Selection of appropriate protein assay method for a paper microfluidics platform.
ABSTRACT: Background:Paper-analytical devices (PADs) have gained popularity as a simple and low-cost alternative for determining a wide range of analytes including proteins. Even though several colorimetric PADs methods for protein estimation are reported in literature, they lack justification for the chosen method and parameters therein. Aim:Major aim of this work was to thoroughly evaluate the most commonly used colorimetric protein assays and recommend the most appropriate method for PADs platform. Method:We performed following six colorimetric protein assays on PADs: biuret, lowry, bicinchoninic acid, bradford, bromocresol green, and tetrabromophenol blue. We obtained assay signal by analyzing images of the PADs and then assessed analytical figures of merit. Result:Precision, accuracy, LOD, and LOQ of PADs protein assay methods ranged from 1.2 to 6.4%, 73.3-102.4%, 0.3-3.8 ?mg/mL, and 1.2-12.8 ?mg/mL, respectively. Out of six methods, we determined bromocresol green and tetrabromophenol blue as the best methods for serum and urine samples, respectively based on their optimized parameters and analytical figures of merit. The total average serum and urine protein in human samples were found to be 94.6 ?± ?16.2 ?mg/mL and 2.1 ?± ?1.5 ?mg/mL, respectively using PADs methods. The PADs result on human samples moderately correlated with the results from spectrophotometric determination (r2 ?> ?0.6). Conclusion:Paper-based protein assays were easy to perform and were completed with thousand-fold less volume of samples/reagents without a spectrophotometer compared to conventional assay methods. After testing human samples, we found one protein assay method may not be appropriate for all types of samples.
Project description:Insufficient sensitivity is a general issue of colorimetric paper-based analytical devices (PADs) for trace analyte detection, such as metal ions, in environmental water. This paper demonstrates the colorimetric detection of zinc ions (Zn2+) on a paper-based analytical device with an integrated analyte concentration system. Concentration of Zn2+ ions from an enlarged sample volume (1 mL) has been achieved with the aid of a colorimetric Zn2+ indicator (Zincon) electrostatically immobilized onto a filter paper substrate in combination with highly water-absorbent materials. Analyte concentration as well as sample pretreatment, including pH adjustment and interferent masking, has been elaborated. The resulting device enables colorimetric quantification of Zn2+ in environmental water samples (tap water, river water) from a single sample application. The achieved detection limit of 0.53 ?M is a significant improvement over that of a commercial colorimetric Zn2+ test paper (9.7 ?M), demonstrating the efficiency of the developed analyte concentration system not requiring any equipment.
Project description:An improved paper-based analytical device (PAD) using color screening to enhance device performance is described. Current detection methods for PADs relying on the distance-based signalling motif can be slow due to the assay time being limited by capillary flow rates that wick fluid through the detection zone. For traditional distance-based detection motifs, analysis can take up to 45 min for a channel length of 5 cm. By using a color screening method, quantification with a distance-based PAD can be achieved in minutes through a "dip-and-read" approach. A colorimetric indicator line deposited onto a paper substrate using inkjet-printing undergoes a concentration-dependent colorimetric response for a given analyte. This color intensity-based response has been converted to a distance-based signal by overlaying a color filter with a continuous color intensity gradient matching the color of the developed indicator line. As a proof-of-concept, Ni quantification in welding fume was performed as a model assay. The results of multiple independent user testing gave mean absolute percentage error and average relative standard deviations of 10.5% and 11.2% respectively, which were an improvement over analysis based on simple visual color comparison with a read guide (12.2%, 14.9%). In addition to the analytical performance comparison, an interference study and a shelf life investigation were performed to further demonstrate practical utility. The developed system demonstrates an alternative detection approach for distance-based PADs enabling fast (∼10 min), quantitative, and straightforward assays.
Project description:Nelson-Somogyi and 3,5-dinitrosalicylic acid (DNS) assays are the classical analytical methods for the determination of activity of starch-debranching enzymes, however, they have a narrow detection range and do not adapt to the quantitative measurement of linear polysaccharides. Herein, we developed a simple and accurate colorimetric assay for determining the activity of starch-debranching pullulanase through the modified Tollens' reaction in combination with UV irradiation. Silver nanoparticles (AgNPs) were formed by reducing aldehyde groups in short-chain glucans (SCGs) generated by debranching of waxy maize starch using pullulanase through the modified Tollens' reaction. In addition to providing a reducing moiety to the Tollens' reaction, the debranching product, SCGs, also enhanced the colloidal stability of synthesized AgNPs, of which the amplitude of its surface plasmon resonance (SPR) absorbance peak was proportional to the concentration of SCGs ranging from 0.01-10 mg/mL. The detection limit of this system was 0.01 mg/mL, which was found to be 100 times higher than that of the conventional DNS assay. The purification of SCGs by recrystallization and gelatinization improved the selectivity of this colorimetric assay for debranching products, which provides a simple and accurate means of monitoring the debranching process and characterizing the activity of starch-debranching enzymes.
Project description:We developed a new nanozyme-based electrochemical immunoassay method for the monitoring of glycated albumin (GA) known to reflect short-term glycaemic levels. For this study, we synthesized urchin-like Pt nanozymes (uPtNZs) and applied them to colorimetric and electrochemical assays for sensitive determination of GA in total human serum albumin (tHSA) using 3,3',5,5'-tetramethylbenzidine (TMB) and thionine as substrates, respectively. The uPtNZs showed peroxidase-mimic activity in the presence of hydrogen peroxide. Boronic acid (BA)-agarose bead was used to capture GA through specific cis-diol interactions. uPtNZs were modified with GA antibody (GA-Ab) to form sandwich complexes with GA/BA-agarose bead. The amount of Ab-uPtNZ/GA/BA-agarose bead complex increased with increasing percentage of GA in 50?mg/mL tHSA. The colorimetric assay exhibited linearity from 0.02 to 10% (10?µg/mL - 5?mg/mL) GA with an LOD of 0.02% (9.2?µg/mL). For electrochemical assay, GA was detected from 0.01 to 20% (5?µg/mL - 10?mg/mL) with an LOD of 0.008% (3.8?µg/mL). The recovery values of measured GA in human plasma samples were from 106 to 107%. These results indicate that electrochemical assay using uPtNZs is a promising method for determining GA.
Project description:A systematic investigation was conducted to study the effect of paper type on the analytical performance of a series of microfluidic paper-based analytical devices (?PADs) fabricated using a CO2 laser engraver. Samples included three different grades of Whatman chromatography paper, and three grades of Whatman filter paper. According to the data collected and the characterization performed, different papers offer a wide range of flow rate, thickness, and pore size. After optimizing the channel widths on the ?PAD, the focus of this study was directed towards the color intensity and color uniformity formed during a colorimetric enzymatic reaction. According to the results herein described, the type of paper and the volume of reagents dispensed in each detection zone can determine the color intensity and uniformity. Therefore, the objective of this communication is to provide rational guidelines for the selection of paper substrates for the fabrication of ?PADs.
Project description:This paper describes a silica nanoparticle-modified microfluidic paper-based analytical device (?PAD) with improved color intensity and uniformity for three different enzymatic reactions with clinical relevance (lactate, glucose, and glutamate). The ?PADs were produced on a Whatman grade 1 filter paper and using a CO2 laser engraver. Silica nanoparticles modified with 3-aminopropyltriethoxysilane were then added to the paper devices to facilitate the adsorption of selected enzymes and prevent the washing away effect that creates color gradients in the colorimetric measurements. According to the results herein described, the addition of silica nanoparticles yielded significant improvements in color intensity and uniformity. The resulting ?PADs allowed for the detection of the three analytes in clinically relevant concentration ranges with limits of detection (LODs) of 0.63 mM, 0.50 mM, and 0.25 mM for lactate, glucose, and glutamate, respectively. An example of an analytical application has been demonstrated for the semi-quantitative detection of all three analytes in artificial urine. The results demonstrate the potential of silica nanoparticles to avoid the washing away effect and improve the color uniformity and intensity in colorimetric bioassays performed on ?PADs.
Project description:This study describes the colorimetric detection of aflatoxin M1 (Afl M1) in milk samples using a microfluidic paper-based analytical device (µPAD). Fabrication of µPADs was done using a simple and quick approach. Each ?PAD contained a detection zone and a sample zone interconnected by microchannels. The colorimetric assay was developed using unmodified AuNPs as a probe and 21-mer aptamer as a recognition molecule. The free aptamers were adsorbed onto the surface of AuNPs in absence of Afl M1, even at high salt concentrations. The salt induced aggregation of specific aptamers occurred in presence of Afl M1. Under optimum conditions, the analytical linear range was found to be 1 µM to 1 pM with limit of detection 3 pM and 10 nM in standard buffer and spiked milk samples respectively. The proposed aptamer based colorimetric assay was repeatable, quick, selective, and can be used for on-site detection of other toxins in milk and meat samples.
Project description:A simple, sensitive and selective colorimetric biosensor for the detection of Staphylococcal enterotoxin B (SEB) was developed using SEB-binding aptamer (SEB2) as recognition element and unmodified gold nanoparticles (AuNPs) as colorimetric probes. The assay is based on color change from red to purple due to conformational change of aptamer in the presence of SEB, and the phenomenon of salt-induced AuNPs aggregation which could be monitored by naked eye or UV-vis spectrometer. Results showed that the AuNPs can effectively differentiate the SEB induced conformational change of the aptamer in the presence of a given high salt concentration. A linear response in the range of 50 ?g/mL to 0.5 ng/mL of SEB concentration was obtained. The assay was highly specific to SEB as compared to other related toxins. The limit of detection (LOD) of SEB achieved within few minutes was 50 ng/mL visually and spectrometric method improved it to 0.5 ng/mL. Robustness of the assay was tested in artificially spiked milk samples and cross-checked using in house developed sandwich ELISA (IgY as capturing and SEB specific monoclonal as revealing antibody) and PCR. This colorimetric assay could be a suitable alternative over existing methods during biological emergencies due to its simplicity, sensitive and cost effectiveness.
Project description:The concentration of human serum albumin (HSA) indicates the health state of individuals and is routinely measured by UV spectroscopy with bromocresol. However, this method tends to overestimate HSA, and more critically, depends highly on the timing, in seconds, of the measurements. Here, we report an analog of 2',7'-dichlorofluorescein that can be used as a fluorescent sensor to quantify HSA in human sera. The accuracy of this new method proved superior to that of bromocresol when an international standard serum sample was analyzed. This method is more convenient than the bromocresol method because it allows for fluorescence measurements during a >15 min period. Colorimetric analysis was also performed to further investigate the effects of the binding of the sensor to HSA. These spectroscopic studies suggest that absorption and emission changes upon HSA binding may be due to the dehydration of the dye and/or stabilization of the tritylic cation species.
Project description:Natural antioxidants present in several medicinal plants are responsible for inhibiting the harmful effects of oxidative stress. These plants contain polyphenols and flavonoids that act as free radical scavengers and reduce oxidative stress and may be an alternative remedy to cure various harmful human diseases. This study aims to quantify the total phenolic and flavonoid contents (TPC and TFC) and antioxidant properties of methanol extracts of fruits, seeds, and bark of an important medicinal and aromatic plant, Zanthoxylum armatum collected from wild and cultivated populations in Nepal. TPC was determined by Folin-Ciocalteu colorimetric method using gallic acid as standard, and various concentrations of the extract solutions were measured at 760?nm. TFC was calculated by aluminum chloride colorimetric assay. Quercetin was used as standard, and the absorbance was measured at 510?nm. The antioxidant potential of the different extracts was estimated by DPPH free radical scavenging assay, and the absorbance was measured at 517?nm. The highest TPC value was 226.3?±?1.14?mg GAE/g in wild fruits, and the lowest was 137.72?±?4.21?mg GAE/g in cultivated seeds. Similarly, the highest TFC value was 135.17?±?2.02?mg QE/g in cultivated fruits, and the lowest was 76.58?±?4.18?mg QE/g in cultivated seeds. The extracts showed variable antioxidant properties. The fruits exhibited excellent antioxidant properties with IC50 values of 40.62??g/mL and 45.62??g/mL for cultivated and wild fruits, respectively. Similarly, the IC50 values of the bark were 63.39??g/mL and 67.82??g/mL, respectively, for cultivated and wild samples. And the least antioxidant capacity was shown by the seeds extract with IC50 values of 86.75??g/mL and 94.49??g/mL for wild and cultivated seeds, respectively. The IC50 value of the standard ascorbic acid was 36.22??g/mL. Different extracts of Z. armatum contain considerable amount of phenols and flavonoids, including antioxidant properties, suggesting the potential use of this species in pharmacy and phytotherapy as a source of natural antioxidants.