Adsorption enrichment integrated with paper-based devices for detection of trace levels of hexavalent chromium in water samples.

In the present study, a sensitive microfluidic paper-based analytical device (µ-PADs) integrated with adsorption enrichment procedure was developed to analyze Cr(VI) in water samples. The affecting factors, including pH and amounts of reagents were optimized. The limit of detection of 0.0015 mg L-1 and linear range of 0.005-2 mg L-1 were achieved with good intra- and inter-day precision of 5.1 and 7.6% RSD, respectively. The results obtained by the proposed method were validated by inductively coupled plasma-optical emission spectrometry (ICP-OES). The recoveries of the present method and ICP-OES were ranged from 96.3 to 109.0‬% and 106.0 to 109.7%, respectively. The two sets of (µ-PADs and ICP-OES) results were in a good agreement as paired t-test indicated no significant differences. The proposed method could be utilized for analyzing trace levels of Cr(VI) in water samples in the absence of conventional analytical instruments.

Separation and fractionation of glutamic acid and histidine via origami isoelectric focusing.

We demonstrated the fractionation of two amino acids, glutamic acid and histidine, separated via isoelectric focusing (IEF) on filter paper folded and stacked in an origami fashion. Channels for electrophoresis were fabricated as circular zones acquired via wax printing onto the filter paper. An ampholyte solution with amphiphilic samples was deposited on all the circle zones, which was followed by folding to form the electrophoresis channels. IEF was achieved by applying an electrical potential between the anodic and cathodic chambers filled with phosphoric acid and sodium hydroxide solutions, respectively. A pH gradient was formed using either a wide-range ampholyte with a pH of 3 to 10 or a narrow-range version with a pH of 5 to 8, which was confirmed by adding pH indicators to each layer. The origami IEF was used to separate the amino acids, glutamic acid and histidine, by mixing with the ampholytes, which were deposited on the layers. The components in each layer were extracted with water and measured by high-performance liquid chromatography using pre-column derivatization with dansyl chloride. The results indicated that the focus for glutamic acid and that for histidine were at different layers, according to their isoelectric points. The origami isoelectric focusing achieved the fractionation of amino acids in less than 3 min using voltage as low as 30 V.

Development of Pipetteless Paper-Based Analytical Devices with a Volume Gauge.

In this work, we propose a new design for paper-based analytical devices (PADs) that eliminate the need to use a micropipette for sample introduction. With this design, a PAD is equipped with a distance-based detection channel that is connected to a storage channel that indicates the volume of a sample introduced into the PAD. The analyte in the sample solution reacts with a colorimetric reagent deposited into the distance-based detection channel as the sample solution flows into the storage channel where the volume is measured. The ratio of the lengths of the detection channel and that of the storage channel (D/S ratio) are constant for a sample containing a certain concentration, which is independent of the introduced volume. Therefore, the PADs permit volume-independent quantification using a dropper instead of a micropipette because the length of the storage channel plays the role of a volume gauge to estimate the introduced sample volume. In this study, the D/S ratios obtained with a dropper were comparable to those obtained with a micropipette, which confirmed that precise volume control is unnecessary for this PAD system. The proposed PADs were applied to the determinations of iron and bovine serum albumin using bathophenanthroline and tetrabromophenol blue as colorimetric reagents, respectively. The calibration curves showed good linear relationships with coefficients of 0.989 for iron and 0.994 for bovine serum albumin, respectively.

Optical collection of extracellular vesicles in a culture medium enhanced by interactions with gold nanoparticles.

Extracellular vesicles (EVs) exist in biological fluids such as blood, urine, and cerebrospinal fluid and are promising cancer biomarkers. Attempts to isolate and analyze trace EVs, however, have been a challenge for researchers studying their functions and secretion mechanisms, which has stymied the options for diagnostic application. This study demonstrated a collection of EVs that was enhanced by gold nanoparticles (AuNPs) via the use of optical force. The collection system consists of an inverted microscope equipped with a CCD camera, a square capillary connected with a PTFE tube, and an Nd:YAG laser that generates optical force. The laser beam was focused on a capillary wall in which a cell culture medium containing EVs flowed continuously. Control of the surface charges on both the capillary wall and the AuNPs achieved the collection and retention of EVs on the capillary wall. The positively charged capillary wall retained EVs even after the laser irradiation was halted due to the negative charges inherent on the surface of EVs. Conversely, positively charged AuNPs had a strong electrostatic interaction with EVs and enhanced the optical force acting on them, which made collecting them a much more efficient process.

Dispersive liquid-liquid microextraction coupled with microfluidic paper-based analytical device for the determination of organophosphate and carbamate pesticides in the water sample.

A microfluidic paper-based analytical device (µ-PAD) is a promising new technology platform for the development of extremely low-cost sensing devices. However, it has low sensitivity that might not enable to measure maximum allowable concentration of various pollutants in the environment. In this study, a dispersive liquid-liquid microextraction (DLLME) was developed as a preconcentration method to enhance the sensitivity of the µ-PAD for trace analysis of selected pesticides. Four critical parameters (volume of n-hexane and acetone, extraction time, NaCl amount) that affect the efficiency of DLLME have been optimized using response surface methodology. An acceptable mean recovery of 79-97% and 83-93% was observed at 1 µg L-1 and 5 µg L-1 fortification level, respectively, with very good repeatability (2.2-6.01% RSD) and reproducibility (5.60-10.41% RSD). Very high enrichment factors ranging from 317 to 1471 were obtained. The limits of detection for the studied analytes were in the range of 0.18-0.41 µg L-1 which is much lower than the WHO limits of 5-50 µg L-1 for similar category of analytes. Therefore, by coupling DLLME with µ-PAD, a sensitivity that allows to detect environmental threat and also that surpassed most of the previous reports have been achieved in this study. This implies that the preconcentration step has a paramount contribution to address the sensitivity problem associated with µ-PAD.

Microfluidic Paper Based Analytical Devices for the Detection of Carbamate Pesticides.

Microfluidic paper-based analytical devices (µ-PADs) are a new technology platform for the development of extremely low-cost sensing applications. In this study, µ-PADs has been developed for quantitative determination of carbamate pesticides. Key experimental parameters including concentration and volume of acetylcholinesterase, acetylthiocholine iodide and 5,5'-dithiobis-(2-nitrobenzoic acid), incubation time and image capturing time were systematically optimized. Under optimal conditions, the method showed wide range of linearity (0.25-16 mg/L), repeatability (4%-5% RSD) and intermediate precision (7%-10% RSD). Limit of detection was observed to be 0.4, 0.24 and 0.46 mg/L for carbaryl, carbosulfan and furathiocarb, respectively. An acceptable mean recovery (87% to 94%) was observed for the three pesticides at 1 mg/L fortification level. The results reveal that the developed method requires minimal reagents, simple and is easy to handle. It can be used for the quantification of carbamate pesticides in resource limited laboratories without the need for the conventional analytical instruments.

Dip-and-Read, Organic Solvent-Compatible, Paper-Based Analytical Devices Equipped with Chromatographic Separation for Indole Analysis in Shrimp.

We developed an organic solvent-compatible paper-based analytical device (PAD) for the quantitative analysis of indole, which is an indicator of shrimp freshness. Although indole is insoluble in water, ethyl acetate is a suitable solvent to dissolve and extract indole from shrimp. The PADs are fabricated using a cutting method that allows the use of an organic solvent because no hydrophobic barrier is needed to form fluidic channels. Ehrlich's reagent consists of 4-(dimethylamino)benzaldehyde and p-dimethylaminobenzaldehyde and was deposited onto the reaction zone of the PAD followed by lamination to prevent evaporation of the ethyl acetate. Samples are introduced into the PAD via immersion in organic sample solutions. When the PAD is immersed into an indole solution of ethyl acetate in a closed bottle, the sample solution penetrates the channel of the PAD and successively flows into the detection zone to form a hydrophilic colored product. The PADs provide a linear relationship between the logarithm of the indole concentration and the color intensity within a range of 1.0-20 ppm with correlation coefficients of r2 > 0.99. The limits of detection and quantification are 0.36 and 0.71 ppm, respectively. Relative standard deviations for both the intraday (n = 2) and interday (n = 3) precision were less than 2.5%. In the indole analysis of shrimp, the PADs separated the interfering orange-colored astaxanthin in the extract from the colored product of indole via the paper chromatographic principle. We used the PADs to investigate the degradation of shrimp, and the results showed a rapid increase in the indole level after 7 days. High-performance liquid chromatography verified the accuracy of the PADs by showing good agreement with the obtained indole levels.

N-Benzoyl leucomethylene blue as a novel substrate for the assays of horseradish peroxidase by spectrophotometry and capillary electrophoresis-laser-induced fluorometry.

Horseradish peroxidase (HRP) is an enzyme that is frequently employed in various assays because HRP catalyzes the oxidation reactions of chromogenic and fluorogenic compounds to produce chromophores and fluorophores, respectively. The results of this study show that N-benzoyl leucomethylene blue (BLMB) is an excellent substrate for enzyme assay using HRP. In the presence of hydrogen peroxide (H2O2), HRP catalyzed an oxidation reaction of BLMB that produced methylene blue with a deep blue color. Thus, absorption spectrophotometry and capillary electrophoresis-laser-induced fluorometry (CE-LIF) could be used to easily determine the produced methylene blue. Under the optimum conditions, absorption spectrophotometry showed a linear calibration curve that ranged from 25 to 500 µg mL-1. The reaction conditions were also applicable to CE-LIF, showing a linear range of from 25 to 500 µg mL-1 with limits of detection and quantification at 2 and 6 µg mL-1, respectively.

Microfluidic paper-based analytical devices coupled with coprecipitation enrichment show improved trace analysis of copper ions in water samples.

The present study focused on improving sensitivity to trace levels of Cu(II) by subjecting microfluidic paper-based analytical devices (µ-PADs) to a preconcentration process via coprecipitation using aluminum hydroxide. The experimental conditions were optimized for the pH of the coprecipitation, centrifugation, and amounts of reagents that were deposited onto µ-PADs for the Cu(II) assay. The resultant limit of detection reached as low as 0.003 mg L-1 with a linear range of 0.01-2.00 mg L-1. The relative standard deviations for intra- and inter-day precision were 3.2 and 4.6%, respectively (n = 9). Spiked water samples were analyzed using the µ-PADs after coprecipitation preconcentration. The results were verified by comparing them with those of inductively coupled plasma-optical emission spectrometry (ICP-OES). Recoveries ranged from 97.1 to 104% and from 98.7 to 105% using the present method and ICP-OES, respectively. These results suggest that the simple, highly sensitive, and inexpensive proposed method would be helpful for analyzing trace levels of Cu(II) in water samples in poorly equipped laboratories.

Speciation of chromium in water samples using microfluidic paper-based analytical devices with online oxidation of trivalent chromium.

Speciation of chromium (Cr) was demonstrated using microfluidic paper-based analytical devices (µ-PADs) that permit the colorimetric determination of hexavalent chromium (Cr(VI)) and trivalent chromium (Cr(III)) via online oxidation. The µ-PADs consist of left and right channels that allow the simultaneous measurements of Cr(VI) and total Cr based on the colorimetric reaction of Cr(VI) with 1,5-diphenylcarbazide (DPC). For the determination of Cr(VI), a sample solution was directly reacted with DPC in the left channels whereas total Cr was determined in the right channels, which permitted online oxidation in the pretreatment zone containing cerium (IV) (Ce(IV)) followed by a colorimetric reaction with DPC. We found that the online oxidation of Cr(III) proceeded 100% whereas Ce(IV) inhibited the reaction of Cr(VI) with DPC. Therefore, speciation can be achieved by measuring the Cr(VI) and total Cr in the left and right channels followed by the subtraction of Cr(VI) from total Cr. The limits of detection and quantification were 0.008 and 0.02 mg L-1 for Cr(VI) and 0.07 and 0.1 mg L-1 for Cr(III) or total Cr, respectively. The linear dynamic ranges were 0.02-100 mg L-1 and 0.1-60 mg L-1 for Cr(VI) and Cr(III), respectively. The RSDs were less than 7.5%. The results obtained using µ-PADs were in good agreement with those obtained via ICP-OES with recoveries of 92-108% for Cr(III) and 108-110% for Cr (VI) using µ-PADs, and 106-110% for total Cr using ICP-OES. Thus, the µ-PADs could potentially be utilized for the speciation of chromium in developing countries where environmental pollution and the availability of sophisticated instruments are significant problems.

Simultaneous separation of 17 anions by capillary electrophoresis with the addition of an organic solvent.

Seventeen inorganic and organic anions, that normally are insufficiently separated via ion chromatography, were completely separated by the addition of an organic solvent to a solution of BGE combined with an adjustment of the apparent pH via CE in combination with indirect UV absorbance detection. Methanol, ethanol, and acetonitrile were examined for their utility in manipulating the selective separation of anions. Methanol and acetonitrile were better modifiers than ethanol at enhancing the resolution of anions comigrating in an aqueous solution of BGE. Methanol was selected as the modifier that provided the largest separation window that could achieve a complete separation of the target analytes. Via the use of methanol, manipulation of the selectivity between inorganic anions and that between inorganic and organic anions was enhanced, but the separation between organic anions remained difficult when only methanol was used. By varying the apparent pH of the BGE in the presence of 10% v/v methanol, however, the separation selectivity between organic anions was substantially improved. Eventually, 7 inorganic and 10 organic anions were simultaneously separated using BGE at a pH of 6.3 in the presence of 10% v/v methanol.

Remote Investigation of Total Chromium Determination in Environmental Samples of the Kombolcha Industrial Zone, Ethiopia, Using Microfluidic Paper-based Analytical Devices.

Microfluidic paper-based analytical devices (µ-PADs) fabricated in Japan were employed for the determination of total chromium (Cr) in water, soil, and lettuce irrigated with wastewater in Ethiopia. The µ-PADs, which were printed by wax printing in Japan, were transported to Ethiopia and prepared for the determination of total Cr by adding appropriate reagents to the pretreatment and detection zones. Soil and lettuce samples were determined by the µ-PADs and a UV-Vis spectrophotometer in Ethiopia. A paired t-test showed that the mean total Cr concentrations determined in the soil and lettuce samples were not significantly different between µ-PADs and UV-Vis spectrophotometric analysis at the 5% level of significance. This implies that the µ-PADs have good accuracy and reliability, and could be employed to monitor Cr in environmental samples. We found that the total Cr concentrations in all soil and lettuce samples were above the permissible limit. Moreover, evaluating Cr contamination level using the geo-accumulation index indicated that the soils were contaminated with Cr moderately to heavily. Thus, the present work successfully demonstrated the potential of remote investigations of pollution in a less-equipped laboratory by transporting the µ-PADs fabricated in another laboratory.

On-site analysis of paraquat using a completely portable photometric detector operated with small, rechargeable batteries.

This work describes a methodology that can be used to achieve on-site analysis of paraquat in water samples by using a miniaturized portable photometer consisting of a couple of light-emitting diodes (LEDs). Paraquat produces a colored radical via a redox reaction with sodium dithionite, which is unstable against oxygen in solution. The steps taken to stabilize the reagent solution included control of the pH and the addition of organic solvents, but the most effective was the formation of an oil layer. Together, these steps stabilized the reagent solution for two days. An increase in the duration of reagent stability, however, is necessary in order to transport the reagent for on-site applications in remote locales. For the time being, an excess amount of solid sodium dithionite can be added directly to sample solutions because the unreacted dithionite shows no influence on absorbance of the paraquat radical. Orange LEDs with a maximum emission wavelength of 609 nm were employed in the portable photometer to measure the absorbance of paraquat radical produced by a redox reaction that has an absorption maximum of 603 nm. The developed photometer showed excellent performance with a linear range of from 2.0 mg L-1 to 40.0 mg L-1 and a linear regression (r2 = 1). The limits of detection and quantification were 0.5 mg L-1 and 1.5 mg L-1, respectively, intra-day precision (n = 3) and inter-day precision (n = 5) were both less than 5%, and accuracy based on the percentage of sample recovery ranged from 89 ± 0 to 105 ± 0% (n = 3). The proposed method was applied to the analysis of paraquat in water samples taken from rice fields. The results showed no paraquat in all thirteen samples, which could have been due to strong adsorption of paraquat by soil particles and/or to complications with the sampling conditions. To confirm the adsorption onto soil of paraquat contained in water, we constructed an artificial rice field where water containing paraquat was impounded above the soil layer. The results showed that paraquat in water gradually decreased within three days and could be measured in the soil on the fourth day. These results were confirmed by HPLC analysis, which underscores the utility of this portable photometer for the on-site monitoring of paraquat in water samples.

Indirect capillary electrophoresis immunoassay of membrane protein in extracellular vesicles.

Extracellular vesicles (EVs) exist in biological fluids such as blood, urine, and cerebrospinal fluid, and these have shown promise for use as biomarkers of cancers. Conventional methods for determination of EVs include direct detection via enzyme-linked immunosorbent assay and detection of their membrane proteins via western blotting. These techniques, however, have individual shortcomings in terms of the need for large sample consumption, processes that are time-consuming, and a lack of the capacity for quantification. In this study, we developed a method to determine the EV membrane protein, CD63, by coupling capillary electrophoresis immunoassay with laser-induced fluorescence (CEIA-LIF). In this process, the EVs were isolated from a culture medium and were subsequently reacted with a fluorescently labeled anti-CD63 antibody to form a CD63 complex localized on the surface of EVs. After removing the EVs containing the CD63 immune complex by centrifugation, the supernatant containing the free fluorescent antibody was injected into a capillary to serve as a sample. A decrease in the peak area of the free fluorescent antibody became apparent when the amount of EVs was increased while that of the fluorescent antibody remained constant. The peak areas were decreased proportionally against the increased amounts of EVs. The concentration of the CD63 could then be estimated based on the slope of the linear relationship. This study is the first to quantify CD63 immobilized on EVs via CEIA-LIF, which is a novel method with the potential to determine membrane proteins localized on the surface of EVs.

Direct counting of exosomes in a cell culture medium using neither isolation nor preconcentration.

Exosomes are expected to be biomarkers of cancer since they contain information about the cells that excrete them. In this study we developed a method to count the exosomes secreted from cancer cells in a culture medium without the need for isolation and/or preconcentration. This detection system consists of a square capillary on which a laser beam is focused in a sheet shape via the use of two cylindrical lenses. A fluorescently labeled anti-CD63 antibody is used to mark the exosomes that are then flowed into the square capillary. In this study, individual exosomes were observed on a trajectory when passing through the laser beam sheet and were counted for 10 min at a constant flow velocity. The total analysis time was less than 1.5 h including the steps required to remove large particles and allow reaction with the antibody. The results for two samples prepared with and without the isolation of exosomes showed a loss of exosomes in the isolation step. We also determined the number of the exosomes secreted by the cells to a culture medium during cultivation. As expected, the total number of exosomes in a culture medium increased with an increase in the cultivation time, and the number of exosomes released every 12 h either remained constant or showed no more than a slight increase for as long as 72 h. It was unclear whether the number exosomes was dependent on the cell population at confluences of 10-60%.

Long-term stabilization of hydrogen peroxide by poly(vinyl alcohol) on paper-based analytical devices.

Stabilizing reagents that can be deposited onto paper is an important issue for researchers who depend on paper-based analytical devices (PADs), because long-term stability of the devices is essential in point-of-care testing. Here, we found that poly(vinyl alcohol) (PVA) would stabilize hydrogen peroxide placed on a paper substrate following exposure to air. Horseradish peroxidase was employed as a sample in colorimetric measurements of PADs after hydrogen peroxide and 3,3',5,5'-tetramethylbenzidine were deposited as substrates in an enzymatic reaction. The addition of PVA to hydrogen peroxide significantly suppressed its degradation. Concentrations of PVA that ranged from 0.5 to 2%, increased the duration of the stability of hydrogen peroxide, and the results for a PVA concentration of 1% approximated those of 2% PVA. Storage of the PADs at 4 °C in a refrigerator extended the stability of the hydrogen peroxide containing 2% PVA by as much as 30 days. The stability of hydrogen peroxide without PVA was degraded after one day under room temperature.

Chromium speciation using paper-based analytical devices by direct determination and with electromembrane microextraction.

In this study, we developed and compared three different methods for chromium speciation in water samples using microfluidic paper-based analytical devices (µPADs). In all methods, detection was based on the complexation reaction of Cr(VI) with diphenylcarbazide on the µPADs. Cr(III) ions were oxidized to Cr(VI) by Ce(IV) prior to colorimetric detection on the µPADs. In the first method, oxidization of Cr(III) to Cr(VI) in the solution containing both trivalent and hexavalent chromium was performed using a batch procedure to obtain total chromium. A dual electromembrane extraction (DEME) technique for simultaneous preconcentration and extraction of chromium species and a single electromembrane extraction (SEME) for preconcentration and extraction of Cr(VI)/total chromium [quantified as Cr(VI) content after oxidation of Cr(III) ions to Cr(VI)] were used in the second and third methods, respectively. The electromembrane extraction was based on the electrokinetic migration of cationic Cr(III) and anionic Cr(VI) toward the cathode and anode, respectively, into the two different hollow fibres. Octanol-1 and bis(2-ethylhexyl) phosphate (DEHP) in octanol-1 (0.7% v/v) were the most suitable supported liquid membranes for extraction of Cr(VI) and Cr(III), respectively. Among these methods, SEME showed the lowest limits of detection for both analytes. Under optimized conditions, linear calibrations were obtained for Cr(III) from 3 to 30 µg L-1 and for Cr(VI) from 3 to 70 µg L-1. The detection limits were 1.0 µg L-1 and 0.7 µg L-1 for Cr(III) and Cr(VI), respectively. Our developed method was applied to analyse water samples spiked with different concentrations of Cr(III) and Cr(VI) at the parts-per-billion (ppb) level. The statistical evaluation showed that the proposed method agreed well with the validation method, i.e., inductively coupled plasma atomic emission spectroscopy (ICP-AES).

Characterization of Pieces of Paper That Form Reagent Containers for Use as Portable Analytical Devices.

Reagent-deposited pieces of paper were characterized by the use of a compact conductometer, a compact pH sensor, and a conventional spectrophotometer to assess their suitability for use as reagent containers. The pieces of paper were fabricated by wax printing to form a limited hydrophilic area to which a consistent volume of an aqueous reagent could be added. The pieces of paper without the reagent increased the conductivity of water gradually because of the release of sodium salts, whereas pH of NaOH decreased because of the acidity of the functional groups in the paper. Three reagents, sulfamic acid as an acid, Na2CO3 as a base, and BaCl2 as a metal salt, were deposited on the pieces of paper to evaluate their ability to release from the pieces of paper. Sulfamic acid and Na2CO3 were released in quantities of 58 and 73% into water after 420 s, whereas 100% of BaCl2 was released after 480 s. The conductometric titrations of NaOH, HCl, and Na2SO4, and the spectrophotometry of Fe2+ were examined using the pieces of paper that contained sulfamic acid, Na2CO3, BaCl2, and 1,10-phenanthroline. Titrations using the pieces of paper suggested that the reagents were quantitatively released into the titrant, which resulted in a linear relationship between the endpoints and the equivalent points. In 120 s of soaking time, 60-70% of the reagents were released. The spectrophotometric measurements of Fe2+ indicated that when an excess amount of the reagents was deposited onto the pieces of paper, they nonetheless sufficiently fulfilled the role of a reagent container.

Miniaturized Potentiometric Titration for Improving Portability and Accuracy in the Determination of Total Acid in Squeezed Fruit Juice.

To determine the total acidity in freshly squeezed fruit juice, we miniaturized the potentiometric titrations and achieved better accuracy compared with titrations from a conventional pH probe. The improvement was the result of a higher jump in pH at the endpoint due to a reduction in the dilutions of both the titrand and titrant. A conventional pH probe requires more than 50 mL of titrand, which can lead to a 25000-fold dilution of the titrant when adding the titrant at 2 µL intervals. Conversely, when the volume of the titrand can be reduced to 1 mL, the dilution is only 500-fold, which results in a higher jump in pH at the endpoint. The concentration of the titrant, NaOH, was optimized by titrating sample solutions containing 25 and 50 mM of citric acid. The addition of 5 M NaOH in intervals of 2 µL led to a more accurate endpoint for both 25 and 50 mM citric acid solutions. Miniaturization of the titration process is advantageous in terms of portability, accuracy, and in requiring less consumption of a sample, thereby simplifying the process of repeat measurements that are helpful in evaluating the precision of analytical results. Practical samples of squeezed fruit juices were titrated via three methods that showed no significant differences: classic titrimetry with an indicator, conventional potentiometry, and miniaturized potentiometry. This process would be effective for use in the field and in developing countries. PRACTICAL APPLICATION: The total acidity of fruits and fruit juices is an important indicator of quality and is generally expressed in terms of the citric acid content. However, a standard potentiometric titration requires a large sample volume, which makes it difficult to assess dispersion of the acidity for individual fruits. The results of this study indicate that the use of miniaturized potentiometric titration could benefit food chemistry in many developing countries in addition to opening new fields of food chemistry such as on-site quality control of citrus fruit and evaluation of variations in quality.

Development of a miniaturized photometer with paired emitter-detector light-emitting diodes for investigating thiocyanate levels in the saliva of smokers and non-smokers.

A simple, small and inexpensive photometer that uses a pair of light-emitting diodes (LEDs) and a simple operational amplifier was developed for investigating thiocyanate levels in saliva obtained from smokers and non-smokers. The photometer is based on paired emitter-detector diodes (PEDDs), and the entire system can be purchased for less than a hundred US dollars. The PEDD-based photometer can measure the transmittance of a solution in a 1-cm disposable polystyrene cuvette using only rechargeable dry-cell batteries, which makes it suitable for analysis outside of equipped laboratories. The metal complex formation between Fe (III) and thiocyanate ions in an acidic condition permits colorimetric detection of thiocyanate ions using LEDs emitting at 465 nm, because the complex shows maximum absorption at 457 nm. The developed photometer exhibits excellent performance with linearity ranging from 0.05 mmol L-1 to 0.75 mmol L-1 and a correlation coefficient (r2) > 0.999. The limits of detection and quantification were 0.01 mmol L-1 and 0.05 mmol L-1, respectively. Both intra- and inter-day precision were obtained with relative standard deviations (RSD) of less than 1% in the determination of thiocyanate. The proposed method is simple, facile, and sensitive enough to investigate the levels of thiocyanate in the saliva samples of smokers and non-smokers with centrifugation being the only special treatment for samples. The results showed that the concentrations of thiocyanate were approximately 5-fold higher in smokers than in non-smokers.