Colorimetric Reagent Research Articles

Single-point calibration and standard addition assays on calibrant-loaded paper-based analytical devices

This article describes a new, simplified approach for performing quantitative colorimetric assays on paper-based analytical devices that uses calibrant-loaded paper devices to perform external calibration and standard addition calibration using one calibration point. Calibrant-loaded devices consist of sensing areas pre-loaded with a colored product which is produced from the reaction of a standard solution of the analyte with the appropriate colorimetric reagents. When the sample is added into the calibrant-loaded sensing zone the analytical signal (i.e. color intensity) increases proportionally to the concentration of the analyte in the tested sample. The total measured signal corresponds to the sum of the concentration of the analyte in the sample and the standard solution pre-stored in the device and is used to calculate the concentration of the analyte in the sample based on the principles of linear calibration. The applicability of this approach was benchmarked in three colorimetric assays (i.e., for the determination of iron, nickel, and amino acids) that use different reaction chemistries. This work demonstrates a simplified calibration-free approach for assays performed on paper-based analytical devices that requires minimum experimental and computational effort, it can be used for either external or standard addition calibration and can be used to identify and eliminate the presence of interferences in a sample.

Design and Optimization of Colorimetric Paper-Based Analytical Device for Rapid Detection of Allopurinol in Herbal Medicine.

Traditional herbal medicine in Indonesia is still in great demand and popular in society. The Indonesian government regulations state that herbal medicine should not contain chemical drug due to the toxic effect of uncontrolled consumption. Allopurinol is one of the drugs commonly added to herbal medicine for the treatment of chronic gout. Paper-based analytical device is one of the latest forms of analysis that has been widely used for the identification of chemical elements, environmental contamination, bacteria, and many more. In this study, experiments were conducted using Whatman filter paper No. 1, No. 2, and No. 4 and Whatman chromatography as a paper, and 9 colorimetric reagents were tested for allopurinol detection in herbal medicine. There were 5 specific reagents that reacted positively with allopurinol and only 3 reagents that can be applied to the paper, that is, Folin-Ciocalteu, Tollens, and p-DAB reagent. The results of the optimization show that the most optimal immersion time was 60 minutes with a drying time of 30 minutes at 50°C. Each filter paper has different characteristic; however, there was no significant difference when all of the papers were used as PAD for allopurinol detection.

Double-layered microfluidic paper-based device with multiple colorimetric indicators for multiplexed detection of biomolecules

In this work, a double-layered microfluidic paper-based device (μPAD) with multiple colorimetric indicators was presented for the sensitive simultaneous detection of four kinds of small biomolecules, including glucose, uric acid, lactate, and choline in mixture. The double-layered μPAD were composed of the top detection layer and the bottom auxiliary layer to construct 3D microfluidic channels. The detection zones on the top detection layer were modified with various colorimetric reagents, different kinds of oxidase (glucose oxidase, uricase, lactate oxidase or choline oxidase) and horseradish peroxidase (HRP). The immobilized chromogenic reagents could be oxidized by the generated H2O2 from the specific enzymatic reactions between the oxidases and their corresponding substrates to form different colored products with co-immobilized HRP as catalyst. As a result, four linear calibration curves were obtained for the determination of glucose (0.01–10.0 mmol/L), uric acid (0.01–5.0 mmol/L), lactate (0.04–10.0 mmol/L), and choline (0.04–24.0 mmol/L), respectively. The proposed double-layered μPAD with multiple colorimetric indicators exhibited good colorimetric performance, effectively improved sensitivity, and extended linear range, which was further applied to the simultaneously determination of glucose, uric acid, lactate, and choline in serum samples.

Activity Analysis of Iron in Water Using a Simple LED Spectrophotometer

Access to clean water is a vitally important part of the lives of all people, yet there is often a concern about the contamination of water by heavy metals. The detection and measurement of heavy metals in water can be performed using colorimetric reagents paired with ultraviolet–visible light (UV–vis) spectrophotometry. The presented activity includes construction of a simple, inexpensive single-wavelength spectrophotometer using LEDs as the light source and detector. Using a colorimetric reagent, 2,4,6-tripyridyl-s-triazine, the concentration of iron can be measured by the LED spectrophotometer. Multiple natural water samples were collected and analyzed by this technique, and the performance of the LED spectrophotometer was compared to that of a conventional UV–vis spectrophotometer. The LED spectrophotometer had comparable results to those of the conventional UV–vis spectrophotometer while teaching students about simple circuit design, spectroscopy, and water quality measurements.

Arsenic Monitoring in Water by Colorimetry Using an Optimized Leucomalachite Green Method.

Arsenic contamination of drinking water is a global concern. Standard laboratory methods that are commonly used for arsenic detection in water, such as atomic absorption spectroscopy and mass spectroscopy, are not suitable for mass monitoring purposes. Autonomous microfluidic detection systems combined with a suitable colorimetric reagent could provide an alternative to standard methods. Moreover, microfluidic detection systems would enable rapid and cost efficient in situ monitoring of water sources without the requirement of laborious sampling. The aim of this study is to optimize a colorimetric method based on leucomalachite green dye for integration into a microfluidic detection system. The colorimetric method is based on the reaction of arsenic (III) with potassium iodate in acid medium to liberate iodine, which oxidizes leucomalachite green to malachite green. A rapid colour development was observed after the addition of the dye. Beer’s law was obeyed in the range between 0.07–3 µg mL−1. The detection limit and quantitation limit were found to be 0.19 and 0.64 µg mL−1, respectively.

Detection of Disulfides in Protein Extracts ofArabidopsis thaliana using Monobromobimane (mBB).

Thiol-disulfide exchange is a key posttranslational modification, determining the folding process of intra- and inter-protein structures. Thiols can be detected by colorimetric reagents, which are stoichiometrically reduced by free thiols, and by fluorescent adducts, showing fluorescence only after thioester formation. We adapted a simple three-step method for detection of disulfide bonds in proteins. After irreversible blocking of protein thiols, disulfide bonds are reduced, followed by the detection of thiols. The approach presented here provides an economical procedure that can be used to obtain a global overview of the thiol-disulfide status of proteins in plants. This method allows the detection of modifications in samples on a gel and can be used for semi-quantitative analysis.

Activities of blood superoxide dismutase, glutathione peroxidase and serum vitamin E level in dogs with age-related cataract

Purpose: Oxidative mechanisms play a major role in the etiology and pathogenesis of cataract, especially age-related cataract (ARC). Our study aimed to investigate the serum vitamin E level and the activities of systemic antioxidant enzymes: superoxide dismutase (SOD) and glutathione peroxidase (GPX) in the blood of dogs with age-related cataract but without any general diseases. Material and methods: The population examined consisted of 40 dogs of various breeds and both sexes, aged 10-16 years, diagnosed with age-related cataract. Cataract was diagnosed by complete ocular examination. Blood was collected during clinical examination from the external jugular vein in dogs weighing less than 20 kg or from the small saphenous vein in dogs weighing over 20 kg. The control group consisted of 10 healthy dogs of different breeds and sexes, aged 8-12 years, without cataract. SOD and GPX were measured by a colorimetric reagent kit Randox SOD and Randox GPX. The level of vitamin E was determined using a Perkin-Elmer LS 30 luminescent spectrometer. This apparatus determines the vitamin E level by the fluorometric method of Hansen and Warwick . Results: The results showed a significantly lower activity of SOD as well as a lower activity of GPX and a lower level of the vitamin E in dogs with ARC compared to healthy dogs. Conclusions: The results obtained indicate a weakening of the general antioxidant defence system in the course of ARC in dogs. These results suggest that such conditions may have effect on the development of canine age-related cataract and are consistent with the free radical theory of age-related cataract development.

Printable QR code paper microfluidic colorimetric assay for screening volatile biomarkers

We present a QR code paper microfluidic colorimetric assay that can exploit the hardware and software on mobile devices, and circumvent sample preparation by directly targeting volatile biomarkers. Our platform is a printable microarray of well-defined reaction regions, which outputs an instant diagnosis by directing the user to a URL containing their test result, while simultaneously storing epidemiological data for remote access and bioinformatics. To assist in the rapid identification of Escherichia coli in bloodstream infections, we employed an existing colorimetric reagent (p-dimethylaminocinnamaldehyde) and adapted its use to detect volatile indole, a biomarker produced by E. coli. Our assay was able to quantitatively detect indole in the headspace of E. coli culture after 12 h of growth (27.0 ± 3.1 ppm), assisting in species-level identification hours earlier than existing methods. Results were confirmed with headspace solid-phase microextraction (HS-SPME) two-dimensional gas chromatography time-of-flight mass spectrometry (GC×GC-ToFMS), which estimated indole concentration in E. coli culture to average 32.3 ± 5.2 ppm after 12 h of growth. This QR paper microfluidic platform represents a novel development in both telemedicine and diagnostics using volatile biomarkers. We envision that our QR code platform can be extended to other colorimetric assays for real-time diagnostics in low-resource environments.

Two-dimensional ammonium distribution in sediment pore waters using a new colorimetric diffusive equilibration in thin-film technique

This study presents a new gel based technique to describe the pore water ammonium distribution through the sediment-water interface in two dimensions at a millimeter scale. The technique is an adaptation of the classical colorimetric method based on the Berthelot's reaction. After the thin film of the gel probe was equilibrated by diffusion either in standard solutions or in pore waters, a colorimetric reagent gel was set on the gel probe, allowing development of the characteristic green color. A flatbed scanner and subsequent densitometry image analysis allowed to determine the concentration distribution of ammonium. The gel probe was tested in the laboratory for two media, deionized water and seawater, within the range 0–3000 μM in NH4+. Detection limit is about 20 μM and accuracy about ±25 μM. The field validation was realized in a tidal mudflat of the French Atlantic coast by comparison with conventional pore water extraction and colorimetric analysis. The large range of concentrations and its applicability in continental and marine sediments suggest a wide range of applications of the technique for a reasonable cost.

Experimental and first principles investigations on the photoisomerization and electrochemical properties of chlorophosphonazo III

Photoresponsive molecules have been widely applied to solar cells and molecular devices. In this manuscript, we report the photoresponsive behaviours of chlorophosphonazo III, a cheap commercially available molecule and previously only used as a colorimetric reagent for earth metals, focusing on the photoisomerization properties in solution or on TiO2 substrates. Apart from the optomechanical properties, the chlorophosphonazo dye is found to generate a weak but non-negligible photocurrent under the visible light in aqueous environment. Density functional theory calculations are used to understand the structural, electronic and optical properties of the functional molecule. Possible routes to enhance the optoelectronic and optomechanical performance of chlorophosphonazo III toward multiple functions in integrated devices where both of the optomechanical and optoelectronic performances are desired are suggested.

Development of a Cost-Effective Sensing Platform for Monitoring Phosphate in Natural Waters

A sensing platform for the in situ, real-time analysis of phosphate in natural waters has been realised using a combination of microfluidics, colorimetric reagent chemistries, low-cost LED-based optical detection and wireless communications. Prior to field deployment, the platform was tested over a period of 55 days in the laboratory during which a total of 2682 autonomous measurements were performed (854 each of sample, high standard and baseline, and 40 × 3 spiked solution measurements). The platform was subsequently field-deployed in a freshwater stream at Lough Rea, Co., Galway, Ireland, to track changes in phosphate over a five day period. During this deployment, 165 autonomous measurements (55 each of sample, high standard, and baseline) were performed and transmitted via general packet radio service (GPRS) to a web interface for remote access. Increases in phosphate levels at the sampling location coincident with rainfall events (min 1.45 µM to max 10.24 µM) were detected during the deployment. The response was found to be linear up to 50 µM PO43−, with a lower limit of detection (LOD) of 0.09 µM. Laboratory and field data suggest that despite the complexity of reagent-based analysers, they are reasonably reliable in remote operation, and offer the best opportunity to provide enhanced in situ chemical sensing capabilities. Modifications that could further improve the reliability and scalability of these platforms while simultaneously reducing the unit cost are discussed.

Determination of pyrrolizidine alkaloids in dietary sources using a spectrophotometric method

Pyrrolizidine alkaloids (PAs) are a class of toxic compounds found in the composition of more than 6000 plants. People can be exposed to PAs by consuming phytotherapeutic products, food from crops contaminated with seeds of some species with high content of PAs, and/ or contaminated animal products like bee products. For this reason we developed and validated a method for quantitative determination of PAs, from the most frequently contaminated food sources, honey and flour. Colorimetric Ehrlich reagent method was used with standard addition (1mg/kg senecionine). The extraction solvent was methanol 50% acidified with citric acid to pH 2-3, as this solvent can be used for alkaloids and N-oxides. We found that, in extracting the alkaloid only once from the dietary sources, the percent of recovery is low (52.5% for honey, and 45.75% for flour). Using successive extractions, three times with the same solvent, the senecionine retrieval percentage increased to 86.0% for honey and 76.0% for flour. The method was validated using the following parameters: selectivity, linearity (0,25- 20 mg/ mL senecionine), accuracy (average recovery 93.5 - 107.93%) and precision (RSD 3,26-4.55%.). The calculated limit of quantification (0.174 mg/ mL) makes this method applicable for determining Pas occurring at toxic levels for consumers.

Sensitive assay of pharmaceutical amines in environmental samples and formulations by oxygenated graphene quantum dots

Oxygenated graphene quantum dots (GQDs) were prepared and used as a new colorimetric reagent for analysis of pharmaceutical amines. Citric acid was thermalized to prepare GQDs with functional groups along their edges. The prepared GQDs were used as a coupling reagent in an azo dye formation for dapsone (DAP) and metoclopramide (MET) determination. The characterization was done by scanning electron microscopy, transmission electron microscopy, atomic force microscopy, X-ray diffraction spectroscopy, Energy dispersive X-ray spectroscopy, EDX-mapping, dynamic light scattering, photoluminescence spectroscopy, Raman and FT-IR spectroscopy. DAP and MET were diazotized by nitrite to their related diazonium cations in acidic condition through a flow injection manifold. Then they were coupled to GQDs to produce the yellow colored products. Detection of the products was performed by spectrophotometry at 423 nm. Various chemical and instrumental parameters were optimized by a univariate procedure and a central composite design (CCD) chemometrics approach. The analytical method showed the linear calibrations for DAP and MET in the ranges of 0.03–20 and 0.1–40 µg mL−1, respectively. The limit of detection for both DAP and MET was of 0.01 µg mL−1, respectively. The inter-day experiments showed a satisfactory robustness of the method for determination of DAP and MET. The sample throughput found in this method was about 60 h−1. Lake, river and well water samples as well as soil samples along with some pharmaceutical formulations were analyzed.

Phenolphthalein-Conjugated Hydrogel Formation under Visible-Light Irradiation for Reducing Variability of Colorimetric Biodetection.

Polymerization-based signal amplification (PBA) is a colorimetric biodetection method for use in resource-limited settings. It uses phenolphthalein as a colorimetric reagent, but leaching of the dye from a resultant hydrogel can lead to variability in PBA results. In this work, we use polymerizable phenolphthalein in PBA to produce a phenolphthalein-conjugated hydrogel. Considering the photoinitiation mechanism of eosin Y and triethanolamine in air with an aqueous monomer solution, we determine reaction conditions for modified PBA that maintain a rapid polymerization response. Using the optimized conditions, we demonstrate that modified PBA can detect malaria antigen, PfHRP2, with reduced variability.

A paper platform for colorimetric determination of aluminum hydrochloride in antiperspirant samples

A simple, fast, low-cost, portable, and eco-friendly method using a spot test on a paper platform, together with diffuse reflectance spectroscopy, was developed and validated for the quantification of aluminum hydrochloride, a potential neurotoxic agent, in antiperspirant samples. The determination of aluminum hydrochloride was performed at a wavelength of 615 nm, by measuring consumption of the purple colorimetric reagent Alizarin S, due to reaction with aluminum. The linear range was from 10.0 to 125.0 mg L−1 and could be described by the equation: AR = 0.4479 − 0.002543 CAl (R = 0.999). The limits of detection (LOD) and quantification (LOQ) were 3.06 and 10.2 mg L−1, respectively. The method was specific, accurate, and repeatable, with relative standard deviation (RSD) <5.0%. The recovery was between 92.2 and 103.4%. The method was successfully used for the determination of aluminum hydrochloride in commercial antiperspirant samples, revealing concentrations below the maximum permitted by current legislation.

Method Development for Quantification of Bromochloramine Using Membrane Introduction Mass Spectrometry.

During chloramination of bromide-containing waters, the main brominated amine formed is bromochloramine (NHBrCl). To date, there is no analytical method, free of interference, allowing its accurate quantification. The major reason is that it is not possible to produce a pure NHBrCl solution. In this study, we report a method allowing the accurate quantification of NHBrCl with membrane introduction mass spectrometry (MIMS). First, the molar absorption coefficient for NHBrCl was determined by quantifying NHBrCl as 2,4,6-tribromophenol by HPLC-UV and comparing the results with the direct UV response at 320 nm. A molar absorption coefficient of 304 M-1cm-1 was obtained. The results obtained by direct UV measurements were compared to the MIMS signal recorded at m/ z 131 corresponding to the mass of the molecular ion and used to establish a calibration curve. A limit of detection of 2.9 μM (378 μg/L) was determined. MIMS is the only method enabling the unambiguous quantification of NHBrCl, as it is based on m/ z 131, while with other analytical techniques, other halamines can interfere, i.e., overlapping peaks with direct UV measurements and reaction of several halamines with colorimetric reagents or phenols. While the detection limit is not quite low enough to measure NHBrCl in actual drinking water, this analytical method will benefit the scientific community by allowing further mechanistic studies on the contribution of NHBrCl to the formation of toxic disinfection by-products.

Design of Optical Sensor Membrane Based on Polymer Poly(methyl methacrylate) for Paracetamol Detection in Traditional Herbal Medicine.

Generally, regulation states that herbal medicines are remedies containing plants or preparation of plants as active ingredients only. Paracetamol is one of the drugs that is frequently added in herbal medicine to enhance the effect as an analgesic. The government regulation disallows chemical drugs contained in herbal medicine due to the toxic effect of uncontrolled consumption. On this study, the optical sensor membrane from polymer poly(methyl methacrylate) (PMMA) was synthesized by phase inversion method and was used to detect paracetamol in herbal medicine. PMMA was made in three different concentrations 5%, 7.5%, and 10% and was mixed with ferric chloride (FeCl3), Folin-Ciocalteu, and Nessler reagent as specific colorimetric reagents for paracetamol detection, with a ratio of solvent:reagent was 6:4; 7:3; and 8:2. The result of the experiment shows that PMMA-FeCl3 7.5% (7:3), PMMA-Folin 5% (6:4), and PMMA-Nessler 5% (6:4) give the best performance for paracetamol detection. Real herbal medicine samples were analyzed to confirm the practical application of this sensor, and the result shows good agreement with UV-Vis data. The results show that optical sensor membrane which has been developed can be used as new detection method of paracetamol for community application.

Salicylamide derivatives for iron and aluminium sequestration. From synthesis to complexation studies

This paper presents an easy, fast and economic synthesis of chelating agents for medical, environmental and analytical applications, and the evaluation of the stability of their complexes with Fe3+ and Al3+. Complex formation equilibria with Cu2+ and Zn2+ metal ions were also studied to evaluate if the chelating agents can perturb the homeostatic equilibria of these essential metal ions. Effective chelating agents for metal ions, in addition to their well-known medical uses, find an increasing number of applications in environmental remediation, agricultural applications (supplying essential elements in an easily available form), and in analytical chemistry as colorimetric reagents. Besides the stability of the complexes, the lack of toxicity and the low cost are the basic requisites of metal chelating agents. With these aims in mind, we utilized ethyl salicylate, a cheap molecule without toxic effects, and adopted a simple synthetic strategy to join two salicylate units through linear diamines of variable length. Actually, the mutual position of the metal binding oxygen groups, as well as the linker length, affected protonation and complex formation equilibria. A thorough study of the ligands is presented. In particular, the complex formation equilibria of the three ligands toward Fe3+, Al3+, Zn2+ and Cu2+ ions were investigated by combined potentiometric and spectrophotometric techniques. The results are encouraging: all the three ligands form stable complexes with all the investigated metal ions, involving the oxygen donor atoms from the 2-hydroxybenzamido unit, and nitrogen atoms in copper and zinc coordination.

Natural ionophore functionalized nanobiosensor capable of staining Cr3+ in live brine shrimp Artemia

We report the fabrication of gold nanoparticles using a sulfur containing microbial ionophore aeruginic acid (H2L), functioning as a reducing as well as stabilizing agent, to develop a nanobiosensor HL-AuNPs which showed highly selective colorimetric detection of Cr3+ in aqueous (100% water) HEPES buffer at physiological pH. On exposing HL-AuNPs to the aqueous solution of Cr3+, a red shift in the surface plasmon resonance (SPR) band of the probe is observed along with bare eye observable color change of the solution. Mechanistic studies revealed attachement of carboxylate moiety to the gold nanosurface. On addition of Cr3+ to the solution of HL-AuNPs, aggregation occurs as a result of coordination of the phenolate O and imine N with the octahedral Cr3+ center. The nanosensor with lower detection limit (LOD) of 0.5 μM can also be utilized for colorimetric detection and removal of Cr3+ in environmental samples. Being non-toxic and permeable in nature, HL-AuNPs can function as a colorimetric staining reagent for Cr3+ in living whole brine shrimp Artemia. Use of functionalized AuNPs as imaging reagent for Cr3+ in living organim is unprecedented.

Expanding Quantification of Arsenic in Water to 0 μg L−1 with a Field Test Kit: Substituting 0.4% M/V Silver Nitrate as the Colorimetric Reagent; Employing Digital Image Analysis

This study confronts the questionable reliability and accuracy of field test kits distributed globally to determine arsenic in drinking water. Because kits are the primary method of arsenic analysis in the areas most affected, an alternate, nontoxic formulation is needed to provide accurate results. Hypothesizing that introducing silver nitrate as the reagent in test kits could successfully substitute for restricted mercuric bromide, the study found that the reformulated kits provided reliable, precise, and accurate results over a broader range of contamination. Digital image analysis was used to examine the blue color value produced when arsine reacts with silver nitrate impregnated test strips. An optimal concentration of AgNO3 exhibiting the greatest linearity was determined by graphical comparison and the color intensity of the strips observed to be inversely proportional to the concentration of As (III). Adapted field test kits were then constructed to examine water samples ranging in arsenic contamination from 0 to 50 μg L−1. A series of reactions was completed to demonstrate reproducibility and test for the accuracy of the procedure. Statistical examination of colorimetrically quantified results confirmed the hypothesis that silver nitrate can reliably and precisely expand the kit’s range of detection while maintaining its low cost, quick assessment, and uncomplicated technique. This new method, using 0.4% m/v AgNO3 as a reagent for the Gutzeit reaction, was able to distinguish between concentrations of 0, 5, 10, 15, and 50 μg L−1 at the 95% confidence level.