ng?mL^?1.

     

Direct electrochemical detection of kanamycin based on peroxidase-like activity of gold nanoparticles

An enzyme-free, ultrasensitive electrochemical detection of kanamycin residue was achieved based on mimetic peroxidase activity of gold nanoparticles (AuNPs) and target-induced replacement of the aptamer. AuNPs which were synthesized using tyrosine as a reducing and capping agent, exhibited mimetic peroxidase activity. In the presence of kanamycin-specific aptamer, however, the single-stranded DNA (ssDNA) adsorbed on the surface of AuNPs via the interaction between the bases of ssDNA and AuNPs, and therefore blocked the catalytic site of AuNPs, and inhibited their peroxidase activity. While in the presence of target kanamycin, it bound with the adsorbed aptamer on AuNPs with high affinity, exposed the surface of AuNPs and recovered the peroxidase activity. Then AuNPs catalyzed the reaction between H₂O₂ and reduced thionine to produce oxidized thionine. The latter exhibited a distinct reduction peak on gold electrode in differential pulse voltammetry (DPV), and could be utilized to quantify the concentration of kanamycin. Under the optimized conditions, the proposed electrochemical assay showed an extremely high sensitivity towards kanamycin, with a linear relationship between the peak current and the concentration of kanamycin in the range of 0.1–60 nM, and a detection limit of 0.06 nM. Moreover, the established approach was successfully applied in the detection of kanamycin in honey samples. Therefore, the proposed electrochemical assay has great potential in the fields of food quality control and environmental monitoring.     

A high sensitive assay platform based on surface-enhanced Raman scattering for quantification of protease activity

In this study, a new, sensitive, and rapid assay was developed to quantitatively measure the proteolytic enzyme activity using the surface-enhanced Raman scattering (SERS) probe. Two different shapes of gold nanoparticles, gold nanosphere and nanorod particles were produced. SERS label, comprising self-assembled monolayers (SAMs) of Raman reporter molecule (5,5-Dithiobis (2-Nitrobenzoic acid), DTNB), was coated on the surface of the nanoparticles. Two different SERS-based analysis platforms were designed using gold-coated glass slide and polystyrene microtiter plate. The calibration curves were obtained by plotting the intensity of the SERS signal of symmetric NO₂ stretching of DTNB at 1326 cm⁻¹vs. the protease concentration. The effects of nanoparticle geometry and assay platform on the protease assay were investigated and the best working combination of the parameters was selected as rod shaped SERS probe and gold-coated glass slide. The correlation between the protease activity and SERS signal was found to be linear within the range of 0.1-2 mU/mL (R² = 0.979). The limit of detection (LOD) and limit of quantification (LOQ) values of the validated method were found as 0.43 and 1.30 mU/mL, respectively. The intra-day and inter-day precisions of the method, as relative standard deviation (RSD), were determined as 2.5% and 3.6%, respectively. The developed method was successfully applied for quantitative analysis of the commercial enzyme preparate that is used in cheese making process. It was also used for investigation of substrate specificity of protease enzyme towards the casein and bovine serum albumin. The proposed method has a flexibility to try different substrates for the detection of various enzyme activities.     

Colorimetric detection of sulfide based on target-induced shielding against the peroxidase-like activity of gold nanoparticles

Colorimetric recognition and sensing of sulfide with high sensitivity was proposed based on target-induced shielding against the peroxidase-like activity of bare gold nanoparticles. Significant features of the new assay system are its simplicity and cost-effectiveness. The recognition of sulfide by bare gold nanoparticles can be fulfilled in a few seconds and the assay can be accomplished in about 10 min. Furthermore, the new assay system does not require surface modification of GNPs to obtain the specificity for sulfide, and a salt-induced aggregation step is not needed. The detection limit of this method for sulfide was 80 nM. These features make this sensor a potentially powerful tool for the quantitative determination of sulfide in water samples.     

Antioxidant activity assay based on laccase-generated radicals

A novel antioxidant activity assay was developed using laccase-oxidized phenolics. In a three-step approach, phenolic compounds were first oxidized by laccase. Laccase was then inhibited using 80% (v/v) methanol which also stabilized the oxidized phenolics which were then used to measure antioxidant activities of ascorbic acid and Trolox. From a number of laccase-oxidized phenolics screened for potential use in the measurement of antioxidant activities, syringaldazine emerged the best, giving results comparable to the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, which is currently used in conventional methods. Like DPPH radicals, two moles of stoichiometric oxidized syringaldazine were reduced by one mole of either ascorbic acid or Trolox. For the first time we show that antioxidant activity can be correlated to oxygen consumption by laccase. Reduction of one molecule of oxygen corresponded to oxidation of four molecules of syringaldazine which in turn is reduced by two molecules of Trolox or ascorbic acid. This study therefore demonstrates the great potential of using laccase-oxidized syringaldazine for the measurement of antioxidant activity.     

A microchip electrophoretic assay for DNA methyltransferase activity based on methylation‐sensitive endonuclease DpnⅡ

DNA methylation is a significant epigenetic modification and the methods for the detection of DNA methyltransferase (MTase) activity are important due to aberrant methylation closely related to the occurrence of cancer. In this study, a simple and rapid microchip electrophoresis (ME) coupled with LED‐induced fluorescence (LEDIF) method was presented for the detection of Dam MTase activity. This strategy was based on methylation‐sensitive endonuclease DpnⅡ which could recognize the same specific site 5′‐GATC‐3′ with Dam MTase in double‐stranded DNA (dsDNA). The adenines in the specific site could be methylated by Dam MTase, then the special site could not be digested by DpnⅡ. Both methylated dsDNA and unmethylated dsDNA could be analyzed by ME‐LEDIF after stained by SYBR gold. The results showed the fluorescence intensities of methylated dsDNA were directly proportional to Dam MTase activities in the range of 0.5–20 U/mL with a detection limit of 0.12 U/mL. Furthermore, the method could successfully be applied to evaluation experiments of Dam MTase inhibitors. The results confirmed the ME‐LEDIF method is a promising approach for inhibitors screening of DNA MTase and development of anticancer drugs     

Highly sensitive colorimetric enzyme-linked oligonucleotide assay based on cyclodextrin-modified polymeric surfaces

In this paper, we describe the development of an enzyme-linked oligonucleotide assay for the detection of a human leukocyte antigen allele associated with celiac disease based on cyclodextrin-modified polymeric surfaces. The surface of maleimide-pre-coated plates was modified with a layer of thiolated cyclodextrin polymer and used for the supramolecular capture of adamantane or ferrocene-modified carboxymethylcellulose polymers bearing DNA probes. The assay was optimised in terms of incubation time, temperature, and surface chemistry and applied to the highly sensitive and selective detection of HLA sequences with a limit of detection of 0.7 nM. A real sample analysed using this platform showed an excellent correlation with maleimide-activated plates using thiolated DNA probes.     

A heme-peptide metalloenzyme mimetic with natural peroxidase-like activity

Mimicking enzymes with alternative molecules represents an important objective in synthetic biology, aimed to obtain new chemical entities for specific applications. This objective is hampered by the large size and complexity of enzymes. The manipulation of their structures often leads to a reduction of enzyme activity. Herein, we describe the spectroscopic and functional characterization of Fe(III)-mimochrome VI, a 3.5 kDa synthetic heme-protein model, which displays a peroxidase-like catalytic activity. By the use of hydrogen peroxide, Fe(III)-mimochrome VI efficiently catalyzes the oxidation of several substrates, with a typical Michaelis-Menten mechanism and with several multiple turnovers. The catalytic efficiency of Fe(III)-mimochrome VI in the oxidation of 2,2'-azino-di(3-ethyl-benzothiazoline-6-sulfonic acid (ABTS) and guaiacol (k(cat)/K(m)=4417 and 870 mM(-1) s(-1), respectively) is comparable to that of native horseradish peroxidase (HRP, k(cat)/K(m)=5125 and 500 mM(-1) s(-1), respectively). Fe(III)-mimochrome VI also converts phenol to 4- and 2-nitrophenol in the presence of NO(2) (-) and H(2) O(2) in high yields. These results demonstrate that small synthetic peptides can impart high enzyme activities to metal cofactors, and anticipate the possibility of constructing new biocatalysts tailored to specific functions.     

Highly sensitive assay for PZ-peptidase activity by high-performance liquid chromatography

A rapid and highly sensitive assay method for measuring PZ-peptidase activity in newborn rat brain is described. The method is based on monitoring the absorption at 320 nm of PZ-Pro-Leu enzymatically formed from the substrate, PZ-L-Pro-L-Leu-Gly-L-Pro-D-Arg, after separation by high-performance liquid chromatography using a reversed-phase column. This method is sensitive enough to measure PZ-Pro-Leu at concentrations as low as 5 pmol, and is able to make the column ready for the next injection within 10 min after the preceding injection. By using this method, PZ-peptidase activity was discovered in clonal osteoblastic cells derived from newborn mouse calvaria.     

Magnetic assistance highly sensitive protein assay based on surface-enhanced resonance Raman scattering

A simple and effective surface-enhanced Raman scattering (SERS)-based protocol for the detection of protein-small molecule interactions has been developed. We employed silver-coated magnetic particles (AgMNPs), which can provide high SERS activity as a protein carrier to capture a small molecule. Combining magnetic separation and the SERS method for protein detection, highly reproducible SERS spectra of a protein-small molecule complex can be obtained with high sensitivity. This time-saving method employs an external magnetic field to induce the AgMNPs to aggregate to increase the amount of atto610-biotin/avidin complex in a unit area with the SERS enhancement. Because of the contribution of the AgMNP aggregation to the SERS, this protocol has great potential for practical high-throughput detection of the protein-small molecule complex and the antigen-antibody immunocomplex.     

基于铜纳米线放大的汞离子高灵敏比色检测法

将滚环扩增技术与铜纳米线相结合进行信号放大,建立高选择性、高灵敏的汞离子比色检测新方法。以链霉亲和素修饰的磁珠为探针捕获和分离基质,将生物素修饰的引物链固定到其表面。汞离子存在时,模板链将通过T-Hg^2+-T作用与引物链结合。加入T4连接酶及DNA聚合酶引发滚环扩增反应形成超长单链DNA。与短单链DNA互补形成的长双链DNA可作为铜纳米线沉积模板,加入盐酸释放出大量铜离子催化底物氧化显色。在0.005~1.0 nmol/L范围,汞离子浓度与吸收信号呈良好线性关系,检出限低至3.7 pmol/L。     

Structural effects of Fe3O4 nanocrystals on peroxidase-like activity

The catalytic activity of nanocrystal catalysts depends strongly on their structures. Herein, we report three distinct structures of Fe(3)O(4) nanocrystals, cluster spheres, octahedra, and triangular plates, prepared by a similar hydrothermal procedure. Additionally, the three Fe(3)O(4) nanostructures were used as peroxidase nanomimetics and the correlation between the catalytic activities and the structures was first explored by using 3,3',5,5'-tetramethylbenzidine and H(2)O(2) as peroxidase substrates. The results showed that the peroxidase-like activities of the Fe(3)O(4) nanocrystals were structure dependent and followed the order cluster spheres>triangular plates>octahedra; this order was closely related to their preferential exposure of catalytically active iron atoms or crystal planes. Such investigation is of great significance for peroxidase nanomimetics with enhanced activity and utilization.     

单原子铂纳米酶的简便构建及其类过氧化物酶活性

采用浸渍吸附法,以多孔碳纳米笼(CNC)作为载体,简便构建了单原子铂/CNC(SA-Pt/CNC)纳米酶。通过透射电子显微镜(TEM)、高分辨透射电子显微镜(HRTEM)和X射线光电子能谱(XPS)深入解析了SA-Pt/CNC的微观结构。酶活性测试表明,SA-Pt/CNC表现出优异的类过氧化物酶活性,能高效催化过氧化氢氧化各种底物分子。