O-Phthalaldehyde Derivatization for the Paper-Based Fluorometric Determination of Glutathione in Nutritional Supplements.

Herein, a new, direct paper-based fluorimetric method is described for the quantitative determination of glutathione (GSH) molecules in nutritional supplements. Briefly, the proposed analytical method is based on the fluorescence emission resulting from the direct and selective chemical reaction of GSH molecules with the derivatization reagent that is o-phthalaldehyde (OPA) in acidic conditions at room temperature. The intensity of the emitted fluorescence on the surface of the analytical paper devices after irradiation with a lamp at 365 nm is proportional to the concentration of GSH and is measured using a smartphone as the detector. This methodology, which is suitable for measurements in laboratories with limited resources, does not require specialized instrumentation or trained personnel. The protocol governing the proposed method is simple and easily applicable. Essentially, the chemical analyst should adjust the value of pH on the surface of the paper by adding a minimal amount of buffer solution; then, after adding a few microliters of the derivatization reagent, wait for the surface of the paper to dry and, finally, add the analyte. Subsequently, the irradiation of the sensor and the measurement of the emitted fluorescence can be recorded with a mobile phone. In the present study, several parameters affecting the chemical reaction and the emitted fluorescence were optimized, the effect of interfering compounds that may be present in dietary supplements was examined, and the stability of these paper sensors under different storage conditions was evaluated. Additionally, the chemical stability of these paper devices in various maintenance conditions was studied, with satisfactory results. The detection limit calculated as 3.3 S/N was 20.5 µmol L-1, while the precision of the method was satisfactory, ranging from 3.1% (intra-day) to 7.3% (inter-day). Finally, the method was successfully applied to three different samples of dietary supplements.

High-throughput and green optical sensing of thiocyanate in human saliva based on microplates and an overhead book scanner as detector.

An instrumental-free, high-throughput assay has been developed for the quantification of thiocyanate in human saliva. The proposed green method is based on the rapid reaction of the analyte with Fe(III) under acidic pH in a microplates format to form a colored complex that is captured as an image by an overhead book scanner. Optimization included the effects of the amount concentration of Fe(III), acidity and reaction time / complex stability using a total volume of 300 µL per well. Validation towards the matrix effect was focused on the specific application and was performed using both artificial and human saliva. The linearity of the developed assay was up to 500 µM thiocyanate offering a lower limit of quantification (LLOQ) of 30 µM. The green potentials were evaluated by both the Green Analytical Procedure (GAPI) and Blue Applicability Grade (BAGI) indexes. The thiocyanate content in the saliva of non-smoking volunteers ranged between 750 and 1350 µΜ, while elevated concentrations were verified in smoking individuals (1860-3080 µΜ). Statistical agreement with a corroborative method was assessed using the Bland-Altman plot.

Smartphone-Based High-Throughput Fluorimetric Assay for Histidine Quantification in Human Urine Using 96-Well Plates.

A high-throughput fluorimetric assay for histidine was developed, using a 96-well plates platform. The analyte reacts selectively with o-phthalaldehyde under mild alkaline conditions to form a stable derivative. Instrumental-free detection was carried out using a smartphone after illumination under UV light (365 nm). The method was proved to be linear up to 100 µM histidine, with an LLOQ (lower limit of quantification) of 10 µM. The assay was only prone to interference from glutathione and histamine that exist in the urine samples at levels that are orders of magnitude lower compared to histidine. Human urine samples were analyzed following minimum treatment and were found to contain histidine in the range of 280 to 1540 µM. The results were in good agreement with an HPLC corroborative method.

A Novel Equipment-Free Paper-Based Fluorometric Method for the Analytical Determination of Quinine in Soft Drink Samples.

A simple, equipment-free, direct fluorometric method, employing paper-based analytical devices (PADs) as sensors, for the selective determination of quinine (QN) is described herein. The suggested analytical method exploits the fluorescence emission of QN without any chemical reaction after the appropriate pH adjustment with nitric acid, at room temperature, on the surface of a paper device with the application of a UV lamp at 365 nm. The devices crafted had a low cost and were manufactured with chromatographic paper and wax barriers, and the analytical protocol followed was extremely easy for the analyst and required no laboratory instrumentation. According to the methodology, the user must place the sample on the detection area of the paper and read with a smartphone the fluorescence emitted by the QN molecules. Many chemical parameters were optimized, and a study of interfering ions present in soft drink samples was carried out. Additionally, the chemical stability of these paper devices was considered in various maintenance conditions with good results. The detection limit calculated as 3.3 S/N was 3.6 mg L-1, and the precision of the method was satisfactory, being from 3.1% (intra-day) to 8.8% (inter-day). Soft drink samples were successfully analyzed and compared with a fluorescence method.

A Low-Cost Colorimetric Assay for the Analytical Determination of Copper Ions with Consumer Electronic Imaging Devices in Natural Water Samples.

This study reports a new approach for the determination of copper ions in water samples that exploits the complexation reaction with diethyldithiocarbamate (DDTC) and uses widely available imaging devices (i.e., flatbed scanners or smartphones) as detectors. Specifically, the proposed approach is based on the ability of DDTC to bind to copper ions and form a stable Cu-DDTC complex with a distinctive yellow color detected with the camera of a smartphone in a 96-well plate. The color intensity of the formed complex is linearly proportional to the concentration of copper ions, resulting in its accurate colorimetric determination. The proposed analytical procedure for the determination of Cu2+ was easy to perform, rapid, and applicable with inexpensive and commercially available materials and reagents. Many parameters related to such an analytical determination were optimized, and a study of interfering ions present in the water samples was also carried out. Additionally, even low copper levels could be noticed by the naked eye. The assay performed was successfully applied to the determination of Cu2+ in river, tap, and bottled water samples with detection limits as low as 1.4 µM, good recoveries (89.0-109.6%), adequate reproducibility (0.6-6.1%), and high selectivity over other ions present in the water samples.

Analyte-mediated formation and growth of nanoparticles for the development of chemical sensors and biosensors.

The cornerstone of nanomaterial-based sensing systems is the synthesis of nanoparticles with appropriate surface functionalization that ensures their stability and determines their reactivity with organic or inorganic analytes. To accomplish these requirements, various compounds are used as additives or growth factors to regulate the properties of the synthesized nanoparticles and their reactivity with the target analytes. A different rationale is to use the target analytes as additives or growth agents to control the formation and properties of nanoparticles. The main difference is that the analyte recognition event occurs before or during the formation of nanoparticles and it is based on the reactivity of the analytes with the precursor materials of the nanoparticles (e.g., metal ions, reducing agents, and coatings). The transition from the ionic (or molecular) state of the precursor materials to ordered nanostructured assemblies is used for sensing and signal transduction for the qualitative detection and the quantitative determination of the target analytes, respectively. This review focuses on assays that are based on analyte-mediated regulation of nanoparticles' formation and differentiate them from standard nanoparticle-based assays which rely on pre-synthesized nanoparticles. Firstly, the principles of analyte-mediated nanomaterial sensors are described and then they are discussed with emphasis on the sensing strategies, the signal transduction mechanisms, and their applications. Finally, the main advantages, as well as the limitations of this approach, are discussed and compared with assays that rely on pre-synthesized nanoparticles in order to highlight the major advances accomplished with this type of nano-sensors and elucidate challenges and opportunities for further evolving new nano-sensing strategies.

Development of an equipment free paper based fluorimetric method for the selective determination of histidine in human urine samples.

A direct fluorimetric method, employing µicro-analytical paper-based devices (µ-PADs) for the selective determination of histidine (HIS) is described. The suggested method exploits the fluorescence emission of histidine after its rapid reaction with o-phthalaldehyde (OPA) at a basic medium (pH = 10) on the surface of a paper device with the application of a UV lamp at 354 nm. The devices are inexpensive and are composed of chromatographic paper and wax barriers. The analytical protocol is easily applicable with minimal technical expertise and without the need of expensive experimental apparatus. The user has to add a test sample, illuminate the device with a UV lamp, and read the fluorescence of the sensing area using a simple imaging device such as a cell-phone camera. The method is free from common interferences likely to affect the measurement of histidine and is selective among all other amino acids. This analytical procedure was optimized and validated, paying special attention to its intended application. The detection limits are as low as 1.8 µM with very satisfactory precision ranging from 6.4% (intra-day) to 8.9% (inter-day). Random urine samples from adult volunteers (n = 5) were successfully analyzed and HIS content ranged between 260 and 1114 µmol L-1 with percentage recoveries in the range of 78.2 and 124.6%.

Development of a Paper-Based Analytical Method for the Colorimetric Determination of Calcium in Saliva Samples.

A novel, rapid, and facile method for the colorimetric determination of calcium using micro-analytical paper-based devices (µ-PADs) was developed. The proposed analytical method utilizes the color differences developing, after the addition of calcium, on the surface of the devices because of the complexation reaction of calcium with Methylthymol Blue (MTB) at room temperature, in alkaline pH. The devices were manufactured with chromatographic paper, using wax barriers, and the analytical protocol was easily implemented without the need of any experimental apparatus except for a simple imaging device. The user must regulate the pH, add the solutions on the paper, and measure the color intensity of the formed Ca(II)-MTB complex with a flatbed scanner. The experimental conditions for optimum color development, the possible interfering substances, and the reliability of the paper devices in different preserving conditions were optimized, with satisfactory results. The method exhibited acceptable detection limits (2.9 mg L-1) with sufficiently good precision, which varied from 4.2% (intra-day) to 6.4% (inter-day). Saliva samples from healthy volunteers were successfully analyzed, and the calcium levels were calculated in the range of 30.71 to 84.15 mg L-1.

Development of a sequential extraction and speciation procedure for assessing the mobility and fractionation of metal nanoparticles in soils.

This study describes the development of a sequential extraction procedure for the evaluation of metal nanoparticle mobility and bioaccessibility in soils. The procedure, that was developed using gold nanoparticles (AuNPs) as model species, relies on the fractionation of nanoparticles by sequentially dissolving soil matrix components (carbonates, metal oxides, organic matter and mineral phases) in order to release the entrapped nanoparticle species in the extract solution. By summing up the concentration of AuNPs recovered in each fraction it was found that 93.5% of the spiked AuNP concentration could be recovered which satisfactorily represents the nominal AuNP concentration in the soil. The efficiency of the procedure was found to depend on several procedural artifacts related to the separation of AuNPs from soil colloids and the reactivity of the extraction reagents with AuNPs and their precursor metal ions. Based on the results obtained a protocol for the speciation of the AuNPs and Au ions in the soil sample was also developed. The results of the study show that both AuNPs and Au ions are mainly associated with soil organic matter, which significantly reduces their mobility, while a small amount (<10%) is associated with metal oxides which are more mobile and potentially bioaccessible. The developed procedure provides a springboard for further development of sequential extraction procedures of metal nanoparticles in soils that could be used to assess both the exposure and release of metal nanoparticles and their precursor metal ions in the environment (as total extractable concentration) as well as provide evidence regarding their bioaccessibility and potential bioavailability by determining the concentration of nanoparticles in each specific soil fraction.

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.

Determination of silver nanoparticles by atomic absorption spectrometry after dispersive suspended microextraction followed by oxidative dissolution back-extraction.

This study presents a liquid phase microextraction method for the quantification of silver nanoparticles (AgNPs) in environmental samples by means of directly suspended microextraction and atomic absorption spectrometry. The method is based on the dispersion of octanol into fine droplets where AgNPs are partitioned and extracted from the bulk aqueous phase. Then octanol is re-dispersed again in a strongly acidic and oxidizing aqueous medium in order to selectively decompose AgNPs to silver ions. The aqueous extract is then analyzed by atomic absorption spectrometry. Under the working conditions, the method offers high selectivity for AgNPs since most nanoparticle species cannot be extracted into the octanol phase, while the selective back-extraction procedure ensures that only AgNPs are dissolved to the final extract medium. The method works equally well for AgNPs of different size and coatings, thus enabling the determination of the total concentration AgNPs in real samples. Method application in different water samples was used to evaluate the utility of the method in the analysis of real samples of variable complexity with satisfactory results in terms of detection limit (less than 100 femto-mole of AgNPs), recoveries (90-104%) and reproducibility (lower than 7%).

Paper-based devices for biothiols sensing using the photochemical reduction of silver halides.

This study describes the development of paper-based devices for the determination of biothiols. The devices are inexpensive (composed of paper and silver halide particles), and the analytical protocol is easily executable with minimum technical expertise and without the need of specialized equipment; the user has to add a test sample, illuminate the device with a UV lamp, and read the color change of the sensing area using a simple imaging device (i.e., cell-phone camera) or a bare eye. The detection mechanism of the assay is based on the biothiols-mediated photoreduction of nanometer-sized silver chloride particles deposited on the surface of paper; photoreduced silver chloride particles have a grayish coloration that depends on the concentration of biothiols in the tested solution. This is the first time that the UV-mediated photoreduction of solid silver halides particles is used for analytical purposes. The performance of the devices has been tested on the detection of total biothiols content of artificial body fluids and protein-free human blood plasma samples, and the results were satisfactory in terms of sensitivity, selectivity, recoveries and reproducibility.

Generic Assay of Sulfur-Containing Compounds Based on Kinetics Inhibition of Gold Nanoparticle Photochemical Growth.

This work describes a new, equipment-free, generic method for the determination of sulfur-containing compounds that is based on their ability to slow down the photoreduction kinetics of gold ions to gold nanoparticles. The method involves tracking the time required for a red coloration to appear in the tested sample, indicative of the formation of gold nanoparticles, and compare the measured time relative to a control sample in the absence of the target analyte. The method is applicable with minimal and simple steps requiring only two solutions (i.e., a buffer and a gold solution), a source of light (UV or visible), and a timer. The method responds to a large variety of sulfur-containing compounds including thiols, thioesters, disulfides, thiophosphates, metal-sulfur bonds, and inorganic sulfur and was therefore applied to the determination of a variety of compounds such as dithiocarbamate and organophosphorous pesticides, biothiols, pharmaceutically active compounds, and sulfides in different samples such as natural waters and wastewater, biological fluids, and prescription drugs. The analytical figures of merit of the method include satisfactory sensitivity (quantitation limits at the low µM levels), good recoveries (from 93 to 109%), and satisfactory reproducibility (from 4.8 to 9.8%). The method is easily adoptable to both laboratory settings and nonlaboratory conditions for quantitative and semiquantitative analysis, respectively, is user-friendly even for the minimally trained user, and can be performed with limited resources at low cost.

Low-cost colorimetric assay of biothiols based on the photochemical reduction of silver halides and consumer electronic imaging devices.

This work describes a new approach for the determination of free biothiols in biological fluids that exploits some of the basic principles of early photographic chemistry - that was based on silver-halide recording materials - and uses broadly-available imaging devices (i.e. flatbed scanners) as detectors. Specifically, the proposed approach relies on the ability of biothiols to bind to silver ions and dissociate the silver halide crystals thus changing the photosensitivity of silver halide crystal suspension. The changes induced by biothiols on the light intensity transmitted through the silver halide suspension, after photochemical reduction, were measured with a simplified photometric approach that employs a flatbed scanner operating in transmittance mode. The overall analytical procedure for the determination of biothiols was easily executable, fast and could be applied with inexpensive and commercially available materials and reagents. What is more, physiologically relevant biothiol levels could be inspected even by the unattended eye. The developed assay was successfully applied to the determination of biothiols in urine and blood plasma samples with detection limits as low as 10µM, satisfactory recoveries (92-97%), good reproducibility (6.7-8.8%) and high selectivity against other major components of biological fluids. The utility of the method to the determination of reduced/oxidized thiol ratio's as well as its application under natural light illumination, without external energy sources, was also demonstrated and is discussed with regard to point-of need applications in facility-limited settings.

Intrinsic peroxidase-like activity of rhodium nanoparticles, and their application to the colorimetric determination of hydrogen peroxide and glucose.

The intrinsic peroxidase-like activity of rhodium nanoparticles (RhNPs) and their use as catalytic labels for sensitive colorimetric assays is presented. RhNPs catalyze the oxidation of the peroxidase substrate 3,3,5,5-tetramethylbenzidine (TMB) in the presence of H2O2 to produce a blue reaction product with a maximum absorbance at 652 nm. Kinetic studies show catalysis to follow Michaelis-Menten kinetics and a "ping-pong" mechanism. The calculated kinetic parameters indicate high affinity of RhNPs for both the substrate TMB and H2O2. In fact, they are better than other peroxidase mimicking nanomaterials and even the natural enzyme horseradish peroxidase. On the other hand, RhNPs exhibit no reactivity towards saccharides, thiols, amino acids and ascorbic acid. Based on these findings, a sensitive and selective colorimetric method was worked out for the determination of H2O2 in real samples with a linear response in the 1-100 µM concentration range. By employing glucose oxidase, the glucose assay has a linear range that covers the 5 to 125 µM glucose concentration range. The detection limits are <0.75 µM for both species. The methods were applied to the determination of H2O2 in spiked pharmaceutical formulations, and of glucose in soft drinks and blood plasma. Figures of merit include (a) good accuracy (with errors of <6%), (b) high recoveries (96.5-103.7%), and (c) satisfactory reproducibility (<6.3%). Graphical abstract Rhodium nanoparticles catalyze the oxidation of 3,3,5,5-tetramethylbenzidine (TMB) in the presence of H2O2 to produce a blue reaction product. The effect is exploited in photometric assays for hydrogen peroxide and glucose.

Determination of gold nanoparticles in environmental water samples by second-order optical scattering using dithiotreitol-functionalized CdS quantum dots after cloud point extraction.

This work presents a new method for the sensitive and selective determination of gold nanoparticles in water samples. The method combines a sample preparation and enrichment step based on cloud point extraction with a new detection motif that relies on the optical incoherent light scattering of a nano-hybrid assembly that is formed by hydrogen bond interactions between gold nanoparticles and dithiotreitol-functionalized CdS quantum dots. The experimental parameters affecting the extraction and detection of gold nanoparticles were optimized and evaluated to the analysis of gold nanoparticles of variable size and surface coating. The selectivity of the method against gold ions and other nanoparticle species was also evaluated under different conditions reminiscent to those usually found in natural water samples. The developed method was applied to the analysis of gold nanoparticles in natural waters and wastewater with satisfactory results in terms of sensitivity (detection limit at the low pmolL-1 levels), recoveries (>80%) and reproducibility (<9%). Compared to other methods employing molecular spectrometry for metal nanoparticle analysis, the developed method offers improved sensitivity and it is easy-to-operate thus providing an additional tool for the monitoring and the assessment of nanoparticles toxicity and hazards in the environment.

In-situ suspended aggregate microextraction of gold nanoparticles from water samples and determination by electrothermal atomic absorption spectrometry.

This work describes a new method for the extraction and determination of gold nanoparticles in environmental samples by means of in-situ suspended aggregate microextraction and electrothermal atomic absorption spectrometry. The method relies on the in-situ formation of a supramolecular aggregate phase through ion-association between a cationic surfactant and a benzene sulfonic acid derivative. Gold nanoparticles are physically entrapped into the aggregate phase which is separated from the bulk aqueous solution by vacuum filtration on the surface of a cellulose filter in the form of a thin film. The film is removed from the filter surface and is dissociated into an acidified methanolic solution which is used for analysis. Under the optimized experimental conditions, gold nanoparticles can be efficiently extracted from water samples with recovery rates between 81.0-93.3%, precision 5.4-12.0% and detection limits as low as 75femtomolL(-1) using only 20mL of sample volume. The satisfactory analytical features of the method along with the simplicity indicate the efficiency of this new approach to adequately collect and extract gold nanoparticle species from water samples.

Programming fluid transport in paper-based microfluidic devices using razor-crafted open channels.

Manipulating fluid transport in microfluidic, paper-based analytical devices (µPADs) is an essential prerequisite to enable multiple timed analytical steps on the same device. Current methods to control fluid distribution mainly rely on controlling how slowly the fluid moves within a device or by activating an on/off switch to flow. In this Article, we present an easy approach for programming fluid transport within paper-based devices that enables both acceleration as well as delay of fluid transport without active pumping. Both operations are programmed by carving open channels either longitudinally or perpendicularly to the flow path using a craft-cutting tool equipped with a knife blade. Channels are crafted after µPADs fabrication enabling the end user to generate patterns of open-channels on demand by carving the porous material of the paper without cutting or removing the paper substrate altogether. Parameters to control the acceleration or delay of flow include the orientation, length, and number of open channels. Using this method, accelerated as well as reduced fluid transport rates were achieved on the same device. This methodology was applied to µPADs for multiple and time-programmable assays for metal ion determination.

Ultratrace determination of silver, gold, and iron oxide nanoparticles by micelle mediated preconcentration/selective back-extraction coupled with flow injection chemiluminescence detection.

A new method has been developed for the ultrasensitive determination of silver, gold, and iron oxide nanoparticles in environmental samples. Cloud point extraction was optimized and used as a means to extract and preconcentrate all nanoparticle species simultaneously from the same sample. The extracted nanoparticles were sequentially isolated from the surfactant-rich phase by a new selective back-extraction procedure and dissociated into their precursor metal ions. Each ion solution was injected in a flow injection analysis (FIA) manifold, accommodating the chemiluminogenic oxidation of luminol, in order to amplify chemiluminescence (CL) emission in a manner proportional to its concentration. Under the optimum experimental conditions, the detection limits were brought down to the picomolar and femtomolar concentration levels with satisfactory analytical features in terms of precision (2.0-13.0%), selectivity against dissolved ions, and recoveries (74-114%). The method was successfully applied to the determination of iron oxide, silver, and gold nanoparticles in environmental samples of different complexity, ranging from unpolluted river water to raw sewage. The developed method could also serve as a basis for future deployment of molecular spectrometry detectors for the selective determination and speciation analysis of nanoparticles in environmental applications.

Ligand-free gold nanoparticles as colorimetric probes for the non-destructive determination of total dithiocarbamate pesticides after solid phase extraction.

In this work, we describe a simple and sensitive non-destructive method for the determination of the total concentration of dithiocarbamate fungicides (DTCs) in real samples. The proposed method combines for the first time the benefits of an extraction method for sample clean-up and preconcentration with a sensitive colorimetric assay based on gold nanoparticle probes. In this two-step procedure, the target DTCs are isolated from the matrix and preconcentrated by solid phase extraction onto commercially available C18 sorbents. Following elution, the extract containing the target dithiocarbamates, free from most interferences and matrix components, is delivered into an aqueous dispersion of plain citrate-capped gold nanoparticles (AuNPs) which aggregate in response to DTCs coordination on AuNPs surface through multiple gold thiolate bonds. This aggregation is evidenced by changes in the spectral properties of the solution involving a decrease in the original absorbance of Au nanoparticles at 522 nm and the appearance of a new absorption band above 700 nm. An ensuing chromatic shift of the solution from wine-red to purple-blue is observed which is visual by naked eye at concentrations as low as 50 µg L(-1). Further improvement in the detection limits can be accomplished by scaling-down the method to micro-volume conditions alleviating the need to preconcentrate larger sample volumes. Overall, by combining sample clean-up and preconcentration with the strong affinity of DTC thiol group for the gold surface, the total concentration of dithiocarbamate pesticides was successfully determined in various water samples at the low and ultra-low µg L(-1) levels without resorting to destructive techniques, sophisticated instrumentation or post-synthetic modification of gold nanoparticles. Method application in real samples showed good analytical features in terms of recoveries (81.0-94.0%), precision (5.6-8.9%) and reproducibility (~9%) rendering the method as an attractive alternative to current methodologies for the determination of DTC fungicide residues in samples of environmental interest.