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.

Utilizing Paper-Based Devices for Antimicrobial-Resistant Bacteria Detection.

Antimicrobial resistance (AMR), the ability of a bacterial species to resist the action of an antimicrobial drug, has been on the rise due to the widespread use of antimicrobial agents. Per the World Health Organization, AMR has an estimated annual cost of USD 34 billion in the US and is predicted to be the number one cause of death worldwide by 2050. One way AMR bacteria can spread, and by which individuals can contract AMR infections, is through contaminated water. Monitoring AMR bacteria in the environment currently requires that samples be transported to a central laboratory for slow and labor intensive tests. We have developed an inexpensive assay using paper-based analytical devices (PADs) that can test for the presence of ß-lactamase-mediated resistance. To demonstrate viability, the PAD was used to detect ß-lactam resistance in wastewater and sewage and identified resistance in individual bacterial species isolated from environmental water sources.

Colorimetric and Electrochemical Bacteria Detection Using Printed Paper- and Transparency-Based Analytic Devices.

The development of transparency-based electrochemical and paper-based colorimetric analytic detection platforms is presented as complementary methods for food and waterborne bacteria detection from a single assay. Escherichia coli and Enterococcus species, both indicators of fecal contamination, were detected using substrates specific to enzymes produced by each species. ß-galactosidase (ß-gal) and ß-glucuronidase (ß-glucur) are both produced by E. coli, while ß-glucosidase (ß-gluco) is produced by Enterococcus spp. Substrates used produced either p-nitrophenol (PNP), o-nitrophenol (ONP), or p-aminophenol (PAP) as products. Electrochemical detection using stencil-printed carbon electrodes (SPCEs) was found to provide optimal performance on inexpensive and disposable transparency film platforms. Using SPCEs, detection limits for electrochemically active substrates, PNP, ONP, and PAP were determined to be 1.1, 2.8, and 0.5 µM, respectively. A colorimetric paper-based well plate system was developed from a simple cardboard box and smart phone for the detection of PNP and ONP. Colorimetric detection limits were determined to be 81 µM and 119 µM for ONP and PNP respectively. While colorimetric detection methods gave higher detection limits than electrochemical detection, both methods provided similar times to positive bacteria detection. Low concentrations (101 CFU/mL) of pathogenic and nonpathogenic E. coli isolates and (100 CFU/mL) E. faecalis and E. faecium strains were detected within 4 and 8 h of pre-enrichment. Alfalfa sprout and lagoon water samples served as model food and water samples, and while water samples did not test positive, sprout samples did test positive within 4 h of pre-enrichment. Positive detection of inoculated (2.3 × 102 and 3.1 × 101 CFU/mL or g of E. coli and E. faecium, respectively) sprout and water samples tested positive within 4 and 12 h of pre-enrichment, respectively.

Development of a Quasi-Steady Flow Electrochemical Paper-Based Analytical Device.

An electrochemical paper-based analytical device (ePAD) was developed for quasi-steady flow detection at microwire electrodes, for the first time. The device implements a fan shaped geometry connected to an analysis channel whereby solution is pulled from an inlet, through a channel, and into the steadily increasing capillary network of the fan. The network counteracts the decrease in solution flow rate associated with increasing viscosity within the channel, generating quasi-steady flow within the analysis channel. Microwire electrodes were embedded between two paper layers within the analysis channel, such that solution flow occurred on both sides of the wire electrodes. The quasi-steady flow ePAD increased the current by 2.5 times and 0.7 times from a saturated channel with no flow and from a single-layer paper device with flow, respectively. Amperometric detection was used for flow injection analysis (FIA) of multiple analytes at both Au and Pt microwire working electrodes, both of which provided similar sensitivity (ca. 0.2 mM-1) when normalized to the same standard. The two-layer paper devices provided a detection limit of 31 µM for p-aminophenol (PAP) using Pt electrodes and was also used to detect enzyme activity for the reaction of ß-galactosidase with p-aminophenyl-galactopyranoside (PAPG). Measured enzyme kinetics provided similar Vmax (0.079 mM/min) and Km (0.36 mM) values as those found in the literature. This device shows great promise toward use in enzyme-linked immunosorbent assays or other analytical techniques where flow or washing steps are necessary. The developed sensor provides a simple and inexpensive device capable of performing multiple injection analysis with steady-flow and online detection that would normally require an external pump to perform.

Electrochemical detection in paper-based analytical devices using microwire electrodes.

Microwire electrodes are presented as an alternative to screen-printed electrodes for detection in electrochemical paper-based analytical devices (ePADs). Compared to carbon ink electrodes, microwire electrodes offer lower resistance and a significant increase in current density relative to carbon ink electrodes. Various microwire compositions and diameters, including 30 µm Pt, 25 µm Au, 18 µm Pt with 8% W, and 15 µm Pt with 20% Ir, were tested and compared to theoretically predicted behavior. The measured current in static solution was below predicted levels for cylindrical microelectrodes but greater than levels predicted for hemi-cylindrical electrodes most likely as a result of the proximity of the electrode to the paper surface. Furthermore, the current response was indicative of semi-thin layer behavior, likely due to the confined solution volume in the paper. After electrode characterization, a device was developed for the non-enzymatic detection of glucose, fructose, and sucrose using a Cu electrode in alkaline solution. The limits of detection for glucose, fructose, and sucrose were 270 nM, 340 nM, and 430 nM, respectively, which are significantly below sugar concentrations found in sweetened beverages or glucose levels in serum.

Colorimetric paper-based detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from large volumes of agricultural water.

This protocol describes rapid colorimetric detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from large volumes (10 L) of agricultural waters. Here, water is filtered through sterile Modified Moore Swabs (MMS), which consist of a simple gauze filter enclosed in a plastic cartridge, to concentrate bacteria. Following filtration, non-selective or selective enrichments for the target bacteria are performed in the MMS. For colorimetric detection of the target bacteria, the enrichments are then assayed using paper-based analytical devices (µPADs) embedded with bacteria-indicative substrates. Each substrate reacts with target-indicative bacterial enzymes, generating colored products that can be detected visually (qualitative detection) on the µPAD. Alternatively, digital images of the reacted µPADs can be generated with common scanning or photographic devices and analyzed using ImageJ software, allowing for more objective and standardized interpretation of results. Although the biochemical screening procedures are designed to identify the aforementioned bacterial pathogens, in some cases enzymes produced by background microbiota or the degradation of the colorimetric substrates may produce a false positive. Therefore, confirmation using a more discriminatory diagnostic is needed. Nonetheless, this bacterial concentration and detection platform is inexpensive, sensitive (0.1 CFU/ml detection limit), easy to perform, and rapid (concentration, enrichment, and detection are performed within approximately 24 hr), justifying its use as an initial screening method for the microbiological quality of agricultural water.

Development of a paper-based analytical device for colorimetric detection of select foodborne pathogens.

Foodborne pathogens are a major public health threat and financial burden for the food industry, individuals, and society, with an estimated 76 million cases of food-related illness occurring in the United States alone each year. Three of the most important causative bacterial agents of foodborne diseases are pathogenic strains of Escherichia coli , Salmonella spp., and Listeria monocytogenes , due to the severity and frequency of illness and disproportionally high number of fatalities. Their continued persistence in food has dictated the ongoing need for faster, simpler, and less expensive analytical systems capable of live pathogen detection in complex samples. Culture techniques for detection and identification of foodborne pathogens require 5-7 days to complete. Major improvements to molecular detection techniques have been introduced recently, including polymerase chain reaction (PCR). These methods can be tedious; require complex, expensive instrumentation; necessitate highly trained personnel; and are not easily amenable to routine screening. Here, a paper-based analytical device (µPAD) has been developed for the detection of E. coli O157:H7, Salmonella Typhimurium, and L. monocytogenes in food samples as a screening system. In this work, a paper-based microspot assay was created by use of wax printing on filter paper. Detection is achieved by measuring the color change when an enzyme associated with the pathogen of interest reacts with a chromogenic substrate. When combined with enrichment procedures, the method allows for an enrichment time of 12 h or less and is capable of detecting bacteria in concentrations in inoculated ready-to-eat (RTE) meat as low as 10(1) colony-forming units/cm(2).

Photodynamics simulations of thymine: relaxation into the first excited singlet state.

Ab initio nonadiabatic dynamics simulations are reported for thymine with focus on the S(2) --> S(1) deactivation using the state-averaged CASSCF method. Supporting calculations have been performed on vertical excitations, S(1) and S(2) minima, and minima on the crossing seam using the MS-CASPT2, RI-CC2, MR-CIS, and MR-CISD methods. The photodynamical process starts with a fast (<100 fs) planar relaxation from the S(2) pipi* state into the pi(O)pi* minimum of the S(2) state. The calculations demonstrate that two pi-excited states (denoted pipi* and pi(O)pi*) are actually involved in this stage. The time in reaching the S(2)/S(1) intersections, through which thymine can deactivate to S(1), is delayed by both the change in character between the states as well as the flatness of the S(2) surface. This deactivation occurs in an average time of 2.6 ps at the lowest-energy region of the crossing seam. After that, thymine relaxes to the npi* minimum of the S(1) state, where it remains until the transfer to the ground state takes place. The present dynamics simulations show that not only the pi(O)pi* S(2) trapping but also the trapping in the npi* S(1) minimum contribute to the elongation of the excited-state lifetime of thymine.