Even though they were introduced less than a decade ago, electrochemical paper‐based devices (ePADs) have attracted widespread attention because of their inherent advantages in many applications. ePADs combine the advantages of microfluidic paper‐based devices (low cost, ease of use, equipment free pumping, etc.) for sample handling and processing with the advantages of sensitive and selective detection provided by electrochemistry. As a result, ePADs provide simplicity, portability, reproducibility, low cost and high selectivity and sensitivity for analytical measurements in a variety of applications ranging from clinical diagnostics to environmental sensing. Herein, recent advances in ePAD development and application are reviewed, focusing on electrode fabrication techniques and examples of applications specially focused on environmental monitoring, biological applications and clinical assays. Finally, a summary and prospective directions for ePAD research are also provided.

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. β-galactosidase (β-gal) and β-glucuronidase (β-glucur) are both produced by E. coli, while β-glucosidase (β-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 (10 CFU/mL) of pathogenic and nonpathogenic E. coli isolates and (10 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 × 10 and 3.1 × 10 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.

Self-pumping porous microfluidic devices have attracted significant interest because of their low cost and broad applicability in point-of-care and low resource settings. One limitation of many of the devices is sensitivity and selectivity for detection. Electrochemistry can provide a sensitive, selective detection method while still using low cost, portable instrumentation as typified by handheld glucometers. Here, the development of electrochemical paper-based analytical devices (ePADs) is reviewed. Given the importance of electrode geometry and composition, fabrication methods are reviewed first. This is followed by a review of example applications demonstrated for ePADs. Finally, major accomplishments and future directions are summarized.

Salmonella causes over a million foodborne illnesses per year in the United States resulting in more hospitalizations and deaths than any other foodborne bacterial pathogen. To help prevent outbreaks, a rapid, portable, sensitive, and reliable method for onsite detection of bacteria that can be used in different sample matrices would be beneficial. Herein, we present a colorimetric paper-based analytical device (PAD) combined with immunomagnetic separation (IMS) for detecting Salmonella typhimurium. IMS anti-Salmonella coated magnetic beads were applied to capture and separate bacteria from the sample matrix and preconcentrate it into small volumes before testing on paper. To directly detect S. typhimurium after IMS, a sandwich immunoassay was implemented into the procedure with β-galactosidase (β-gal) as the detection enzyme. Using the antibody/enzyme complex, we performed a colorimetric assay with chlorophenol red-β-d-galactopyranoside (CPRG) for bacteria quantification. The method was confirmed to be highly specific to S. typhimurium without interference from other pathogenic bacteria like Escherichia coli. Using this system, the limit of detection of S. typhimurium was found to be 10 CFU mL in culturing solution without any pre-enrichment. In addition, distance-based detection where the concentration is read as the length of colored band formed on the reaction was also demonstrated. This assay had a detection limit of 10 CFU mL for S. typhimurium, providing an instrument-free quantitative analysis alternative to spot tests, which require image analysis. Finally, the proposed platform was applied for detection of S. typhimurium in inoculated Starling bird fecal samples and whole milk with detection limits of 10 CFU g and 10 CFU mL, respectively, and this is the first published paper-based detection method for S. typhimurium in bird feces and whole milk.

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 $34B in the US, and is predicted to be the number one cause of death worldwide by 2050. One way AMR bacteria can spread, and where individuals can contract AMR infections, is through contaminated water. Monitoring environment AMR bacteria currently requires 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 β-lactamase-mediated resistance as a form of AMR. To demonstrate viability, the PAD was used to detect β-lactam resistance in wastewater and sewage, and identified resistance in individual bacteria species isolated from environmental water sources.

Easy-to-use rotary manifold enables an immunomagnetic separation sandwich immunoassay for foodborne pathogen detection at the point-of-need.

Falsified and substandard antibiotics are a growing worldwide problem that leads to increased patient mortality and decreased trust in healthcare, and contributes to antimicrobial resistance. Monitoring falsified antibiotics is difficult because most falsified pharmaceuticals are most commonly found in developing countries, where detecting the active ingredient is difficult due to lack of access to complex instrumentation. Herein, we describe the development and optimization of a microfluidic paper-based analytical device (μPAD) to detect the active ingredient in the most falsified class of antibiotics, β-lactams. The assay is based on enzyme competition, making it the first demonstrated competitive enzyme assay reported in paper-based devices. The assay uses nitrocefin, a chromogenic substrate, to compete with β-lactam antibiotics in a reaction with β-lactamase. A yellow color indicates legitimate drugs, while a color change from yellow to red indicates falsified drugs. In addition to testing for the active ingredient, another section of the device was added to test the sample pH to further verify results and identify common falsified ingredients like aspirin or baking soda. Calibration curves for four different antibiotics, including cefazolin, have been generated making it the first paper-based device capable of detecting both cephalosporin and penicillin antibiotics. The μPAD has also been tested with common falsified ingredients and four antibiotics in tablet or injectable form, demonstrating its potential for in-field falsified antibiotic testing.

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 β‐lactamase‐mediated resistance. To demonstrate viability, the PAD was used to detect β‐lactam resistance in wastewater and sewage and identified resistance in individual bacterial species isolated from environmental water sources.