Foodborne illnesses, particularly in milk, pose significant public health risks, especially in low-income regions where access to reliable testing is limited. Lateral-flow immunoassays (LFAs) offer rapid detection but suffer from small volume capacity and low sensitivity. To address these challenges, we developed a fully automated device that utilizes immunomagnetic separation (IMS), where magnetic nanoprobes (MNPs) selectively capture Escherichia coli (E. coli) in large volumes of milk. The device then removes excess sample and adds a buffer to minimize matrix effects before LFA application. Our device achieves a limit of detection (LOD) of 5 × 103 colony-forming units per milliliter (cfu/mL), a 20-fold improvement over the standard LFA setup. The device is composed of inexpensive 3D-printed materials and hobby electrical components and has a time to result of 40 min, requiring only sample input and a single button press to complete the entire workflow. This novel device offers a widely accessible and user-friendly solution for small-scale milk distributors for mitigating food contamination outbreaks.

We are the first to integrate H2O2 degradation and gold nanorod (GNR) etching on paper with the lateral-flow immunoassay (LFA) in a 3D-printed device to achieve semi-quantitative results for therapeutic drug monitoring of digoxin by correlating different color outputs to different digoxin concentrations.

Two major water pollutant classes are microorganisms and organic chemicals. Although laboratory-based assays effectively detect water contaminants, they are costly, require trained personnel, and take a long time to result. This limits the continuous monitoring required for preventing widespread use of contaminated water. An alternative is the lateral-flow immunoassay (LFA), which is rapid, inexpensive, and easy to use. However, LFAs can yield false negatives when the target is present at low concentrations, such as with water contaminants. Aqueous two-phase systems (ATPSs) can preconcentrate targets into one phase before application to the LFA, and the fold-concentration depends on the volume ratio (VR) between the top and bottom phases. Since a user typically adds a set volume to LFAs, the limit of detection (LOD) of the assay can vary, as the VR, and thus the preconcentration, can vary depending on the water samples' salt content. To address this, we recently applied the entire target-containing phase to an LFA and demonstrated that the same LOD can be achieved, irrespective of the VR. This method, unfortunately, required multiple user steps. To enable this approach to be used at the point of need (PON), we describe the development of the first fully battery-operated and automated device for detecting the ATPS interface regardless of the VR, extracting the entire target-containing phase, and dispensing it onto an LFA. This device yields a ten-fold improvement in the LOD for both Escherichia coli and atrazine, providing results within 30 min and offering a solution for monitoring water quality.

In this work, we developed a smart drug delivery system composed of poly (ethylene glycol)-block-poly (ε-caprolactone) (PEG-PCL)-based polymersomes (Ps) loaded with doxorubicin (DOX) and vemurafenib (VEM). To enhance targeted delivery to malignant melanoma cells, these drug-loaded nanovesicles were conjugated to the oxalate transferrin variant (oxalate Tf) and incorporated into three-dimensional chitosan hydrogels. This innovative approach represents the first application of oxalate Tf for the precision delivery of drug-loaded polymersomes within a semi-solid dosage form based on chitosan hydrogels. These resulting semi-solids exhibited a sustained release profile for both encapsulated drugs. To evaluate their potency, we compared the cytotoxicity of native Tf-Ps with oxalate Tf-Ps. Notably, the oxalate Tf-Ps demonstrated a 3-fold decrease in cell viability against melanoma cells compared to normal cells and were 1.6-fold more potent than native Tf-Ps, indicating the greater potency of this nanoformulation. These findings suggest that dual-drug delivery using an oxalate-Tf-targeting ligand significantly enhances the drug delivery efficiency of Tf-conjugated nanovesicles and offers a promising strategy to overcome the challenge of multidrug resistance in melanoma therapy.

Lateral-flow immunoassays (LFAs) can be used to diagnose urinary tract infections caused by Escherichia coli (E. coli) at the point of care. Unfortunately, urine samples containing dilute concentrations of E. coli can yield false negative results on LFAs. Our laboratory was first to implement aqueous two-phase systems (ATPSs) to preconcentrate samples into smaller volumes prior to their application on LFAs. This is achieved by manipulating the ratio of the volume of the top phase to that of the bottom phase (volume ratio; VR) and concentrating biomarkers in the bottom phase which, when applied to LFAs in fixed volumes, leads to corresponding improvements in sensitivity. This work is the first demonstration that the same LOD can be achieved irrespective of the VR when the entire bottom phase is added to LFAs. A custom 3D-printed device was also developed to decrease liquid handling steps. Across different VRs expected from patient urine variability, this diagnostic workflow successfully detected E. coli concentrations down to 2 × 105 colony-forming units (cfu) mL-1 in synthetic urine, demonstrating consistent 10-fold improvements in sensitivity compared to trials conducted without ATPS preconcentration. This method successfully addresses the variability of patient samples while remaining easy to use at the point of care.

The lateral-flow immunoassay (LFA) has proven to be an effective point-of-care (POC) diagnostic for the rapid detection of target analytes in patient samples. However, due to its reliance on a binary, visual output, the LFA cannot be used to quantify target analyte concentrations, nor is it accessible to blind or visually impaired patients. To address these limitations, we developed the quantitative LFA interpretation device (qLFAID), which operates on the Raspberry Pi 4 Model B. The qLFAID uses a trained neural network in conjunction with a nonlinear regression model to analyze the test line intensity of any LFA to determine the target analyte concentration. In addition, qLFAID’s electronic components provide auditory and tactile signals for blind and visually impaired users to interpret their results. As a proof of concept, the device was applied to analyze an LFA we designed to detect for digoxin in human serum. Our device successfully correlated the LFA test line intensity to the sample digoxin concentration, providing a quantitative output. Overall, qLFAID has the potential to be robust in POC settings and, to our knowledge, is the first assistive technology that can both enable the quantitative capability of all LFAs and increase diagnostic accessibility for blind or visually impaired individuals.

Polymer-salt aqueous two-phase systems (ATPSs) represent liquid-liquid extraction systems that have been used in diagnostics to preconcentrate biomarkers. A novel technology known as the ATPS-automated concentration and enhancement of the lateral-flow immunoassay (ACE-LFA) was recently developed, combining the preconcentration capabilities of ATPS with nanozyme signal enhancement to improve the sensitivity of the lateral-flow immunoassay (LFA), a paper-based diagnostic. The device automatically and sequentially delivers reagents to the detection zone by relying on ATPS macroscopic phase separation on paper. This technology critically relies on the selective partitioning of a relatively hydrophobic signal enhancement reagent and the target biomolecule to opposite phases of the ATPS, which ensures that the enhancement reagent is only delivered to the detection zone after the target biomolecule has bound to the test line as this minimizes premature signal enhancement and improves the test line intensity. Consequently, the ideal combination of enhancement reagent and ATPS is one in which the reagent partitions selectively into the lagging, polymer-rich phase of the ATPS. We therefore chose to predict this partitioning behavior by applying the Universal Quasichemical Functional-Group Activity Coefficient (UNIFAC) thermodynamic model. Using this model with no fitted parameters, the partition coefficients of two signal enhancement substrates, 3,3',5,5'-tetramethylbenzidine (TMB) and 3,3 '-diaminobenzidine (DAB), in a polymer-salt ATPS were predicted and compared with our experimentally measured values, demonstrating the ability to predict which enhancement reagent partitions more favorably into the polymer-rich phase. We also confirmed that when the enhancement reagent partitions more extremely into the polymer-rich phase, it results in improved ACE-LFA tests with darker test lines. Accordingly, this model has the potential for screening ATPS and enhancement reagent combinations for the design of point-of-care diagnostics.

In this work, we present a novel point-of-care hydrogel-based diagnostic device for the rapid detection of elevated bicarbonate levels in serum for the diagnosis of mild to severe cases of metabolic alkalosis. Our system consists of hydrogel beads composed of calcium alginate and the nonionic polymer dextran. This assay utilizes the reaction of sodium bicarbonate and citric acid to produce citrate, a metal chelator capable of competitively binding to calcium cations in the gel matrix to trigger hydrogel degradation. This results in successful detection of elevated bicarbonate concentrations in less than one hour. Specifically, critically high bicarbonate concentrations of 50, 45, and 40 mmol L-1 in human serum were detected in as little as 10, 15, and 20 min, respectively. To demonstrate the assay's feasibility for use in resource-limited settings, we developed a simple electronic device that achieved similar results and could be used by untrained individuals with no lab equipment and minimal power. To our knowledge, this is the first demonstration of the use of nonionic polymers to synthesize and improve the morphology of calcium alginate hydrogel beads using a simple processing method that involves minimal labor and equipment. The simplified bead synthesis protocol combined with the user-friendly device allows for the rapid detection of metabolic alkalosis at the point of care.

We are the first to combine the lateral-flow immunoassay (LFA) with gold nanorod (GNR) etching to achieve a multicolor readout where the color produced was correlated with digoxin concentrations in human serum in the relevant range for therapeutic drug monitoring of 0.5-3.0 ng mL-1.

Malaria is an infectious disease that can cause severe sickness and death if not diagnosed and treated in a timely manner. The current gold standard technique for malaria diagnosis is microscopy, which requires a dedicated laboratory setting and trained personnel and can have a long time to result. These requirements can be alleviated using paper-based diagnostic devices that enable rapid and inexpensive diagnosis at the point of care, which can allow patients to receive treatment before their symptoms progress when used for early detection of diseases. The lateral-flow immunoassay (LFA) is one such device, but currently available LFAs are susceptible to false negative results caused by low parasite density. To improve sensitivity and detection, we utilized the aqueous two-phase system (ATPS) to concentrate and purify the sample, and nanozyme signal enhancement to increase the intensity of the visible signal on the test strip. We were able to achieve a limit of detection (LOD) of 0.01 ng/mL for the malaria biomarker Plasmodium lactate dehydrogenase (pLDH) in human serum using a multi-step assay combining the LFA format with the ATPS and nanozyme signal enhancement.

We developed an innovative 3D printed casing that incorporates a lateral-flow immunoassay, dehydrated signal enhancement reagents, and a sealed buffer chamber. With only the push of a button for signal enhancement, our device detected the SARS-CoV-2 N-protein in 40 min at concentrations as low as 0.1 ng mL-1 in undiluted serum.

Foodborne illness is a major public health issue that results in millions of global infections annually. The burden of such illness sits mostly with developing countries, as access to advanced laboratory equipment and skilled lab technicians, as well as consistent power sources, is limited and expensive. Current gold standards in foodborne pathogen screening involve labor-intensive sample enrichment steps, pathogen isolation and purification, and costly readout machinery. Overall, time to detection can take multiple days, excluding the time it takes to ship samples to off-site laboratories. Efforts have been made to simplify the workflow of such tests by integrating multiple steps of foodborne pathogen screening procedures into a singular device, as well as implementing more point-of-need readout methods. In this review, we explore recent advancements in developing point-of-need devices for foodborne pathogen screening. We discuss the detection of surface markers, nucleic acids, and metabolic products using both paper-based and microfluidic devices, focusing primarily on developments that have been made between 2015 and mid-2020.

We generated stable amphiphilic copolymer-based polymeric micelles (PMs) with temperature-responsive properties utilizing Pluronic® L35 and a variety of ionic liquids (ILs) to generate different aqueous two-phase micellar systems (ATPMSs). The partitioning of the hydrophobic model compound curcumin (CCM) into the PM-rich phase and the drug delivery capabilities of the PMs were investigated. ATPMSs formed using more hydrophobic ILs (i.e., [Ch][Hex] ≈ [Ch][But] > [Ch][Pro] > [Ch][Ac] ≈ [Ch]Cl) were the most effective in partitioning (KCCM) and recovering (RECRich) CCM into the PM-rich phase (15.2 < KCCM < 22.0 and 90% < RECRich < 95%, respectively). Moreover, using 1.2 M [Ch][But] and 0.2 M [Ch][Hex] ILs yielded higher encapsulation efficiency (EE) (94.1 and 96.0%, respectively) and drug loading (DL) capacity (14.8 and 16.2%, respectively), together with an increase in the average hydrodynamic diameter of the PMs (DH) (42.5 and 45.6 nm, respectively). The CCM-PM formulations were stable at 4.0, 25.0, and 37.0 °C and the release of CCM was faster with the less hydrophobic ILs (i.e., [Ch]Cl and [Ch][Ac]). Furthermore, due to the lower critical solution temperature properties of Pluronic® L35, the PMs exhibit temperature responsiveness at 37.0 °C. In vitro cytotoxicity assays were also performed to determine the potency of CCM-PM formulations, and a 1.8-fold decrease in IC50 values was observed between the CCM-PMs/[Ch][Hex] and CCM-PMs/[Ch]Cl formulations for PC3 cells. The lower IC50 value for the [Ch][Hex] version corresponded to a greater potency compared to the [Ch]Cl version, since a lower concentration of CCM was required to achieve the same therapeutic effect. The ATPMSs investigated in this study serve as a novel platform for Pluronic® L35/PBS buffer (pH 7.4) + IL-based ATPMS development. The unique properties reported here may be useful in applications such as controlled-release drug delivery systems (DDS), encapsulation, and bioseparations.

Early disease detection through point-of-care (POC) testing is vital for quickly treating patients and preventing the spread of harmful pathogens. Disease diagnosis is generally accomplished using quantitative polymerase chain reaction (qPCR) to amplify nucleic acids in patient samples, permitting detection even at low target concentrations. However, qPCR requires expensive equipment, trained personnel, and significant time. These resources are not available in POC settings, driving researchers to instead utilize isothermal amplification, conducted at a single temperature, as an alternative. Common isothermal amplification methods include loopmediated isothermal amplification, recombinase polymerase amplification, rolling circle amplification, nucleic acid sequence-based amplification, and helicase-dependent amplification. There has been a growing interest in combining such amplification methods with POC detection methods to enable the development of diagnostic tests that are well suited for resource-limited settings as well as developed countries performing mass screenings. Exciting developments have been made in the integration of these two research areas due to the significant impact that such approaches can have on healthcare. This review will primarily focus on advances made by North American research groups between 2015 and June 2020, and will emphasize integrated approaches that reduce user steps, reliance on expensive equipment, and the system's time-to-result.

Cerebrospinal fluid (CSF) leaks can occur when there is communication between the intracranial cavities and the external environment. They are a common and serious complication of numerous procedures in otolaryngology, and if not treated, persistent leaks can increase a patient's risk of developing life-threatening complications such as meningitis. As it is not uncommon for patients to exhibit increased secretions postoperatively, distinguishing normal secretions from those containing CSF can be difficult. Currently, there are no proven, available tests that allow a medical provider concerned about a CSF leak to inexpensively, rapidly, and noninvasively rule out the presence of a leak. The gold standard laboratory-based test requires that a sample be sent to a tertiary site for analysis, where days to weeks may pass before results return. To address this, our group recently developed a semiquantitative, barcode-style lateral-flow immunoassay (LFA) for the quantification of the beta-trace protein, which has been reported to be an indicator of the presence of CSF leaks. In the work presented here, we created a rapid diagnostic test kit composed of our LFA, a collection swab, dilution buffers, disposable pipettes, and instructions. Validation studies demonstrated excellent predictive capabilities of this kit in distinguishing between clinical specimens containing CSF and those that did not. Our diagnostic kit for CSF leak detection can be operated by an untrained user, does not require any external equipment, and can be performed in approximately 20 min, making it well suited for use at the point of care. This kit has the potential to transform patient outcomes.

In previous work, our group discovered a phenomenon in which a mixed polymer-salt or mixed micellar aqueous two-phase system (ATPS) separates into its two constituent phases as it flows within paper. While these ATPSs worked well in their respective studies to concentrate the target biomarker and improve the sensitivity of the lateral-flow immunoassay, different ATPSs can be advantageous for new applications based on factors such as biomarker partitioning or biochemical compatibility between ATPS and sample components. However, since the mechanism of phase separation in porous media is not completely understood, introducing other ATPSs to paper is an unpredictable process that relies on trial and error experiments. This is especially true for polymer-polymer ATPSs in which the characteristics of the two phases appear quite similar. Therefore, our group aimed to develop semiquantitative guidelines for choosing ATPSs that can phase separate in paper. In this work, we evaluated the Washburn equation and its parameters as a potential mathematical framework to describe the flow behavior of polymer-salt and micellar ATPSs in fiberglass paper. We compared bulk phase fluid characteristics and identified the viscosity difference between the phases as a key determinant of the potential for phase separation in paper. We then used this parameter to predict the phase separation capabilities of polyethylene glycol (PEG)-dextran ATPSs in paper and control the composition of the leading and lagging phases. We also, for the first time, successfully demonstrated the phase separation phenomenon in hydrogels, thereby extending its application and potential benefits to an alternative porous medium.

Infectious diseases remain one of the leading causes of deaths in developing countries because of a lack of basic sanitation, healthcare clinics, and centralized laboratories. Paper-based rapid diagnostic tests, such as the lateral-flow immunoassay (LFA), provide a promising alternative to the traditional laboratory-based tests; however, they typically suffer from having a poor sensitivity. Biomarker preconcentration and signal enhancement are two common methods to improve the sensitivity of paper-based assays. While effective, these methods often require multiple liquid handling steps which are not ideal for use by untrained personnel in a point-of-care setting. Our lab previously discovered the phenomenon of an aqueous two-phase system (ATPS) separating on paper, which allowed for the seamless integration of concentration and detection of biomarkers on the LFA. In this work, we have extended the functionality of an ATPS separating on paper to automate the sequential delivery of signal enhancement reagents in addition to concentrating biomarkers. The timing of reagent delivery was controlled by changing the initial composition of the ATPS. We applied this technology to automate biomarker concentration and nanozyme signal enhancement on the LFA, resulting in a 30-fold improvement in detection limit over the conventional LFA when detecting Escherichia coli, all while maintaining a single application step.

OBJECTIVE:A cerebrospinal fluid leak is one of the most serious complications in otolaryngology. It may occur as a result of injury to the skull base, typically traumatic or iatrogenic. While the presence of a leak is often discerned in the emergent setting, distinguishing normal secretions from those containing cerebrospinal fluid can be difficult during postoperative visits in the clinic. As most current laboratory-based assays are labor intensive and require several days to result, we aim to develop a more user-friendly and rapid point-of-care cerebrospinal fluid detection device.STUDY DESIGN:Our laboratory developed a barcode-style lateral-flow immunoassay utilizing antibodies for beta-trace protein, a protein abundant in and specific for cerebrospinal fluid, with a concentration of 1.3 mg/L delineating a positive result.SETTING:Tertiary medical center.SUBJECTS AND METHODS:Tests with known concentrations of resuspended beta-trace protein and the contents of discarded lumbar drains (presumed to contain cerebrospinal fluid) were performed to validate our novel device.RESULTS:Our results demonstrate the ability of our device to semiquantitatively identify concentrations of beta-trace protein from 0.3-90 mg/L, which is within the required range to diagnose a leak, thus making beta-trace protein an excellent target for rapid clinical detection.CONCLUSION:Herein we detail the creation and initial validation of the first point-of-care cerebrospinal fluid detection device. This device is a feasible method to more efficiently and cost-effectively identify cerebrospinal fluid leaks, minimize costs, and improve patient outcomes.