Plug-and-play assembly of paper-based colorimetric and electrochemical devices for multiplexed detection of metals.

Heavy metals are the main pollutants present in aquatic environments and their presence in human organisms can lead to many different diseases. While many methods exist for analysis, colorimetric and electrochemistry are particularly attractive for on-site analysis and their integration on a single platform can improve multiplexed metals analysis. This report describes for the first time a "plug-and-play" (PnP) assembly for coupling a microfluidic paper-based device (µPAD) and a screen-printed electrochemical paper-based device (ePAD) using a vertical and reversible foldable mechanism for multiplexed detection of Fe, Ni, Cu, Zn, Cd and Pb in river water samples. The integration strategy was based on a reversible assembly, allowing the insertion of a pretreatment zone to minimize potential chemical interfering agents and providing a better control of the aspirated sample volume as well as to a lower sample evaporation rate. In comparison with lateral flow and electrochemical assays performed using independent devices, the integrated prototype proved that the reversible coupling mechanism does not interfere on the analytical performance (95% confidence interval). The limit of detection (LOD) values calculated for metals determined varied from 0.1 to 0.3 mg L-1 (colorimetric) and from 0.9 to 10.5 µg L-1 (electrochemical). When compared to other integrated devices based on horizontal designs, the use of a foldable coupling mechanism offered linear response in a lower concentration range and better LOD values for Fe, Ni and Cu. The proposed method successfully measured heavy metals in river water samples with concentrations ranging from 16 to 786 µg L-1, with recovery studies ranging from 76 to 121%. The new method also showed good correlation with conventional atomic absorption spectroscopic methods (95% significance level). Thus, the integration of µPADs and ePADs by a vertical folding mechanism was efficient for multiplexed heavy metal analysis and could be exploited for environmental monitoring.

Thermoplastic electrodes as a new electrochemical platform coupled to microfluidic devices for tryptamine determination.

This study reports a new electrochemical method for tryptamine determination using a paper-based microfluidic device and a thermoplastic electrode (TPE) as an amperometric detector. Tryptamine (Tryp) is a biogenic amine present in drinks and foods. Even though this compound has some beneficial effects on human health, the ingestion of foods with high concentrations of Tryp may be detrimental, which justifies the need for monitoring the Tryp levels. The TPEs were made from 50% carbon black and 50% polycaprolactone and characterized by cyclic voltammetry, demonstrating enhancement in the analytical response compared to other carbon composites. TPEs also showed a better antifouling effect for Tryp compared to conventional glassy carbon electrodes. Once characterized, the electrodes were incorporated into the microfluidic device to determine Tryp in water and cheese samples using amperometry. A linear range was achieved from 10 to 75 µmol L-1 with limits of detection and quantification of 3.2 and 10.5 µmol L-1, respectively. Therefore, this work shows promising findings of the electrochemical determination of Tryp, bringing valuable results regarding the electrochemical properties of thermoplastic composites.

Rapid Analysis in Continuous-Flow Electrochemical Paper-Based Analytical Devices.

A simple and low-cost continuous-flow (CF) electrochemical paper-based analytical device (ePAD) coupled with thermoplastic electrodes (TPEs) was developed. The fast, continuous flow combined with flow injection analysis was made possible by adding two inlet reservoirs to the same paper-based hollow channel flowing over detection electrodes, terminating in a fan-shaped pumping reservoir. The upstream inlet reservoir was filled with buffer and provided constant flow through the device. Sample injections were performed by adding 2 µL of the sample to the downstream sample inlet. Differences in flow resistance resulted in sample plugs displacing buffer as the solution flowed over the working electrodes. The electrodes were fabricated by mixing carbon black and polycaprolactone (50% w/w). CF-TPE-ePADs were characterized with chronoamperometry using ferrocenylmethyl trimethylammonium as the electrochemical probe. Optimized flow rates and injection volumes gave analysis times roughly an order of magnitude faster than those of previously reported flow injection analysis ePADs. To demonstrate applicability, the CF-TPE-ePADs were used to quantify caffeic acid in three different tea samples. The proposed method had a linear range from 10 to 500 µmol L-1 and limits of detection and quantification of 2.5 and 8.3 µmol L-1, respectively. Our approach is promising for fabricating simple, inexpensive, yet high-performance, flow injection analysis devices using paper substrates and easy-to-make electrodes that do not require external mechanical pumping systems or complicated valves.

USB powered microfluidic paper-based analytical devices.

Microfluidic paper-based analytical devices (µPADs) allow user-friendly and portable chemical determinations, although they provide limited applicability due to insufficient sensitivity. Several approaches have been proposed to address poor sensitivity in µPADs, but they frequently require bulky equipment for power and/or read-outs. Universal serial buses (USB) are an attractive alternative to less portable power sources and are currently available in many common electronic devices. Here, USB-powered µPADs (USB µPADs) are proposed as a fusion of both technologies to improve performance without adding instrumental complexity. Two ITP USB µPADs were developed, both powered by a 5 V potential provided through standard USB ports. The first device was fabricated using the origami approach. Its operation was analyzed experimentally and numerically, yielding a two-order-of-magnitude sample focusing in 15 min. The second ITP USB µPAD is a novel design, which was numerically prototyped with the aim of handling larger sample volumes. The reservoirs were moved away from the ITP channel and capillary action was used to drive the sample and electrolytes to the separation zone, predicting 25-fold sample focusing in 10 min. USB µPADs are expected to be adopted by minimally-trained personnel in sensitive areas like resource-limited settings, the point-of-care and in emergencies.

Rapid Bacteria Detection at Low Concentrations Using Sequential Immunomagnetic Separation and Paper-Based Isotachophoresis.

Detecting bacteria is important in the fields of human health, environmental monitoring, and food safety. Foodborne pathogens alone are estimated to cause 420 000 deaths annually, with low-income regions affected most. Despite improvements in bacterial detection, fast, disposable, low-cost, sensitive, and user-friendly methods are still needed. Traditional methods for detecting bacteria rely primarily on cell culturing or polymerase chain reaction (PCR), which require highly trained personnel and a central laboratory and take several hours or even days to deliver results. Low-cost methods like lateral flow immunoassays exist but frequently suffer from poor sensitivity and/or lack quantitative results. Here, a rapid method for detecting bacteria at very low concentrations is presented using two sequential preconcentration steps. In the first preconcentration step, the sample is mixed with antibody-modified magnetic particles and free antibodies conjugated to ß-galactosidase (ß-gal). The target bacteria are isolated and concentrated using immunomagnetic separation. The isolated bacteria are then incubated with chlorophenol red-ß-d-galactopyranoside (CPRG), which reacts with ß-gal to produce chlorophenol red (CPR) in a bacteria concentration-dependent manner. In the second step, CPR and CPRG are separated and focused using an isotachophoretic microfluidic paper-based analytical device, significantly improving the final detection limit relative to paper-based devices lacking the focusing mechanism. Moreover, CPR and CPRG form two visible color bands that act as test and control bands, respectively, improving assay robustness. The method was tested with E. coli DH5-α and successfully detected concentrations as low as 9.2 CFU/mL in laboratory samples and 920 CFU/mL in apple juice samples in ∼90 min.

Uncovering the Formation of Color Gradients for Glucose Colorimetric Assays on Microfluidic Paper-Based Analytical Devices by Mass Spectrometry Imaging.

This study describes the use of mass spectrometry imaging with matrix-assisted laser desorption/ionization (MALDI) and desorption electrospray ionization (DESI) to understand the color gradient generation commonly seen in microfluidic paper-based analytical devices (µPADs). The formation of color gradients significantly impacts assay sensitivity and reproducibility with µPADs but the mechanism for formation is poorly understood. The glucose enzymatic assay using potassium iodide (KI) as a chromogenic agent was selected to investigate the color gradient generated across a detection spot. Colorimetric measurements revealed that the relative standard deviation for the recorded pixel intensities ranged between 34 and 40%, compromising the analytical reliability. While a variety of hypotheses have been generated to explain this phenomenon, few studies have attempted to elucidate the mechanisms associated with its formation. Mass spectrometry imaging using MALDI and DESI was applied to understand the nonuniform color distribution on the detection zone. MALDI experiments were first explored to monitor the spatial distribution of the glucose oxidase and horseradish peroxidase mixture, before and after lateral flow assay with and without KI. MALDI(+)-TOF data revealed uniform enzyme distribution on the detection spots. On the other hand, after the complete assay DESI(-) measurements revealed a heterogeneous shape indicating the presence of iodide and triiodide ions at the zone edge. The reaction product (I3-) is transported by lateral flow toward the zone edge, generating the color gradient. Mass spectrometry imaging has been used for the first time to prove that color gradient forms as result of the mobility small molecules and not the enzyme distribution on µPAD surface.

A paper-based colorimetric spot test for the identification of adulterated whiskeys.

A colorimetric point-of-care paper-based analytical device (PAD) is developed for detecting adulterated beverages using whiskey falsified with caramel color as a model. Combining principal component analysis and calibration curves facilitated identification of adulteration in samples seized by the Brazilian Federal Police, at only ∼$0.02 per sample.

Versatile fabrication of paper-based microfluidic devices with high chemical resistance using scholar glue and magnetic masks.

Simple methods have been developed for fabricating microfluidic paper-based analytical devices (µPADs) but few of these devices can be used with organic solvents and/or aqueous solutions containing surfactants. This study describes a simple fabrication strategy for µPADs that uses readily available scholar glue to create the hydrophobic flow barriers that are resistant to surfactants and organic solvents. Microfluidic structures were defined by magnetic masks designed with either neodymium magnets or magnetic sheets to define the patter, and structures were created by spraying an aqueous solution of glue on the paper surface. The glue-coated paper was then exposed to UV/Vis light for cross-linking to maximize chemical resistance. Examples of microzone arrays and microfluidic devices are demonstrated. µPADs fabricated with scholar glue retained their barriers when used with surfactants, organic solvents, and strong/weak acids and bases unlike common wax-printed barriers. Paper microzones and microfluidic devices were successfully used for colorimetric assays of clinically relevant analytes commonly detected in urinalysis to demonstrate the low background of the barrier material and generally applicability to sensing. The proposed fabrication method is attractive for both its ability to be used with diverse chemistries and the low cost and simplicity of the materials and process.

Construction and electrochemical characterization of microelectrodes for improved sensitivity in paper-based analytical devices.

This work presents a simple, low cost method for creating microelectrodes for electrochemical paper-based analytical devices (ePADs). The microelectrodes were constructed by backfilling small holes made in polyester sheets using a CO2 laser etching system. To make electrical connections, the working electrodes were combined with silver screen-printed paper in a sandwich type two-electrode configuration. The devices were characterized using linear sweep voltammetry, and the results are in good agreement with theoretical predictions for electrode size and shape. As a proof-of-concept, cysteine was measured using cobalt phthalocyanine as a redox mediator. The rate constant (k(obs)) for the chemical reaction between cysteine and the redox mediator was obtained by chronoamperometry and found to be on the order of 10(5) s(-1) M(-1). Using a microelectrode array, it was possible to reach a limit of detection of 4.8 µM for cysteine. The results show that carbon paste microelectrodes can be easily integrated with paper-based analytical devices.

Second virial coefficient determination of a therapeutic peptide by self-interaction chromatography.

Self-interaction of macromolecules has been shown to play an important role in a number of physical processes, including crystallization, solubility, viscosity, and aggregation. Peptide self-interaction is not as well studied as for larger proteins, but should play an equally important role. The osmotic second virial coefficient, B, can be used to quantify peptide and protein self-interaction. B values are typically measured using static light scattering (SLS). Peptides, however, do not scatter enough light to allow such measurements. This study describes the first use of self-interaction chromatography (SIC) for the measurement of peptide B values because SIC does not have the molecular size limitations of SLS. In the present work, SIC was used to measure B for enfuvirtide, a 36-amino acid therapeutic peptide, as a function of salt concentration, salt type, and pH. B was found to correlate strongly with solubility and apparent molecular weight. In general, the solubility of enfuvirtide increases with pH from 6 to 10 and decreases as the salt concentration increases from 0 to 0.5M for three different salts. The effect of peptide concentration on B was also investigated and shown to have a significant effect, but only at high concentrations (>80 mg/mL).