Dithiobis 2-nitrobenzoic Acid Research Articles

Responsive Supramolecular Nanomicelles Formed through Self-Assembly of Acyclic Cucurbit[n]uril for Targeted Drug Delivery to Cancer Cells.

The supramolecular drug delivery systems (SDDSs) based on host-guest recognition through noncovalent interactions, capable of responsive behavior and dynamic switching to external stimuli, have attracted considerable attention in cancer therapy. In this study, a targeted dual-functional drug delivery system was designed and synthesized. A hydrophilic macrocyclic host molecule (acyclic cucurbit[n]uril ACB) was modified with folic acid (FA) as a targeting ligand. The guest molecule consists of a disulfide bond attached to adamantane (DA) and cannabidiol (CBD) at both ends of the response element of glutathione. Recognition and self-assembly of host and guest molecules successfully functionalize supramolecular nanomicelles (SNMs), targeting cancer cells and releasing drugs in a high glutathione environment. The interactions between host and guest molecules were investigated by using nuclear magnetic resonance (NMR), fluorescence titration, Fourier-transform infrared spectroscopy (FT-IR), and thermal analysis (TGA). Transmission electron microscopy (TEM) and dynamic light scattering (DLS) confirmed the nanostructure of the SNMs. Experimentation with 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) demonstrated the responsiveness of SNMs to glutathione (GSH). In vitro cytotoxicity assays demonstrated that SNMs had a greater targeting efficacy for four types of cancer cells (HeLa, HCT-116, A549, and HepG2) compared to normal 293T cells. Cellular uptake studies revealed that HeLa cells more readily absorbed SNMs, leading to their accumulation in the tumor cell cytoplasm. Fluorescence colocalization assays verified that SNMs efficiently accumulated in organelles related to energy metabolism and signaling, including mitochondria and the endoplasmic reticulum, affecting cellular metabolic death. Both flow cytometry and confocal nuclear staining assays confirmed that SNMs effectively induced apoptosis over time, ultimately resulting in the death of cancer cells. These findings demonstrate that SNMs exhibit excellent targeting ability, responsiveness, high bioavailability, and stability, suggesting significant potential in drug delivery applications.

Ellman's Assay on the Surface: Thiol Quantification of Human Cofilin-1 Protein through Surface Plasmon Resonance.

Oxidative stress on cysteine (Cys)-containing proteins has been associated with physiological disorders, as suggested for the human cofilin-1 (CFL-1) protein, in which the oxidized residues are likely implicated in the aggregation process of α-synuclein, leading to severe neuronal injuries. Considering the relevance of the oxidation state of cysteine, quantification of thiols may offer a guide for the development of effective therapies. This work presents, for the very first time, thiol quantification within CFL-1 in solution and on the surface following classic and adapted versions of Ellman's assay. The 1:1 stoichiometric Ellman's reaction occurs between 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB), and the free thiol of the cysteine residue, producing two 2-nitro-5-thiobenzoate (TNB2-) ions, one of which is released into the medium. While in solution, the thiol concentration was determined by the absorbance of the released TNB2-, on the surface, the mass of the attached TNB2- ion to the protein allowed the quantification by means of the multiparametric surface plasmon resonance (MP-SPR) technique. The SPR angle change after the interaction of DTNB with immobilized CFL-1 gave a surface coverage of 26.5 pmol cm-2 for the TNB2- ions (ΓTNB2-). The ratio of this value to the surface coverage of CFL-1, ΓCFL-1 = 6.5 ± 0.6 pmol cm-2 (also determined by MP-SPR), gave 4.1 as expected for this protein, i.e., CFL-1 contains four Cys residues in its native form (reduced state). A control experiment with adsorbed oxidized protein showed no SPR angle change, thus proving the reliability of adapting Ellman's assay to the surface using the MP-SPR technique. The results presented in this work provide evidence of the heterogenization of Ellman's assay, offering a novel perspective for studying thiol-containing species within proteins. This may be particularly useful to ensure further studies on drug-like molecules that can be carried out with validated oxidized or reduced CFL-1 or other analogous systems.

A dual-mode immunochromatographic sensor with photothermal and surface-enhanced Raman scattering for sensitive detection of aflatoxin B1

Multimodal lateral flow immunoassay has displayed the great potential to improve the flexibility and practicality of point-of-care testing. Herein, this study developed a dual-mode photothermal (PT) and surface-enhanced Raman scattering (SERS) immunochromatographic sensor for sensitive detection of aflatoxin B1 (AFB1). The bifunctional waxberry-like core-satellite nanoparticles loaded with 5,5′-Dithiobis (2-nitrobenzoic acid) (DTNB) were prepared and coupled with antibody to form PT@SERS nanoprobes for qualitative and quantitative detection of AFB1. The photothermal conversion efficiency and SERS enhancement factor of the nanoprobes were 42.11 % and 1.59 × 107, respectively. Under the optimal conditions, the limit of detection of PT assay was 0.033 ng/mL with a linear range of 0.05–10 ng/mL (R2 = 0.997); the limit of detection of SERS assay was 0.0073 ng/mL with a linear range of 0.005–10 ng/mL (R2 = 0.998). The results of the specificity analysis indicated no cross-reactions with the other toxins. The recoveries of the spiked corn and peanut were from 85.39 % to 112.15 % (PT assay) and 80.04 % to 106.57 % (SERS assay), respectively. The assay demonstrated that the developed dual-mode sensor provided a promising option for achieving the rapid detection of AFB1.

MRI-Guided Tumor Therapy Based on Synergy of Ferroptosis, Immunosuppression Reversal and Disulfidptosis.

Triple negative breast cancer (TNBC) cells have a high demand for oxygen and glucose to fuel their growth and spread, shaping the tumor microenvironment (TME) that can lead to a weakened immune system by hypoxia and increased risk of metastasis. To disrupt this vicious circle and improve cancer therapeutic efficacy, a strategy is proposed with the synergy of ferroptosis, immunosuppression reversal and disulfidptosis. An intelligent nanomedicine GOx-IA@HMON@IO is successfully developed to realize this strategy. The Fe release behaviors indicate the glutathione (GSH)-responsive degradation of HMON. The results of titanium sulfate assay, electron spin resonance (ESR) spectra, 5,5'-Dithiobis-(2-nitrobenzoicacid (DTNB) assay and T1-weighted magnetic resonance imaging (MRI) demonstrate the mechanism of the intelligent iron atom (IA)-based cascade reactions for GOx-IA@HMON@IO, generating robust reactive oxygen species (ROS). The results on cells and mice reinforce the synergistic mechanisms of ferroptosis, immunosuppression reversal and disulfidptosis triggered by the GOx-IA@HMON@IO with the following steps: 1) GSH peroxidase 4 (GPX4) depletion by disulfidptosis; 2) IA-based cascade reactions; 3) tumor hypoxia reversal; 4) immunosuppression reversal; 5) GPX4 depletion by immunotherapy. Based on the synergistic mechanisms of ferroptosis, immunosuppression reversal and disulfidptosis, the intelligent nanomedicine GOx-IA@HMON@IO can be used for MRI-guided tumor therapy with excellent biocompatibility and safety.

Extracellular organic disulfide reduction by Shewanella oneidensis MR-1.

Microbial reduction of organic disulfides affects the macromolecular structure and chemical reactivity of natural organic matter. Currently, the enzymatic pathways that mediate disulfide bond reduction in soil and sedimentary organic matter are poorly understood. In this study, we examined the extracellular reduction of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) by Shewanella oneidensis strain MR-1. A transposon mutagenesis screen performed with S. oneidensis resulted in the isolation of a mutant that lost ~90% of its DTNB reduction activity. Genome sequencing of the mutant strain revealed that the transposon was inserted into the dsbD gene, which encodes for an oxidoreductase involved in cytochrome c maturation. Complementation of the mutant strain with the wild-type dsbD partially restored DTNB reduction activity. Because DsbD catalyzes a critical step in the assembly of multi-heme c-type cytochromes, we further investigated the role of extracellular electron transfer cytochromes in organic disulfide reduction. The results indicated that mutants lacking proteins in the Mtr system were severely impaired in their ability to reduce DTNB. These findings provide new insights into extracellular organic disulfide reduction and the enzymatic pathways of organic sulfur redox cycling.IMPORTANCEOrganic sulfur compounds in soils and sediments are held together by disulfide bonds. This study investigates how Shewanella oneidensis breaks apart extracellular organic sulfur compounds. The results show that an enzyme involved in the assembly of c-type cytochromes as well as proteins in the Mtr respiratory pathway is needed for S. oneidensis to transfer electrons from the cell surface to extracellular organic disulfides. These findings have important implications for understanding how organic sulfur decomposes in terrestrial ecosystems.

Enzymatic characterization of five thioredoxins and a thioredoxin reductase from Myxococcus xanthus.

Thioredoxin (Trx) is a disulfide-containing redox protein that functions as a disulfide oxidoreductase. Myxococcus xanthus contains five Trxs (Trx1-Trx5) and one Trx reductase (TrxR). Trxs typically have a CGPC active-site motif; however, M. xanthus Trxs have slightly different active-site sequences, with the exception of Trx4. The five Trxs of M. xanthus exhibited reduced activities against insulin, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), cystine, glutathione disulfide (GSSG), S-nitrosoglutathione (GSNO), and H2O2 in the presence of TrxR. Myxococcus xanthus adenylate kinase and serine/threonine phosphatase activities, which were increased by the addition of dithiothreitol, were activated by the addition of Trxs and TrxR. Among these, Trx1, which has a CAPC sequence in its active site, exhibited the highest reducing activity with the exception of GSNO. Myxococcus xanthus TrxR showed weak reducing activity towards DTNB, GSSG, GSNO, and H2O2, suggesting that it has broad substrate specificity, unlike previously reported low-molecular-weight TrxRs. TrxR reduced oxidized Trx1 as the best substrate, with a kcat/Km value of 0.253 min-1 µM-1, which was 10-28-fold higher than that of the other Trxs. These results suggest that all Trxs possess reducing activity and that Trx1 may be the most functional in M. xanthus because TrxR most efficiently reduces oxidized Trx1.

Purification of thioredoxin reductase from Spirulina platensis by affinity chromatography and investigation of kinetic properties

The thioredoxin system consists of thioredoxin (Trx), thioredoxin reductase (TrxR) and nicotinamide adenine dinucleotide phosphate (NADPH). Spirulina platensis, which is one of the blue-green algae in the form of spiral rings, belongs to the cyanobacteria class. Spirulina platensis can produce Trx under stress conditions. If it can produce Trx, it also has TrxR activity. Therefore, in this study, the TrxR enzyme was purified for the first time from Spirulina platensis, an algae the most grown and also used as a nutritional supplement in the world. A two-step purification process was used: preparation of the homogenate and 2′,5′-ADP sepharose 4B affinity chromatography. The enzyme was purified with a purification fold of 1059.51, a recovery yield of 9.7 %, and a specific activity of 5.77 U/mg protein. The purified TrxR was tested for purity by SDS-PAGE. The molecular weight of its subunit was found to be about 45 kDa. Optimum pH, temperature and ionic strength of the enzyme were pH 7.0, 40 °C and 750 mM in phosphate buffer respectively. The Michaelis constant (Km) and maximum velocity of enzyme (Vmax) values for NADPH and 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) are 5 μM and 2.2 mM, and 0.0033 U/mL and 0.0044 U/mL, respectively. Storage stability of the purified enzyme was determined at several temperatures. The inhibition effects of Ag+, Cu2+, Al3+ and Se4+ metal ions on the purified TrxR activity were investigated in vitro. While Se4+ ion increased the enzyme activity, other tested metal ions showed different type of inhibitory effects on the Lineweaver–Burk graphs.

Purification and characterization of thioredoxin reductase enzyme from commercial Spirulina platensis tablets by affinity chromatography and investigation of the effects of some chemicals and drugs on enzyme activity.

Thioredoxin reductase (TrxR, enzyme code [E.C.] 1.6.4.5) is a widely distributed flavoenzyme that catalyzes nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reduction of thioredoxin and many other physiologically important substrates. Spirulina platensis is a blue-green algae that is often used as a dietary supplement. S. platensis is rich in protein, lipid, polysaccharide, pigment, carotenoid, enzyme, vitamins and many other chemicals and exhibits a variety of pharmacological functions. In the present study, a simple and efficient method to purify TrxR from S. platensis tablets is reported. The extractions were carried out using two different methods: heat denaturation and 2',5'-adenosine diphosphate Sepharose 4B affinity chromatography. The enzyme was purified by 415.04-fold over the crude extract, with a 19% yield, and specific activity of 0.7640U/mg protein. Optimum pH, temperature and ionic strength of the enzyme activity, as well as the Michaelis constant (Km ) and maximum velocity of enzyme (Vmax ) values for NADPH and 5,5'-dithiobis(2-nitrobenzoic acid) were determined. Tested metal ions, vitamins, and drugs showed inhibition effects, except Se4+ ion, cefazolin sodium, teicoplanin, and tobramycin that increased the enzyme activity in vitro. Ag+ , Cu2+ , Mg2+ , Ni2+ , Pb2+ , Zn2+ , Al3+ , Cr3+ , Fe3+ , and V4+ ions; vitamin B3 , vitamin B6 , vitamin C, and vitamin U and aciclovir, azithromycin, benzyladenine, ceftriaxone sodium, clarithromycin, diclofenac, gibberellic acid, glurenorm, indole-3-butyric acid, ketorolac, metformin, mupirocin, mupirocin calcium, paracetamol, and tenofovir had inhibitory effects on TrxR. Ag+ exhibited stronger inhibition than 1-chloro-2,4-dinitrobenzene (a positive control).

Express formation and characterization of SERS-active substrate from a non-degradable Ag-Nb-N-O film

In this work, we propose an original approach for rapid and easy conversion of a thin Ag-Nb-N-O film into surface enhanced Raman scattering (SERS) active substrate. The initial Ag-Nb-N-O substrate has a long shelf life because silver particles are prevalently accumulated inside the film and possess resistivity to degradation. An activation of the substrate is carried out just before the SERS-measurements by a 15-second microwave heating the Ag-Nb-N-O film at 800 W, which facilitates immediate extrusion of internal silver to the surface. This leads to the formation of a dense array of Ag particles that show an improved SERS-activity. We compare SERS-activity of the initial and microwave-treated Ag-Nb-N-O films with that of a control substrate based on Ag nanoparticles grown on the SiO2 surface. Both initial and control substrates are aged in a moist air for 60 days before the SERS-measurements. An intensity of the SERS-signal from 10-5 M 5,5′-Dithiobis(2-nitrobenzoic acid) or DTNB on the activated Ag-Nb-N-O film is approx. 270 higher than that on the control Ag nanoparticles. The TEM and EDS studies of the initial Ag-Nb-N-O film prove its stability to sulfidization while the control Ag nanoparticles are found to bind to sulfur from the moist air.

Magnetic Mesoporous Silica Nanoparticles Functionalized with 5,5′-Dithiobis(2-Nitrobenzoic Acid) for Highly Efficient Removal of Organic Dyes from Contaminated Water

The removal of organic pollutants has become an increasingly important environmental concern. In recent years, there has been significant research into the use of nanomaterials for removing organic dyes in single-component systems. In this study, magnetic mesoporous silica nanoparticles (magnetic MSNs) were prepared with an average particle size of 170 nm. Iron oxide nanoparticles (20 nm) were embedded within the mesoporous silica structure. These nanoparticles were functionalized with amine, derivatized with thiol, and then reacted with 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB). The presence of DTNB molecules attached to the surface was confirmed by FTIR spectra, as evidenced by the appearance of peaks at ∼1528 cm−1 and ∼1365 cm−1. The nanoadsorbents demonstrated high removal efficiency for bromothymol blue (BT) and methyl orange (MO) at a pH below 5, with a maximum adsorption capacity of 139.27 mg/g and 101.62 mg/g for BT and MO, respectively. The linear regression coefficient value suggested that the adsorption of BT and MO onto magnetic MSNs-DTNB followed the Langmuir isotherm and second-order process. Overall, these findings suggest that magnetic MSNs-DTNB could be a promising nanoadsorbent for removing organic pollutants from contaminated water sources.

Thiolated chitosan hydrogel-embedded niosomes: A promising crocin delivery system toward the management of aphthous stomatitis

Aphthous stomatitis is a common inflammatory oral disease with challenging management. Crocin is a natural carotenoid that has shown great anti-inflammatory properties. The aim of this study was to develop thiolated chitosan (TCS)-based hydrogels containing niosomes to serve as a mucoadhesive crocin delivery system for aphthous stomatitis. Crocin-loaded niosomes were prepared and the impact of surfactant type, cholesterol content, and lipid to drug ratio on the characteristics of niosomes was evaluated. TCS was synthesized and the success of thiolation was investigated. The optimum niosomal formulation was loaded into the hydrogel and the hybrid system was characterized regarding the morphology, mucoadhesive properties, viscosity, chemical structure, in vitro drug release, and in vivo efficacy. The optimized niosome formulation showed 77% crocin entrapment, a particle diameter of 59 nm, and a zeta potential of −18 mV. The niosome-containing hydrogel exhibited pseudoplastic rheological behavior, mucoadhesive properties, suitable swelling, and sustained release of crocin. In vivo study revealed that the niosome-containing hydrogel improved ulcer healing and decreased the expression of tumor necrosis factor-alpha (TNF-α) and p53 while increasing the expression of vascular endothelial growth factor (VEGF) and alpha-smooth muscle actin (α-SMA). Collectively, TCS hydrogel-embedded crocin-loaded niosomes is a promising therapeutic option for aphthous stomatitis. Chemical compounds studied in this articleCrocin (PubChem CID: 5281233)Chitosan (PubChem CID: 71853)Thioglycolic acid (PubChem CID: 1133)1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (PubChem CID: 2723939)5,5′-dithiobis (2-nitrobenzoic acid) (PubChem CID: 6254)Cholesterol (PubChem CID: 5997)

Enzyme colorimetric cellulose membrane bioactivity strips based on acetylcholinesterase immobilization for inhibitors preliminary screening

To quickly screen the active pharmaceutical ingredient that can be used as acetylcholinesterase inhibitors (AChEIs) to treat Alzheimer's disease, an enzyme colorimetric cellulose membrane bioactivity strip (CBS) was developed for simple and rapid screening of AChEIs. The amino group of acetylcholinesterase (AChE) undergoes Schiff base reaction with the aldehyde group on the oxidized cellulose membranes, then the AChE was covalently cross-linking on the surface of cellulose membranes, enabling the screening based on Ellman's enzyme colorimetric method. When the enzyme activity of AChE was inhibited after incubation with inhibitors, the hydrolysis of S-Acetylthiocholine iodide decreased, consequently, the 5-thio-2-nitrobenzoic acid generated by the reaction with 5,5′-dithiobis (2-nitrobenzoic acid) also decreased, leading to a decreased color intensity. In addition, CBSs had fast chromogenic time, excellent specificity, and extraordinary storage stability. Tacrine and Donepezil were used as representative inhibitors during the detection, while their IC50 and limit of detection were determined.Therefore, our work not only established a platform for effective preliminary screening of AChEIs but also inspired the further development of other cellulose membrane-based biosensors.

Simultaneous determination of intracellular reduced and oxidized glutathiones by the König reaction.

The König reaction is commonly used for the detection of cyanide and its derivatives, including thiocyanate and selenocyanate. We found that this reaction can be used to quantify glutathione fluorometrically, and applied it to the simultaneous determination of reduced and oxidized glutathiones (GSH and GSSG) using a conventional LC system with isocratic elution. The limits of detection were 6.04 nM and 9.84 nM for GSH and GSSG, respectively, and the limits of quantification were 18.3 nM and 29.8 nM, respectively. We also determined GSH and GSSG levels in PC12 cells exposed to paraquat, an oxidative stressor, and observed a decrease in GSH/GSSG ratio, as expected. Total GSH levels quantified by this method and by the conventional colorimetric method with 5,5'-dithiobis(2-nitrobenzoic acid) were comparable. Our new application of the König reaction offers a reliable and useful method to simultaneously quantify intracellular GSH and GSSG.

A SERS aptasensor for rapid detection of aflatoxin B1 in coix seed using satellite structured Fe3O4@Au nanocomposites

Coix seed is a minor cereal that has been used as a functional food in the Chinese diet due to its high nutritional value and health effects. These claimed health benefits include antioxidation, anti-cancer, anti-inflammation and enhancing immunological activity. But coix seed is easily contaminated by various mycotoxin, of which aflatoxin B 1 (AFB 1 ) is one of major threat to human and animal health. For the sake of detecting AFB 1 more effectively, a surface enhanced Raman spectroscopy (SERS) based aptasensor for rapid detection of AFB 1 in coix seed was developed. Fe 3 O 4 @Au nanoparticles (NPs) modified with aptamer which possess the ability of magnetic separation and excellent SERS activity were synthesized and characterized by transmission electron microscope. Ag NPs modified with 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) and complementary DNA (cDNA) were employed as SERS tags. Controllable hot spots were built by the combination of Fe 3 O 4 @Au NPs and Ag NPs through AFB 1 aptamer and cDNA, and also by the interparticle nanogapped between the different size of Au NPs. Based on the principle of competitive binding between AFB 1 and cDNA for aptamer, the rapid quantitative detection of AFB 1 in coix seed was realized. The limit of detection (LOD) was determined to be 0.0060 ng/mL, and a broad linear range between 0.01 and 100 ng/mL was achieved. The spiked recovery rates of coix seed sample were 98.2%–101.2%. The results demonstrated the promising potential of this aptasensor in rapid and effective detection of AFB 1 in foodstuffs. • A SERS aptasensor for the rapid detection of AFB 1 in coix seed was constructed. • Nanogapped Au NPs can provide a lot of controllable hot spots. • GMNPs modified with aptamer exhibited enrichment ability and excellent SERS activity.

A Sensitive and Selective Colorimetric Method Based on the Acetylcholinesterase-like Activity of Zeolitic Imidazolate Framework-8 and Its Applications.

In this study, a simple colorimetric method was established to detect copper ion (Cu2+), sulfathiazole (ST), and glucose based on the acetylcholinesterase (AChE)-like activity of zeolitic imidazolate framework-8 (ZIF-8). The AChE-like activity of ZIF-8 can hydrolyze acetylthiocholine chloride (ATCh) to thiocholine (TCh), which will further react with 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) to generate 2-nitro-5-thiobenzoic acid (TNB) that has a maximum absorption peak at 405 nm. The effects of different reaction conditions (buffer pH, the volume of ZIF-8, reaction temperature and time, and ATCh concentration) were investigated. Under the optimized conditions, the value of the Michaelis-Menten constant (Km) is measured to be 0.83 mM, which shows a high affinity toward the substrate (ATCh). Meanwhile, the ZIF-8 has good storage stability, which can maintain more than 80.0% of its initial activity after 30 days of storage at room temperature, and the relative standard deviation (RSD) of batch-to-batch (n = 3) is 5.1%. The linear dependences are obtained based on the AChE-like activity of ZIF-8 for the detection of Cu2+, ST, and glucose in the ranges of 0.021-1.34 and 5.38-689.66 µM, 43.10-517.24 µM, and 0.0054-1.40 mM, respectively. The limit of detections (LODs) are calculated to be 20.00 nM, 9.25 µM, and 5.24 µM, respectively. Moreover, the sample spiked recoveries of Cu2+ in lake water, ST in milk, and glucose in strawberry samples were measured, and the results are in the range of 98.4-115.4% with the RSD (n = 3) lower than 3.3%. In addition, the method shows high selectivity in the real sample analysis.

Colorimetric test strip cassette readout with a smartphone for on-site and rapid screening test of carbamate pesticides in vegetables

A test strip cassette integrated with a smartphone detector was developed for the colorimetric detection of carbamate pesticides in vegetables. The influence of various experimental conditions was examined for the detection of the optimum analytical performance of the test strip cassette. The test strip cassette detection of carbamate pesticides was carried out by using 140 mM of acetylthiocholine, 13 mM of 5,5′-dithiobis (2-nitrobenzoic acid), temperature at 37 °C and pH of 8.0. The developed test strip cassette platform is rapid within 5 min. Based on the inhibition effect of carbaryl on acetylcholinesterase activity, the inhibition effect of carbaryl was proportional to its concentration ranging from 1 to 50 mg/L with the detection limit of 0.30 mg/L. The limit of quantitative value was determined to be 1.0 mg/L. The spiking carbaryl, the recoveries were within 96–103% and the relative standard deviations were less than 1.56%. The analysis of carbamate pesticides residual in vegetable samples gave the results in good agreement with the most widely used commercial test kit and UPLC-MS/MS method. The developed test strip cassette required only 5 min for an assay. With the simple procedures and tiny size, it showed a great promising for the developed test strip to work on-site for carbamate pesticides detection in vegetables.

Effects of exposure to glyphosate on oxidative stress, inflammation, and lung function in maize farmers, Northern Thailand

BackgroundGlyphosate is a herbicide which is commonly used in agricultural areas. However, previous studies on glyphosate exposure in farmers and their health are still scarce.MethodsA longitudinal pre-post study was performed among maize farmers. Information from questionnaires, urine and blood samples, and lung function were collected a day before and a day after glyphosate application in the morning. The urine samples were analyzed using liquid chromatography-tandem mass spectrometry to detect glyphosate levels. Serum samples were analyzed to detect malondialdehyde (MDA), glutathione (GHS), and C-reactive protein (CRP) levels using thiobarbituric acid, dithiobisnitrobenzoic acid, and nephelometry, respectively. Lung function performances were measured using a spirometer.ResultsA total of 180 maize farmers met the study inclusion criteria. After glyphosate application, it was found that increased urinary glyphosate levels contributed to increased serum MDA (β = 0.024, 95% CI = 0.000, 0.0047) and decreased serum GHS (β = -0.022, 95% CI = -0.037, -0.007), FEV1 (β = -0.134, 95% CI = -0.168, -0.100), FEV1/FVC (β = -0.062, 95% CI = -0.082, -0.042) and PEF (β = -0.952, 95% CI = -1.169, -0.735).ConclusionsExposure to glyphosate during glyphosate application had significant effects on oxidative stress and lung function in maize farmers.

Portable Rapid Paper-Based Device for Analysis of Carbamates and Organophosphates in Water Samples

Traditionally, the analysis of contaminants in water samples is performed using sophisticated techniques, namely HPLC, GC, and MS, requiring qualified personnel and laboratory infrastructure. Such methods are not feasible for the detection of contaminants in the field. In contrast, paper-based portable devices have been demonstrated as versatile, sustainable, and cheap solutions for such purposes. Paper consists of a pressed sheet of entangled hydrophilic cellulose fibres with excellent porosity. Such features enable the capillarity motion of liquids and the immobilization of active substances on the surface.Pesticides are critical pollutants in regions where agricultural activity is intense and represent a serious health risk for rural workers and their communities. These chemicals have been applied in enormous quantities to increase crop production in many countries. Carbamates (CBs) and organophosphates (OPs) are employed as active ingredients in industrial formulations of insecticides. They are considered highly toxic due to their inhibition action in the cholinergic transmission found in the nervous system of living organisms.This work aims to present a low-cost and simple operational paper-based testing device for the fast detection of CBs and OPs in water samples based on Ellman’s method of acetylcholinesterase (AChE) inhibition. We have named the proposed device “dip-and-fold” due to its how-to-use procedure: first, the device’s bottom edge is dipped in the water sample. The liquid moves up by capillarity towards the detection zone, in which the analytes find the AChE previously immobilized on the paper. The top edge of the paper is covered with a thin parafilm layer to avoid leaking.After a 10-minute incubation, the AChE is inhibited in the presence of CBs and/or OPs. Then, the device is folded to release both enzymatic substrate acetylthiocholine iodide (ACThI) and indicator 5,5′-Dithiobis (2-nitrobenzoic acid) (DTNB) from a secondary paper to the detection zone for the colorimetric reaction. The detection zone area remains white in the presence of CBs and/or OPs (inhibited AChE); otherwise, it becomes yellow in the absence of these pesticides (active AChE).It is worthy to emphasize that the dip-and-fold has all required ingredients immobilized onto the paper substrates prior to tests, which excludes the necessity of extra reagents and materials. Besides, the sample can be transferred to the device by capillarity motion when dipping it directly into the contaminated water, eliminating any risk of having contact with the pesticides. Experiment The dip-and-fold device was fabricated using two analytical papers and a backing card as a foldable scaffold. Blotting paper (Whatman GB003) was used as the main device’s component for the sample loading by capillarity as well as for the immobilization of 3 µL AChE (100 U/mL), using chitosan 1% (w/v, in acetic acid 2%), as a crosslinker. The secondary component was the filter paper (Whatman grade 4) in which the 3µL of the substrate ACThI (25mM) and 3 µL of DTNB (10mM) were impregnated on the surface. The detection tests were carried out using standard solutions of carbofuran, propoxur (both carbamates) and chlorpyriphos (an organophosphate) at concentrations between 0.1 and 0.0001 mM. Control samples of DI water were also tested. A minimum volume of methanol was required to dissolve the pesticides in water. Yellow-coloured results of the tests were evaluated by taking digital photos of the devices using a smartphone. The RGB channels of images were analyzed using ImageJ software. Results and Conclusion The result interpretation was performed by comparing the plotted curves of the blue channel (B) values obtained from the images of each tested device for the different analytes and their respective concentrations. The evaluation of the plotted curves confirmed that the yellow colour's intensity is inversely proportional to the concentration of the analytes in the sample, which means, the more intense the yellow, the lower the concentration of pesticides in the sample. The concentrations applied in this study meet the parameters proposed by the Ontario Drinking Water Quality Standards (ODWQS) and may be compared to other paper-based devices described in the literature.Both propoxur and carbofuran presented consistent results that were observed by a steady decrease of the B values related to their concentration. In contrast, the chlorpyrifos presented a non-linear curve, which can be attributed to the higher amount of methanol used as a co-solvent to dissolve it in water due to its extremely poor solubility.The dip-and-fold device presented an exclusive, eco-friendly, and economical testing platform for pesticide detection in water, which offers an attractive solution for water assessment in vulnerable areas exposed to these toxic chemicals. Figure 1

A Multicomponent Butyrylcholinesterase Preparation for Enzyme Inhibition-Based Assay of Organophosphorus Pesticides

A new method of producing butyrylcholinesterase (BChE) preparations, stable in storage and use, has been proposed. The BChE preparation is the enzyme co-immobilized with 0.2 M 5-5′-dithiobis (2-nitrobenzoic acid) in starch or gelatin gel. All experimental preparations retain enzyme activity for at least 300 d. The preparations based on gelatin gel show higher activity but lower sensitivity to the toxicants tested in this study compared to the starch gel-based preparations. A method has been proposed for integrated detection of anti-cholinesterase substances in aqueous solutions using the experimental preparation with immobilized BChE. After the additional incubation of the preparation with the immobilized enzyme in the solution of the analyte, the detection limits of malathion and pirimiphos-methyl determined using the IC20 values were below their maximum allowable concentrations—0.005 µM and 0.03 µM, respectively.

Selective Homocysteine Assay with Cucurbit[7]uril by pH Regulation.

We report the effect of pH on the supramolecular complexation of two biothiols, viz., homocysteine (Hcy) and cysteine (Cys), with cucurbit[7]uril (CB[7]). Under basic pH conditions, Cys did not complex with CB[7], whereas Hcy efficiently complexed with CB[7], as confirmed by 1H NMR spectroscopy and Ellman's reagent (5,5'-dithio-bis(2-nitrobenzoic acid), DTNB) assay. 1H NMR and Raman spectroscopic studies revealed that, in the absence of CB[7], Hcy auto-oxidized slowly (~36 h) to homocystine (HSSH) under basic pH conditions. However, the rate of Hcy oxidation increased by up to 150 fold in the presence of CB[7], as suggested by the DTNB assay. Thus, supramolecular complexation under basic pH conditions led to the formation of a HSSH-CB[7] complex, and not Hcy-CB[7]. The results indicate that Hcy is rapidly oxidized to HSSH under the catalysis of CB[7], which acts as a reaction chamber, in basic pH conditions. Our studies suggest that Hcy concentration, a risk factor for cardiovascular disease, can be selectively and more easily quantified by supramolecular complexation with CB [7].