Visual Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid On-Site Detection of Escherichia coli O157: H7 in Milk Products.

(1) Background: Rapid on-site testing is an effective method for the detection of Escherichia coli O157: H7(E. coli O157: H7) in food ingredients and the environment. (2) Methods: In this study, we developed colorimetric loop-mediated isothermal amplification (LAMP) and immunochromatographic test strips (ICTs) for the rapid and visual detection of E. coli O157: H7. This study designed new specific LAMP primers for E. coli O157: H7 virulence island genes. After the LAMP amplification, the double-stranded DNA target sequence labeled with digoxin and fluorescein isothiocyanate (FITC) at both ends was bound to the anti-digoxin antibody on the gold nanoparticles. Subsequently, it was further bound to the anti-FITC antibody at the T line of the ICTs, forming a positive test result. Hydroxynaphthyl blue dye was directly added to the LAMP amplification product. A blue color indicated positive results, while a purple color indicated negative results. (3) Results: Two visualization methods showed high specificity for the target strains. The visualization tests had sensitivities of 5.7 CFU mL-1, and the detection limit of the Escherichia coli O157: H7 in artificially contaminated milk samples was 5.7 × 102 CFU mL-1, which was consistent with the results of the standard method (LAMP-electrophoresis method) used in commercial inspection. (4) Conclusions: Both methods could be useful in remote and under-resourced areas.

Development of a high-speed and ultrasensitive UV/Vis-CM for detecting total triterpenes in traditional Chinese medicine and its application.

This study proposes a novel colorimetric method based on the ultraviolet/visible spectrophotometry-colorimetric method (UV/Vis-CM) for detecting and quantifying total triterpenoids in traditional Chinese medicine. By incorporating the colourants 2-hydroxy-5-methylbenzaldehyde and concentrated sulfuric acid, triterpenoid compounds colour development became more sensitive, and the detection accuracy was significantly improved. 2-hydroxy-5-methylbenzaldehyde and concentrated sulfuric acid were incorporated in a 1:3 vol ratio at room temperature to react with the total triterpenes for 25 min, incorporated to an ice bath for 5 min, and then detected at the optimal absorption wavelength. The accuracy and reliability of this method were verified by comparison with high-performance liquid chromatography and four other colorimetric methods. Additionally, this approach has the advantages of not requiring heating during operation, high sensitivity, short usage time, low solvent usage, and low equipment costs. This study not only offers a reliable method for detecting total triterpenes in traditional Chinese medicine but also offers a rapid detection tool for on-site testing and large-scale screening, laying a foundation for the modernization of traditional Chinese medicine research, quality control, and drug development.

Urine multi-index intelligent detection based on polymer/paper hybrid microfluidic biochip for hyperuricemia monitoring.

Hyperuricemia (HUA) has gradually become a public health burden as an independent risk factor for a variety of chronic diseases. Herein, a user-friendly point-of-care (POC) detection system (namely "Smart-HUA-Monitor") based on smartphone-assisted paper-based microfluidic is proposed for colorimetric quantification of HUA urinary markers, including uric acid (UA), creatinine (CR) and pH. The detection limits of UA and CR were 0.0178 and 0.5983 mM, respectively, and the sensitivity of pH were 0.1. The method was successfully validated in artificial urine samples and 100 clinical samples. Bland-Altman plots showed a high consistency between µPAD and the testing instruments (HITACHI 7600 Automatic Analyzer, URIT-500B Urine Analyzer and AU5800B automatic biochemical analyzer) in hospital. Smart-HUA-Monitor provides an accurate quantitative, rapid, low-cost and reliable tool for the monitoring and early diagnosis of HUA urine indicators.

Comparison of colorimetric methods for the analysis of total polyphenols in green tea extracts.

Two colorimetric methods are used to determine the total polyphenol contents of tea, namely, "the Folin-Ciocalteu method," defined by the International Organization for Standardization, and the "iron tartrate method," specified in the Standard Tables of Food Composition in Japan. In this study, we compared the Folin-Ciocalteu and iron tartrate methods using green tea extracts. When comparing the 2 methods, the sum of the 4 major catechins measured using high-performance liquid chromatography (HPLC) was regarded as the standard value. The total polyphenol contents obtained using the Folin-Ciocalteu method were closer to the HPLC value than those obtained using the iron tartrate method. However, the iron tartrate method is adequate if the current official method is improved, that is, our results suggest that the coefficients appropriate for common green tea varieties, as well as the degree and duration of cover cultivation, in the official iron tartrate method must be considered.

Indophenol Blue Colorimetric Method to Determine Grain Protein Content of Cereal Plants.

Here, we propose a method to convert the organic nitrogen in maize kernels into ammonia in solution and then chlorinate it to prepare monochloride salts, which can form an oxidatively coupled blue-green mixture with sodium salicylate and sodium dichloroisocyanurate. The concentration of ammonium ions in the blue-green mixture can then be determined in the solution, and finally the protein content in maize kernels can be calculated from the nitrogen content.

Advanced "turn-on" colorimetric uranium platform based on the enhanced nanozyme activity of a donor-acceptor structured covalent organic framework.

BACKGROUND: The increasing uranium containing wastes generated during uranium mining and finishing pose a huge threat to the environment and human health, and thus robust strategies for on-site monitoring of uranium pollutant are of great significance for environmental protection around uranium tailings. RESULTS: Herein, a facile "turn-on" colorimetric platform that can achieve uranium detection by spectrometry and naked eyes was developed based on the uranium-enhanced nanozyme activity of covalent organic framework (JUC-505). Thanks to the extended π-conjugated skeleton and donor-acceptor (D-A) structure, JUC-505 exhibited superior photo-activated nanozyme activity, which would be prohibited when the cyano group in JUC-505 skeleton was transformed to the amidoxime group. Further results elucidated that the coordination of uranium with amidoxime groups led to the electron transfer between uranium and the JUC-505-AO skeleton, and thus significantly restored the nanozymatic activity of JUC-505-AO with the subsequent remarkable color changes. Moreover, the uranium concentrations in uranium tailing wastewater detected by the present "turn-on" colorimetric method were well agreed with those by ICP-MS, demonstrating a high accuracy of the present method in real samples. SIGNIFICANCE: The D-A structured JUC-505 with superior photocatalytic property and nanozymatic activity was applied to facilitate colorimetric detection of uranium, which displays the advantages of low detection limit, excellent selectivity, fast response and simple operation for uranium detection in real samples, and shows a great potential in on-site monitoring of uranium pollutant around uranium tailings as well as nuclear power plant.

Highly selective nanozyme-based glucose sensing platform via construction of artificial recognition sites on gold nanospheres.

Nanozymes have been regarded as the ideal alternatives to natural enzymes in bioassays due to their good stability and low cost. However, their applications in sensing usually suffer from poor selectivity. For example, Au-based nanozymes, as a kind of classical glucose oxidase mimic enzyme, could catalyze diverse monosaccharides. Therefore, it is of great necessity and urgency to endow the Au-based nanozymes with enhanced selectivity for the construction of specific glucose sensing platform. In our study, easily recyclable polydopamine (PDA)-supported Au-based nanozymes (PDA@Au NPs) were successfully prepared and could catalyze diverse monosaccharides including glucose, xylose, mannose, and sucrose. To enhance the selectivity of PDA@Au NPs, molecularly imprinted polymers (MIPs) were constructed on the surface of PDA@Au NPs using glucose and boronic acid derivatives as template and functional monomer. Impressively, the catalytic activity of the obtained molecularly imprinted nanozyme (PDA@Au NPs-MIPs) only shows a slight decrease (6.3%) while their selectivity is obviously enhanced (≥230%). Accordingly, the as-prepared sensor achieved the sensitive and selective detection of glucose in the concentration range of 10 µM-1 mM and a low detection limit (LOD) of 0.227 µM (S/N = 3), avoiding the influence of other monosaccharides exited in the sensing solutions to a great extent. As expected, the as-prepared sensors also showed good recovery, and long-term stability.

Dual-mode sensing strategy based on carbon dots for sensitive and selective detection of molybdate ions.

Two kinds of carbon dots with the maximum fluorescence peak of 492 nm (named as G-CDs) and 607 nm (named as R-CDs) were synthesized. In the presence of MoO42- ions, the fluorescence of R-CDs at 607 nm can be quenched, which can probably be assigned to their aggregation caused by MoO42-, while that of G-CDs at 492 nm remained unchanged. For the first time, a ratiometric fluorescence probe was developed for MoO42- ions detection. In the range 0.25 ~ 100 µM, the fluorescence ratio (F492/F607) of the probe was linearly related to MoO42- concentration, and the detection limit was 61.5 nM, which fully meets the minimum detection requirements of MoO42- ions in drinking water. On the other hand, when MoO42- was introduced, a significant fading phenomenon of R-CDs can be observed with the naked eye; thereby, the colorimetric method can also be proposed. Based on above, the ratiometric fluorometric/colorimetric dual-mode sensing method was established for MoO42- anion quantification. Compared with the traditional analysis methods, the results obtained by multimodal sensing can be mutually verified, which effectively improves the accuracy and reliability. The dual-mode assay proposed in this work provides an alternative scheme to meet the need of sensing target compounds in complex matrices.

Phosphorus Modulated Peroxidase-Like Activity of Carbon Dots for Colorimetric Detection of Acid Phosphatase.

The precise regulation of nanoenzyme activity is of great significance for application to biosensing analysis. Herein, the peroxidase-like activity of carbon dots was effectively modulated by doping phosphorus, which was successfully employed for sensitive, selective detection of acid phosphatase (ACP). Phosphorus-doped carbon dots (P-CDs) with excellent peroxidase-like activity were synthesized by a one-pot hydrothermal method, and the catalytic activity could be easily modulated by controlling the additional amount of precursor phytic acid. P-CDs could effectively catalyze the oxidation of colorless 3,3',5,5'-tetramethylbenzidine (TMB) to blue TMB oxidation products in the presence of hydrogen peroxide. While ACP was able to catalyze the hydrolysis of L-ascorbyl-2-phosphate trisodium salt (AAP) to produce ascorbic acid (AA), which inhibited the peroxidase-like activity of P-CDs, by combining P-CDs nanoenzymes and ACP-catalyzed hydrolysis the colorimetric method was established for ACP detection. The absorbance variation showed a good linear relationship with ACP concentration in the range of 0.4-4.0 mU/mL with a limit of detection at 0.12 mU/mL. In addition, the method was successfully applied to detect ACP in human serum samples with recoveries in the range of 98.7-101.6%. The work provides an effective strategy for regulating nanoenzymes activity and a low-cost detection technique for ACP.

Analytical methods for selectively determining hydrogen peroxide, peroxymonosulfate and peroxydisulfate in their binary mixtures.

Hydrogen peroxide (H2O2), peroxymonosulfate (PMS), and peroxydisulfate (PDS) are key bulk oxidants in many advanced oxidation processes (AOPs) for treating chemically contaminated water. In some systems these peroxides may coexist in solution either through intentional co-addition or their inadvertent formation (especially H2O2) due to reaction chemistry. While many analytical methods to determine these peroxides individually have been established, mutual interference among the peroxides in such methods has seldom been evaluated, and new methods or variants of established methods to selectively determine peroxides in binary mixtures are lacking. We re-examined five established colorimetric methods-the Permanganate, Titanium Oxalate (Ti-oxalate), Iodide, N.N­diethyl-p-phenylenediamine (DPD), and 2,20-azino-bis(3-ethylbenzothiazoline-6-sulfonate (ABTS) methods-for mutual interference among peroxides and devised variants of these methods for selectively quantifying one peroxide in the presence of another. Hydrogen peroxide can be selectively determined by the Permanganate method at short reaction time; by the Ti-oxalate method; by the DPD method with added peroxidase (POD); or by the ABTS method with added POD. PMS can be selectively determined by the Iodide method; by the DPD or ABTS methods with added iodide ion as catalyst; or by the DPD method with added catalase (CAT) (with co-existing H2O2 but not PDS). The DPD method can be used to determine PDS without interference by H2O2 and-provided the sample is pretreated with l-histidine-without interference by PMS. The recommended methods were successfully applied to binary peroxide mixtures in complex waters, including a tap water and a synthetic water. Overall, the new selective methods will assist mechanistic investigation of AOPs based on these peroxides and support efforts to apply them commercially.

Dual-mode self-powered photoelectrochemical and colorimetric determination of procalcitonin accomplished by multienzyme-expressed Ni4Cu2 bimetallic hollow nanospheres and spherical nanoflower-MoS2/Cu2ZnSnS4/Bi2S3.

Bacterial infections, viral infections and autoimmune diseases pose a considerable threat to human health. Procalcitonin (PCT) has emerged as a biomarker for the detection of these diseases. To ensure accurate and reliable results, we propose a dual-mode approach that incorporates self-validation and self-correction mechanisms. Herein, we develop a dual-mode self-powered photoelectrochemical (PEC) and colorimetric sensor to determine PCT. The self-powered PEC sensor was constructed with a photoanode of spherical nanoflower-MoS2/Cu2ZnSnS4/Bi2S3 material and a photocathode of CuInS2 material. Ni4Cu2 bimetallic hollow nanospheres (BHNs) possess superoxide dismutase and catalase performance, which facilitate superoxide anion radical (·O2-) and H2O2 circulating generation, promoting the separation of photogenerated electrons and holes to amplify photocurrent signal. Thus Ni4Cu2 BHNs is used as a marker material for PEC sensor. Meanwhile, in colorimetric mode, Ni4Cu2 BHNs converts blue oxTMB to a colourless TMB for colorimetric detection of PCT. Based on this principle, dual-mode determination of PCT with high sensitivity is achieved. The dual-mode method not only demonstrates outstanding properties and practicability, but also presents an effective, highly efficient and reliable method for detecting PCT.

Mechanistic investigation and dual-mode colorimetric-chemiluminescent detection of glyphosate based on the specific inhibition of Fe3O4@Cu nanozyme peroxidase-like activity.

In this study, a dual-mode colorimetric/CL nanosensor was developed for glyphosate detection based on the specific inhibition of Fe3O4@Cu peroxidase-like activity. Synthesized Fe3O4@Cu exhibited high levels of peroxidase-like activity that triggered the oxidation of luminol/3,3',5,5'-tetramethyl benzidine dihydrochloride (TMB) to excited-state 3-aminophthalic acid/blue oxTMB, thereby delivering a CL signal/visible colorimetric signal, however, the presence of glyphosate inhibited this activity, resulting in a decrease in signal strength. In-depth investigation revealed that this inhibitory mechanism occurs via two pathways: one in which glyphosate chelates with Fe(III)/Cu(II) and occupy the catalytical active sites of Fe3O4@Cu, thereby decreasing the generation of OH, and another in which glyphosate competes with TMB to consume generated OH, thus reducing the oxidation of TMB. This mechanism formed the basis of our novel dual-mode colorimetric/CL glyphosate nanosensor, which achieved limits of detection (LODs) of 0.086 µg/mL and 0.019 µg/mL in tests, thus demonstrating its significant potential for on-site glyphosate monitoring.

Nitrate Reductase Assay for Rapid Determination of Methicillin-Resistant Staphylococcus aureus Clinical Isolates.

OBJECTIVE: To evaluate the performance of nitrate reductase assay (NRA), a rapid, colorimetric method for the determination of methicillin resistance in Staphylococcus aureus isolates obtained from the culture collection of the Akdeniz University Hospital Central Laboratory, Antalya, Türkiye. MATERIALS AND METHODS: Identification for all 290 S aureus isolates at the species level was performed via matrix-assisted laser desorption/ionization-time of flight. Isolates were tested with NRA for methicillin resistance. The cefoxitin broth microdilution (BMD) method recommended by the Clinical and Laboratory Standards Institute was used as the reference method in the study. S aureus ATCC 29213 and S aureus ATCC 43300 strains were used for quality control. RESULTS: According to Food and Drug Administration criteria, the category agreement between NRA and BMD was found to be 100%. The essential agreement between both methods was determined to be 96.20%. There is no minor, major, or extremely major discrepancy between both methods. CONCLUSION: The results show that NRA is a rapid, practical, and reliable colorimetric method for detecting MRSA.

Single-atom Fe nanozymes with excellent oxidase-like and laccase-like activity for colorimetric detection of ascorbic acid and hydroquinone.

Although traditional Fe-based nanozymes have shown great potential, generally only a small proportion of the Fe atoms on the catalyst's surface are used. Herein, we synthesized single-atom Fe on N-doped graphene nanosheets (Fe-CNG) with high atom utilization efficiency and a unique coordination structure. Active oxygen species including superoxide radicals (O2•-) and singlet oxygen (1O2) were efficiently generated from the interaction of the Fe-CNG with dissolved oxygen in acidic conditions. The Fe-CNG nanozymes were found to display enhanced oxidase-like and laccase-like activity, with Vmax of 2.07 × 10-7 M∙S-1 and 4.54 × 10-8 M∙S-1 and Km of 0.324 mM and 0.082 mM, respectively, which is mainly due to Fe active centers coordinating with O and N atoms simultaneously. The oxidase-like performance of the Fe-CNG can be effectively inhibited by ascorbic acid (AA) or hydroquinone (HQ), which can directly obstruct the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB). Therefore, a direct and sensitive colorimetric method for the detection of AA and HQ activity was established, which exhibited good linear detection and limit of detection (LOD) of 0.048 µM and 0.025 µM, respectively. Moreover, a colorimetric method based on the Fe-CNG catalyst was fabricated for detecting the concentration of AA in vitamin C. Therefore, this work offers a new method for preparing a single-atom catalyst (SAC) nanozyme and a promising strategy for detecting AA and HQ.

Rapid and naked-eye colorimetric detection of ultra trace sumatriptan in drinking water, saliva, and human urine samples based on the aggregation of gold nanoparticles.

In this study, the determination of sumatriptan (SUM) was performed using a simple, rapid, and precise colorimetric method based on the surface plasmon resonance (SPR) feature of gold nanoparticles (AuNPs). By adding SUM, the aggregation was observed in AuNPs with red-to-blue color shifts. The size distribution of NPs was estimated before and after adding SUM via dynamic light scattering (DLS), which was found to be 15.34 and 97.45 nm, respectively. Characterization of AuNPs, SUM, and AuNPs in combination with SUM was investigated using transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy (FTIR). Examining the effect of pH, the volume of buffer, the concentration of AuNPs, interaction time, and ionic strength revealed that their optimal values were 6, 100 µL, 5 µM, 14 min, and 12 µg L-1, respectively. The suggested method was able to determine the amount of SUM in a linear range of 10 to 250 µg L-1 with a limit of detection (LOD) and limit of quantification (LOQ) of 0.392 and 1.03 µg L-1, respectively. This approach was successfully applied to determine SUM in drinking water, saliva, and human urine samples with relative standard deviations (RSD) lower than 0.03%, 0.3%, and 1.0%, respectively.

A dual-modal biosensor coupling cooperative catalysis strategy for sensitive detection of AFB1 in agri-products.

Herein, the cooperative catalysis effect between nanocomposite (AgPd NPs/POD-M/PEI-rGO) and horseradish peroxidase (HRP) was applied for the fast and sensitive detection of aflatoxin B1 (AFB1). Upon specific and competitive binding of HRP@DNA and AFB1 to cDNA, the working electrode presented different catalytic capacities for supporting electrolytes (TMB and H2O2). In the redox mechanism of TMB and H2O2, HRP and nanocomposite effectively catalyzed the oxidization of TMB to form the one-electron oxidation intermediate TMB+, and contributed the electrical signals and absorbance signals. Electrochemistry and colorimetric analyses were successfully realized for AFB1 detection with 0.2 pg/mL and 8 pg/mL of detection limits, respectively, which is much lower than that of traditional HPLC methods. Overall, this method had significant reliability and sensitivity, offering a promising potential for conveniently evaluating the quality of agri-products polluted with AFB1. Moreover, this approach provides a new idea for fast and accurate detection of mycotoxin.

Ultrasensitive colorimetric detection of tetracyclines based on in-situ growth of gold nanoflowers.

A colorimetric method based on in-situ generation of gold nanoflowers for the detection of tetracyclines (TCs) was proposed. We found that gold nanoflowers could be formed in the HAuCl4-NH2OH redox reaction directly without the addition of small-sized gold nanoparticles (Au NPs) as seeds when an alkaline borax buffer solution was employed as the reaction medium. Interestingly, the shape and size of the generated gold nanoflowers were regulated with TC. Briefly, large flower-like gold nanoparticles were formed with a low concentration of TC while small spherical gold nanoparticles were generated with a high concentration of TC. The generated gold nanoflowers exhibited different surface plasmon absorption (SPR) properties. Thus, a simple and rapid colorimetric method was established for the detection of TC antibiotics. This method exhibited high sensitivity for the detection of TC, oxytetracycline (OTC), and doxycycline (DC) with detection limits of 2.23 nM, 1.19 nM, and 5.81 nM, respectively. The proposed colorimetric method was applied to the determination of TC in both milk samples and water samples.

[Improvement of Nitrite Analysis Method in Food Products].

The conventional analysis method has problems with extraction efficiency, operability, and reproducibility. In this study, we attempted to solve these problems and improve the analytical method to obtain sufficient extraction efficiency and good operability and accuracy. The conventional method was able to get sufficient extraction in dried meat products, where the extraction efficiency of the conventional method was low, by increasing the concentration of sodium hydroxide solution at the time of homogenization. Suction filtration after adding the defoaming agent was added allowed for accurate volume adjustment. The turbidity of the extract caused by insufficient addition of zinc acetate solution was removed by increasing the amount of zinc acetate solution that was added. Turbidity caused by starch was removed by adding pancreatin. The RSD of the quantitative values was improved by adding sodium hydroxide solution and 80-90℃ water and immediately homogenizing. Furthermore, by changing the dilution factor of the extract solution in the colorimetric method, the inhibition of coloration by reducing substances was suppressed, and more accurate quantitative values could be obtained than with the conventional method. The recovery rate was 78.5-105% (RSD 0.7-5.8%), which was a good result. This method was considered to be a useful analytical method that can contribute to improving the inspection accuracy of nitrite ion analysis.

Dual-modification strategy of Co(II) and g-C3N4 to CuS for efficient colorimetric determination of thioglycolic acid in daily cosmetics.

A conventional colorimetric method based on CuS-catalyzed H2O2 is improved by a dual-modification strategy and employed for thioglycolic acid (TGA) determination. The doping of Co(II) can enhance ion exchange efficiency. Meanwhile, the modification of g-C3N4 can increase specific surface area and decrease unspecific aggregation. The constructed g-C3N4/Co-CuS nanocomposite exhibited a favorable catalytic feature. A Michaelis constant (Km) value of 0.02 mM has been achieved, which is 1/160 of those of CuS and horseradish peroxidase (HRP). The g-C3N4/Co-CuS displays a rapid color response in 3 min and resulted in a stable measurable signal within 10 min. In the determination procedure, the sulfhydryl contained in TGA is capable of preventing TMB oxidation via competing the ·OH produced by catalysis and caused a color distinction that is related to the TGA amount. The distinctions of absorbance (λmax = 652 nm) of different concentrations of TGA are recorded. Linearity is obtained in the ranges of 2.5 - 20 µM and 20 - 160 µM, and the LOD is 0.14 µM. In the real sample assays of perm agent and Qianhu lake water, the recoveries were 96.70 - 106.84% and 100.21 - 101.90%, respectively. This demonstrates that the proposed dual-modification strategy for CuS contributes to highly efficient and convenient determination of TGA in daily cosmetics and water analysis.

The rapid high-throughput screening of ω-transaminases via a colorimetric method using aliphatic α-diketones as amino acceptors.

ω-Transaminases (ω-TAs) are widely available for the production of chiral amines and unnatural amino acids. Herein, a rapid spectrophotometric method was developed for screening ω-TAs based on the colored products that can be generated from transamination reactions between aliphatic α-diketones and amino donors catalyzed by ω-TAs. The possible mechanism of the formation of the colored product was investigated according to LC-Q-TOF-MS analysis. Among seven diketones, 2,3-butanedione was selected as the most suitable amino acceptor for colorimetric screening of ω-TAs with high efficiency, high sensitivity, and low background interference. Meanwhile, the absorbance of the colored product generated by 2,3-butanedione catalyzed by ω-TAs in this method was linearly correlated with the results by HPLC analysis. This method was also confirmed to effectively screen ω-TA mutants with high activity towards isopropylamine.