A Colorimetric Assay to Enable High-Throughput Identification of Biofilm Exopolysaccharide-Hydrolyzing Enzymes

Glycosidase enzymes that hydrolyze the biofilm exopolysaccharide poly-β-(1→6)-N-acetylglucosamine (PNAG) are critical tools to study biofilm and potential therapeutic biofilm dispersal agents. Function-driven metagenomic screening is a powerful approach for the discovery of new glycosidase but requires sensitive assays capable of distinguishing between the desired enzyme and functionally related enzymes. Herein, we report the synthesis of a colorimetric PNAG disaccharide analogue whose hydrolysis by PNAG glycosidases results in production of para-nitroaniline that can be continuously monitored at 410 nm. The assay is specific for enzymes capable of hydrolyzing PNAG and not related β-hexosaminidase enzymes with alternative glycosidic linkage specificities. This analogue enabled development of a continuous colorimetric assay for detection of PNAG hydrolyzing enzyme activity in crude E. coli cell lysates and suggests that this disaccharide probe will be critical for establishing the functional screening of metagenomic DNA libraries.     

Determination of acetylcholinesterase and butyrylcholinesterase activity without dilution of biological samples

Two cholinesterases: acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), are known. The enzymes are important in the body and alteration of their activity has significant use in the diagnosis of poisoning, liver function, etc. Currently available methods for the determination of cholinesterases have some major drawbacks including various interferences and the inability to be used for decreasing the enzyme activity in the presence of reversible inhibitors due to sample dilution; hence, a method for dilution free assay of cholinesterases is desired. Here, microplates were modified with indoxylacetate (100 µL of 10 mmol L^?1 solution) and used for cholinesterases assay after drying at 37°C. The fact that indoxylacetate remains stable in dry state and serves simultaneously as a chromogen and substrate provide good prerequisites for the method. The limit of detection for BChE was 0.71 U while that for AChE was 2.8 U per a 100 µL sample (solution of enzyme or plasma sample). The limit of detection is low enough to allow standard examination of cholinesterasemia. The two cholinesterases can be distinguished from each other using selective inhibitors such as donepezil and iso-OMPA. The new method was also successfully validated for the standard Ellman’s assay using plasma samples with BChE activity adjusted by carbofuran. The new method based on indoxylacetate seems promising for routine tests.     

Development of a MALDI-TOF-MS method to identify and quantify butyrylcholinesterase inhibition resulting from exposure to organophosphate and carbamate pesticides

A novel, proteomics based method was developed for the detection, quantification, and categorization of serum butyrylcholinesterase (BChE) inhibitors, including organophosphates (OPs) and carbamates (CBs). This method was based on the MALDI-TOF-MS analysis of the trypsin generated BChE active site peptide (191-SVTLFGESAGAASVSLHLLSPR-212) previously modified by reaction with an OP or CB. The ionization efficiency of OP modified active site peptides by MALDI was greatly improved by adding diammonium citrate to the MALDI matrix, which made the quantification of OP exposure feasible. Excellent linearity (r2 > 0.98) between the normalized abundance ratios (NARs) and OP concentrations or logarithm of carbaryl concentration was obtained. The accuracy of the developed assay was evaluated by comparison of IC50 and IC100 values from the assay with those determined by the Ellman method. Results from this method were comparable with those from the Ellman method. The advantage of the assay was that both the origin and the extent of pesticide exposure can be determined in one analysis. Our MALDI method can provide critical evidence for the pesticide exposure at low BChE inhibition levels even down to 3%, not available with the Ellman method.     

Rapid and sensitive detection of cholera toxin using gold nanoparticle-based simple colorimetric and dynamic light scattering assay

Herein, a rapid and simple gold nanoparticle based colorimetric and dynamic light scattering (DLS) assay for the sensitive detection of cholera toxin has been developed. The developed assay is based on the distance dependent properties of gold nanoparticles which cause aggregation of antibody-conjugated gold nanoparticles in the presence of cholera toxin resulting discernible color change. This aggregation induced color change caused a red shift in the plasmon band of nanoparticles which was measured by UV–Vis spectroscopy. In addition, we employed DLS assay to monitor the extent of aggregation in the presence of different concentration of cholera toxin. Our assay can visually detect as low as 10 nM of cholera toxin which is lower than the previously reported colorimetric methods. The reported assay is very fast and showed an excellent specificity against other diarrhetic toxins. Moreover, we have demonstrated the feasibility of our method for cholera toxin detection in local lake water.     

Colorimetric determination of hypochlorite with unmodified gold nanoparticles through the oxidation of a stabilizer thiol compound

In this article, we report a colorimetric approach for the determination of hypochlorite (OCl(-)) with gold nanoparticles (Au NPs). The test proceeds as two individual steps and selectivity is developed based on the strong oxidizing ability of hypochlorite. In concentrated phosphate buffer (PB), the red solution of citrate-capped Au NPs could be stabilized by the chemisorption of 11-mercaptoundecanoic acid (MUA), without which the colloidal suspension turned blue because of salt-induced particles aggregation. However, by its oxidizing power, OCl(-) converted the alkanethiol to a sulfonate derivative, which could not protect Au NPs from aggregation, thereby a blue solution was observed after the subsequent addition of Au suspension. With this method and under the optimal conditions (28 nm Au NP, 50 mM PB, pH 7.0, and 10 min for the colorimetric response), 1.5 μM of OCl(-) can be easily visualized by the naked eye. This sensitive and selective colorimetric assay opens up a fresh insight of facile, rapid, and reliable detection of OCl(-), and may find its future application in the monitoring of OCl(-)/HOCl in waters sanitized by chlorine or hypochlorite compounds.     

An electrochemical adaptation of Ellman's test

The electrochemically initiated reaction of thiols with N,N-diethyl-p-phenylenediamine has been coupled with an existing colorimetric sensing reaction developed by Ellman as a means of providing an electrochemical adaptation of the latter whereby the total thiol species present in a sample can be determined. The detection methodology has been proven to be robust with a linear range for cysteine from 2-120 microM, a limit of detection of 1.17 microM and has shown selectivity against a wide range of potential interferences. The efficiency of the methodology has been examined in the determination and recovery of thiol species in three growth tissue media, which contain a number of common biological interferences.     

A colorimetric method for highly sensitive and accurate detection of iodide by finding the critical color in a color change process using silver triangular nanoplates

In this contribution, we demonstrated a novel colorimetric method for highly sensitive and accurate detection of iodide using citrate-stabilized silver triangular nanoplates (silver TNPs). Very lower concentration of iodide can induce an appreciable color change of silver TNPs solution from blue to yellow by fusing of silver TNPs to nanoparticles, as confirmed by UV–vis absorption spectroscopy and transmission electron microscopy (TEM). The principle of this colorimetric assay is not an ordinary colorimetry, but a new colorimetric strategy by finding the critical color in a color change process. With this strategy, 0.1 μM of iodide can be recognized within 30 min by naked-eyes observation, and lower concentration of iodide down to 8.8 nM can be detected using a spectrophotometer. Furthermore, this high sensitive colorimetric assay has good accuracy, stability and reproducibility comparing with other ordinary colorimetry. We believe this new colorimetric method will open up a fresh insight of simple, rapid and reliable detection of iodide and can find its future application in the biochemical analysis or clinical diagnosis.     

一种S-腺苷甲硫氨酸依赖的甲基转移酶活性的检测方法

通过PCR扩增获得了S-腺苷-L-高半胱氨酸核苷酶(SAHN)和S-核糖基高半胱氨酸酶(SRHH)的基因序列, 克隆入表达载体, 转化宿主细胞, 表达纯化得到带有组氨酸标签的重组蛋白, 基于SAHN和SRHH重组酶建立了一种酶偶联分析S-腺苷甲硫氨酸(AdoMet)依赖的甲基转移酶活性的检测方法. 甲基转移酶的共同产物S-腺苷-L-高半胱氨酸(AdoHcy)首先被SAHN酶水解生成腺嘌呤和S-核苷高半胱氨酸, 后者进一步被SRHH酶裂解生成高半胱氨酸, 最后高半胱氨酸与Ellman's试剂反应生成5-硫代-2-硝基苯甲酸(TNB). 实验结果表明, 重组表达获取的2种酶蛋白均具有良好的催化活性, 酶偶联反应生成的TNB与初始AdoHcy浓度呈现显著的线性正相关. 该检测方法克服了S-腺苷甲硫氨酸依赖的甲基转移酶的共同产物AdoHcy的反馈抑制, 使甲基化反应进行完全, 从而保证了对甲基转移酶活性准确有效的定量分析.     

Aptamer-wrapped gold nanoparticles for the colorimetric detection of omethoate

Organophosphorous pesticide(OP) contamination has serious adverse effects on human health and the environment. Due to the toxicity of OPs and the threat presented by their accidental or intentional release in populated areas, the determination and monitoring of these OPs in food products and environment is of great importance. OPs are present in very small quantities and therefore, methods for their detection need to be highly sensitive and selective. Here, we aimed to develop a simple and selective aptamer-based colorimetric assay for the detection of omethoate, which is one of the commonly used OPs. The principle of the assay is that single-stranded DNA(ss DNA)-wrapped gold nanoparticles(Au NPs) are resistant to salt-induced aggregation. By employing an "artificial antibody" organophosphorous pesticide-binding aptamer(OBA) as the recognition element, aptamer-wrapped Au NPs(Au-apta) show high selectivity towards omethoate, resulting in the disconnection of aptamers from Au NPs and the aggregation of Au NPs. As there is a significant color change from the interparticle plasmon coupling during the aggregation of Au NPs, the established assay showed good linearity between 0.1 and 10 μmol/L, with a low detection limit of 0.1 μmol/L. Other OPs such as profenofos, phorate, and isocarbophos would not interfere with the detection of omethoate despite having similar structures. Thus, the colorimetric method shows potential for use in the detection of omethoate in real soil samples.     

Selective, peroxidase substrate based “signal-on” colorimetric assay for the detection of chromium (VI)

Due to the potentially adverse effects of the chromium (VI) on the human health and also on the environment, the quantitative determination of Cr(VI) is of particular interest. This work herein reports a facile, selective and rapid colorimetric determination of Cr(VI) based on the peroxidase substrate-2,2′-azino-bis(3-ethylbenzo-thiazoline-6-sulfonic acid) diammonium salt (ABTS) as the color developing agent. ABTS, which was usually acted as peroxidase substrate for the enzyme linked immunosorbent assay, is used here for the first time to fabricate the “signal-on” colorimetric Assay for Cr(VI). The ABTS was chosen instead of the commonly used 1,5-diphenylcarbazide (DPC) due to its good solubility, stability, sensitivity and low background. This method provided a convenient colorimetric detection of Cr(VI) with a wider linear range from 8.33 μg L⁻¹ to 1.25 mg L⁻¹ by recording the absorption spectra at the wavelength of 419 nm and a low detection limit of 7.87 μg L⁻¹. In addition, the entire detection takes less than 10 min.     

Fabrication of a Colorimetric Carcino-Embryonic Antigen Sensor Using High-Activity DNAzyme as a Catalytic Label

Detection of biomarkers in a biologically complex mixture remains a major challenge. Herein, an ultrasensitive colorimetric sandwich sensor for carcino-embryonic antigen (CEA) detection is introduced. The DNAzyme was tethered to biotinylated monoclonal antibodies (McAbs) which serve as the sensing element to recognize the target protein and was then introduced on to the CEA-McAbs assembled micro plate. The CEA was captured in a sandwich assay by the McAbs. The peroxidase-like DNAzyme catalyzed the oxidation of 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid), which generated a blue-green colorimetric signal. This method detected CEA in a serum-containing medium at a concentration as low as 10 nM. This strategy is a promising tool for bioanalytical and clinical applications.     

Development of a colorimetric inhibition assay for microcystin-LR detection: comparison of the sensitivity of different protein phosphatases

A colorimetric protein phosphatase (PP) inhibition test for the detection of microcystin-LR (MC-LR) has been developed. Three PP2As, one recombinant and two natural versions, as well as one PP1 produced by molecular engineering, were tested. First, assays were performed using the enzymes in solution to compare their sensitivity to MC-LR. The PP2A purchased from ZEU Immunotec and PP1 appeared more sensitive to the toxin than the other enzymes. With PP2A from ZEU Immunotec, the colorimetric test showed a detection limit of 0.0039 μg L⁻¹ and an IC₅₀ value of 0.21 μg L⁻¹. With PP1, the assay gave a detection limit of 0.05 μg L⁻¹ and an IC₅₀ value of 0.56 μg L⁻¹. Therefore, this assay allowed the detection of lower microcystin-LR (MC-LR) concentrations than the maximum level (1 μg L⁻¹) recommended by the World Health Organisation (WHO).The main drawback of this PP-based approach in solution is poor enzyme stabilisation. To overcome this problem, enzymes were entrapped within either a photopolymer or an agarose gel. PP2A from ZEU Immunotec and PP1 were immobilised at the bottom of microwells. The agarose-based tests performed better than the photopolymer-based assay for all of the enzymes. Therefore, the agarose gel is a good candidate to replace the photopolymer, which is generally used in PP-immobilising membranes. The assays based on enzyme-entrapping agarose gels showed detection limits equal to 0.17 μg L⁻¹ and 0.29 μg L⁻¹ with immobilised PP2A from ZEU and PP1, respectively. In view of these performances, these tests can potentially be used for monitoring water quality.     

Studies on the substrate specificity of Escherichia coli galactokinase

In vitro glycorandomization (IVG) technology is dependent upon the ability to rapidly synthesize sugar phosphates. Compared with chemical synthesis, enzymatic (kinase) routes to sugar phosphates would be attractive for this application. This work focuses upon the development of a high-throughput colorimetric galactokinase (GalK) assay and its application toward probing the substrate specificity and kinetic parameters of Escherichia coli GalK. The demonstrated dinitrosalicylic assay should also be generally applicable to a variety of sugar-processing enzymes. [reaction: see text]     

A colorimetric assay for cholesterol based on the encapsulation of multienzyme in leaf-shape crossed ZIF-L

The serum cholesterol level is an important indicator of healthy and there is a great necessity for frequent cholesterol monitoring to some cardiovascular-related diseases, which puts forward higher requirements for point-of-care testing (POCT) of cholesterol. In this work, a cascade catalytic system of cholesterol is developed by encapsulation of cholesterol oxidase (ChOx) and PdCuAu nanoparticles into zeolitic imidazolate framework-L (ChOx/PCA@ZIF-L). Results indicate that ZIF-L carrier can significantly increase the catalytic activity of single or multiple enzymes, due to its high loading capacity and efficient molecular transport. Under the optimal conditions, the absorbance of reaction system performs linear relationships with the concentration of cholesterol in two intervals from 0.0005 mmol/L to 1.0000 mmol/L, with a limit of detection of 0.2176 µmol/L. The proposed colorimetric strategy based on ChOx/PCA@ZIF-L performs a good agreement with the results provided by chemiluminescence method for the serum cholesterol detection. Interestingly, a simple paper-based sensing system is constructed through a pre-reaction-transfer operation, which gets rid of the complex pre-processing requirements of traditional operations on filter paper. The presented strategy allows for the sensitive, convenient, costless assay of serum cholesterol, and paves a new way to design the POCT device for daily monitoring of healthy.     

Self-assembled all-inclusive organic-inorganic nanoparticles enable cascade reaction for the detection of glucose

Traditional colorimetric glucose biosensor generally involves complex assay procedures. Free labile enzymes and peroxidase substrates are used separately for triggering a chromogenic reaction. These limits result in inferior enzyme stability and defective enzymatic catalytic efficiency, making it hard to routinely utilize them for the direct and fast test of glucose. In this work, we provide an all-inclusive substrates/enzymes nanoparticle employed 3,3′5,5′-tetramethylbenzidine (TMB) as chromogenic substrates and glucose oxidase (GOx)/horseradish peroxidase (HRP) as signal amplifier enzymes (TMB-GH NPs) by the molecule self-assembly technique. The “all-inclusive” nanoparticles can realize the tandem colorimetric reactions, and the oxidation product of TMB (ox-TMB) exhibits a strong NIR laser-driven photothermal effect, thus allowing quantitative photothermal detection of glucose. Owing to the restriction of the molecular motion of GOx, HRP, and TMB, the distance of mass transfer between substrates was shortened largely, leading to improved catalytic activity for glucose. Overall, our strategy will simplify the analysis procedure, furthermore, these integrated nanoparticles not only display higher stability and activity than that of the free GOx/HRP system and possesses an excellent performance for colorimetric and photothermal bioassay of glucose simultaneously. We believe that this unique technique will give good inspirations to develop simple and precise methods for bioassay.     

Design,synthesis characterization and in vitro biological activity of a series of 3-aryl-6-(bromoarylmethyl)-7H-thiazolo[3,2-b]-1,2,4-triazin-7-one derivatives as the novel acetylcholinesterase inhibitors

Bromination is used as a strategy to improve biological activity in medicinal chemistry.In order to study on the structure-activity relationships of the novel acetylcholinesterase inhibitors with 7H-thiazolo[3,2-b]-1,2,4-triazin-7-one scaffold,based on our previous work and molecular modeling,a series of novel 3-aryl-6-(bromoarylrnethyl)-7H-thiazolo[3,2-b]-1,2,4-triazin-7-one derivatives were designed by molecular docking,synthesized and characterized by mass spectra,infrared spectra,proton NMR and elemental analyses.The study of AChE inhibitory activity was carried out using the Ellman colorimetric assay with huperzine-A as the positive control.Most of all target compounds exhibited more than 45%inhibition at 10μmol/L.The preliminary structureactivity relationship was the bromine atoms and the hydroxyl group at the phenyl ring at the C6 position of the parent nucleus played significant roles in the AChE inhibitory activity of the target compounds.     

Gold nanoparticle-enabled real-time ligation chain reaction for ultrasensitive detection of DNA

A simple and ultrasensitive colorimetric DNA assay based on the detection of the product of a ligation chain reaction (LCR) and the use of gold nanoparticles (AuNPs) as signal generators has been developed. During LCR, the AuNPs were ligated together, resulting in a distinct color change in real time after a sufficient number of thermal cycles. The cumulative nature of the protocol produced a detection limit of 20 aM with a selectivity factor of 10(3).     

One-step synthesis of silver/dopamine nanoparticles and visual detection of melamine in raw milk

In this work, we propose a simple, sensitive and reliable assay for melamine in raw milk with dopamine-stabilized silver nanoparticles (AgNPs) as a colorimetric reader. Dopamine can reduce Ag(+) and functionalize the produced AgNPs to form monodispersed AgNPs. The coexisting melamine in reaction solution could bind dopamine through Michael addition and Schiff base reactions, which leads to the aggregation of AgNPs and induces a colorimetric response. The one-step assay is simple, rapid and highly sensitive. The color-change is quantitatively correlated with the concentration of melamine in the range of 10 ppb to 1.26 ppm, which is below the safety limit in China (1.0 ppm) and EU (2.0 ppm). The coexisting substances including phenylalanine, dl-leucine, l-glutamate, sulfanilic acid, Mg(2+), galactose, lysine, urea and glucose do not affect the determination of melamine. The colorimetric sensor can be used for rapid monitoring of raw milk quality.     

Au Nanoparticle-DNAzyme Dual Catalyst System for Sensitively Colorimetric Detection of Thrombin

A novel colorimetric aptasensor was developed for thrombin detection with high sensitivity and specificity. The assay takes the advantage of Au nanoparticles-DNAzyme as a dual catalytic system for signal amplification. Au nanparticles were modified with peroxidase mimicking DNAzyme sequence as well as thrombin binding aptamer. And then the thrombin binding aptamer hybridized with its complementary sequence which was immobilized on the surface of the magnetic nanoparticles to construct the colorimetric aptasensor. In the presence of thrombin, the target-induced displacement takes place, resulting in the dissociation of the aptasensor. The DNAzyme functioned Au NPs are released due to the combine ability of thrombin binding aptamer with thrombin. The released Au NPs are capable of catalyzing the colorless 2,2'-azino-bis(3-ethylbenzothiazoline)-6-sulfonic acid(ABTS) conversion into a blue-green product by H₂O₂-mediated oxidation, thus can amplify the colorimetric readout signals of thrombin detection. Such a device can serve as a novel selectivity sensor for thrombin with a detection limit of 0.6 nmol/L.     

Colorimetric bacteria sensing using a supramolecular enzyme-nanoparticle biosensor

Rapid and sensitive detection of pathogens is a key requirement for both environmental and clinical settings. We report here a colorimetric enzyme-nanoparticle conjugate system for detection of microbial contamination. In this approach, cationic gold nanoparticles (NPs) featuring quaternary amine headgroups are electrostatically bound to an enzyme [β-galactosidase (β-Gal)], inhibiting enzyme activity. Analyte bacteria bind to the NP, which releases the β-Gal and restores its activity, providing an enzyme-amplified colorimetric readout of the binding event. Using this strategy, we have been able to quantify bacteria at concentrations of 1 × 10(2) bacteria/mL in solution and 1 × 10(4) bacteria/mL in a field-friendly test strip format.