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

Fluorimetric determination of quinine using a portable system and digital image processing

The development of a portable device created by 3D printing for fluorimetric measurements is an efficient tool for analytical applications in situ or in the laboratory presenting a wide field of applications in the environmental and food field. This device uses a light-emitting diode (LED) as a radiation source and a digital microscope as a detector. Digital images obtained by the interaction between the radiation source and the sample were analyzed with the help of the YouCam software, the images were filed in JPEG format and processed with the Chemostat software.The entire system is connected to a notebook, which serves as an LED and detector power supply without the need for any additional power source. The proposed device was used for the determination in situ of quinine in water and beverage samples, respectively. For the validation of the developed system, the results obtained were compared with a conventional spectrofluorometer with a t-test at a 95% confidence level. The proposed system provides satisfactory precision and accuracy values. The proposed method presented detection and quantification limits of 0.99 and 1.46 mg·L−1, at a 95% confidence interval

Automated Spectrophotometric Multi-Pumping Flow System for the Determination of Total Iron in Wine

[eng] A new automated system based on a multi-pumping flow system (MPFS) for the spectrophotometric determination of total iron in various wine samples is presented. This analytical protocol is based on the complex formation between Fe (II) and 2-(5-bromo-2-pyridylazo)-5-(diethylamino)-phenol (Br-PADAP). The addition of ascorbic acid allows the determination of total iron by reducing the Fe (III) present in the samples to Fe (II). Several physical and chemical parameters, including the mixing coil length, the sample volume, the chromogenic reagent concentration, the pH, and the ascorbic acid concentration have been characterized in order to optimize the analytical conditions. The limits of detection and quantification were 34 mg L 1 and 114 mg L 1 respectively. The developed procedure requires minimal sample preparation

Fluorometric assay of laccase in mushroom extracts and comparisons with absorption spectrophotometry

Laccase is a lignin-degrading enzyme that is expected to move industrial applications to a greener form of biotechnology. Here, we used 2,2'-azinobis(3-ethylbenzthiazolin-6-sulfonic acid) (ABTS) as a mediator and N-benzoyl leucomethylene blue (BLMB) as a substrate to develop a fluorometric assay that we used to measure laccase activity in mushroom extracts. We then compared this novel approach to conventional absorption spectrophotometry. With this novel approach, laccase oxidizes ABTS to produce ABTS radicals that show an absorption maximum at 415 nm. The ABTS radicals oxidize BLMB to generate fluorescent methylene blue that is measured by fluorometry while absorption spectrophotometry directly measures the absorbance of the ABTS radicals at 415 nm. Under the optimal conditions, the fluorometric assay showed a linear calibration curve with limits of detection and quantification of 1.0 × 10-2 mg mL-1 and 3.2 × 10-2 mg mL-1, respectively, and those values are 1.4-fold lower than the results using conventional absorption spectrophotometry to measure ABTS radicals. Laccase activity of extracts from species of mushrooms that include eryngii and shiitake were successfully determined via both fluorometry and absorption spectrophotometry. The eryngii extract showed the highest level of activity, which was followed by the shiitake extract, but laccase activity was not observed in the shimeji extract

Fluorometric assay of laccase in mushroom extracts and comparisons with absorption spectrophotometry

Laccase is a lignin-degrading enzyme that is expected to move industrial applications to a greener form of biotechnology. Here, we used 2,2'-azinobis(3-ethylbenzthiazolin-6-sulfonic acid) (ABTS) as a mediator and N-benzoyl leucomethylene blue (BLMB) as a substrate to develop a fluorometric assay that we used to measure laccase activity in mushroom extracts. We then compared this novel approach to conventional absorption spectrophotometry. With this novel approach, laccase oxidizes ABTS to produce ABTS radicals that show an absorption maximum at 415 nm. The ABTS radicals oxidize BLMB to generate fluorescent methylene blue that is measured by fluorometry while absorption spectrophotometry directly measures the absorbance of the ABTS radicals at 415 nm. Under the optimal conditions, the fluorometric assay showed a linear calibration curve with limits of detection and quantification of 1.0 × 10-2 mg mL-1 and 3.2 × 10-2 mg mL-1, respectively, and those values are 1.4-fold lower than the results using conventional absorption spectrophotometry to measure ABTS radicals. Laccase activity of extracts from species of mushrooms that include eryngii and shiitake were successfully determined via both fluorometry and absorption spectrophotometry. The eryngii extract showed the highest level of activity, which was followed by the shiitake extract, but laccase activity was not observed in the shimeji extract

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

Determination of glutamate using paper-based microfluidic devices with colorimetric detection for food samples

A paper-based device (PAD) capable of colorimetric detection was developed to determine the presence of glutamate in various food samples. The PAD employs an enzymatic reaction with glutamate followed by an oxidation reaction with N-benzoyl leucomethylene blue (BLMB) in the presence of horseradish peroxidase. The designed PAD consists of a sample introduction zone connected to a channel that transports a sample solution to three detection zones. The detection zones contain pre-deposited reagents: glutamate oxidase, horseradish peroxidase, BLMB, a phosphate buffer, and poly(acrylic acid). The PAD is perpendicularly immersed into a sample solution and bent at a right angle using a 3D-printed holder to allow the sample to simultaneously flow into three different detection zones. When the PAD is immersed into a sample containing glutamate, glutamate oxidase produces hydrogen peroxide, which changes the pale blue color of BLMB to a deep blue color in the presence of horseradish peroxidase. Under the optimum conditions, the calibration curve between the logarithm of the glutamate concentrations and the color intensity was linear within a range of from 5 x 10(-6) mol L-1 to 10(-2) and with a correlation coefficient of 0.994. Using this system, the PAD successfully determined glutamate in soup stocks, sauces, snacks, and tomato juice without the need of complicated sample pretreatment. These results agreed with those of a commercially available glutamate assay kit, which was employed as a certification method (t(stat )= 1.95, t(crit )= 2.57). The developed PAD is simple, easy to fabricate, portable, and could be used outside of equipped laboratories to determine the presence of glutamate in food samples