Maximizing flow rate in single paper layer, rapid flow microfluidic paper-based analytical devices.

Small, single-layer microfluidic paper-based analytical devices (µPADs) offer potential for a range of point-of-care applications; however, they have been limited to low flow rates. Here, we investigate the role of laser cutting paper channels in maximizing flow rate in small profile devices with limited fluid volumes. We demonstrate that branching, laser-cut grooves can provide a 59.23-73.98% improvement in flow rate over a single cut, and a 435% increase over paper alone. These design considerations can be applied to more complex microfluidic devices with the aim of increasing the flow rate, and could be used in stand-alone channels for self-pumping. Supplementary Information: The online version contains supplementary material available at 10.1007/s10404-023-02679-8.

Capillary flow-driven immunoassay platform for COVID-19 antigen diagnostics.

Over the last few years, the SARS-CoV-2 pandemic has made the need for rapid, affordable diagnostics more compelling than ever. While traditional laboratory diagnostics like PCR and well-plate ELISA are sensitive and specific, they can be costly and take hours to complete. Diagnostic tests that can be used at the point-of-care or at home, like lateral flow assays (LFAs) are a simple, rapid alternative, but many commercially available LFAs have been criticized for their lack of sensitivity compared to laboratory methods like well-plate ELISAs. The Capillary-Driven Immunoassay (CaDI) device described in this work uses microfluidic channels and capillary action to passively automate the steps of a traditional well-plate ELISA for visual read out. This work builds on prior capillary-flow devices by further simplifying operation and use of colorimetric detection. Upon adding sample, an enzyme-conjugated secondary antibody, wash steps, and substrate are sequentially delivered to test and control lines on a nitrocellulose strip generating a colorimetric response. The end user can visually detect SARS-CoV-2 antigen in 15-20 min by naked eye, or results can be quantified using a smartphone and software such as ImageJ. An analytical detection limit of 83 PFU/mL for SARS-CoV-2 was determined for virus in buffer, and 222 PFU/mL for virus spiked into nasal swabs using image analysis, similar to the LODs determined by traditional well-plate ELISA. Additionally, a visual detection limit of 100 PFU/mL was determined in contrived nasal swab samples by polling 20 untrained end-users. While the CaDI device was used for detecting clinically relevant levels of SARS-CoV-2 in this study, the CaDI device can be easily adapted to other immunoassay applications by changing the reagents and antibodies.

Capillary driven microfluidic sequential flow device for point-of-need ELISA: COVID-19 serology testing.

A capillary-driven microfluidic sequential flow device, designed for eventual at-home or doctor's office use, was developed to perform an enzyme-linked immunosorbent assay (ELISA) for serology assays. Serology assays that detect SARS-CoV-2 antibodies can be used to determine prior infection, immunity status, and/or individual vaccination status and are typically run using well-plate ELISAs in centralized laboratories, but in this format SARs-CoV-2 serology tests are too expensive and/or slow for most situations. Instead, a point-of-need device that can be used at home or in doctor's offices for COVID-19 serology testing would provide critical information for managing infections and determining immune status. Lateral flow assays are common and easy to use, but lack the sensitivity needed to reliably detect SARS-CoV-2 antibodies in clinical samples. This work describes a microfluidic sequential flow device that is as simple to use as a lateral flow assay, but as sensitive as a well-plate ELISA through sequential delivery of reagents to the detection area using only capillary flow. The device utilizes a network of microfluidic channels made of transparency film and double-sided adhesive combined with paper pumps to drive flow. The geometry of the channels and storage pads enables automated sequential washing and reagent addition steps with two simple end-user steps. An enzyme label and colorimetric substrate produce an amplified, visible signal for increased sensitivity, while the integrated washing steps decrease false positives and increase reproducibility. Naked-eye detection can be used for qualitative results or a smartphone camera for quantitative analysis. The device detected antibodies at 2.8 ng mL-1 from whole blood, while a well-plate ELISA using the same capture and detection antibodies could detect 1.2 ng mL-1. The performance of the capillary-driven immunoassay (CaDI) system developed here was confirmed by demonstrating SARS-CoV-2 antibody detection, and we believe that the device represents a fundamental step forward in equipment-free point-of-care technology.

Progress toward a Simplified UTI Diagnostic: Pump-Free Magnetophoresis for E. coli Detection.

Urinary tract infections (UTIs) are one of the most common infections across the world and can lead to serious complications such as sepsis if not treated in a timely manner. Uropathogenic Escherichia coli account for 75% of all UTIs. Early diagnosis is crucial to help control UTIs, but current culturing methods are expensive and time-consuming and lack sensitivity. The existing point-of-care methods fall short because they rely on indirect detection from elevated nitrates in urine rather than detecting the actual bacteria causing the infection. Magnetophoresis is a powerful method used to separate and/or isolate cells of interest from complex matrices for analysis. However, magnetophoresis typically requires complex and expensive instrumentation to control flow in microfluidic devices. Coupling magnetophoresis with microfluidic paper-based analytical devices (µPADs) enables pump-free flow control and simple and low-cost operation. Early magnetophoresis µPADs showed detection limits competitive with traditional methods but higher than targets for clinical use. Here, we demonstrate magnetophoresis using hybrid µPADs that rely on capillary action in hydrophilic polyethylene terephthalate channels combined with paper pumps. We were able to detect E. coli with a calculated limit of detection of 2.40 × 102 colony-forming units per mL.

Paper-based pump-free magnetophoresis.

Microfluidic magnetophoresis is a powerful technique that is used to separate and/or isolate cells of interest from complex matrices for analysis. However, mechanical pumps are required to drive flow, limiting portability and making translation to point-of-care (POC) settings difficult. Microfluidic paper-based analytical devices (µPADs) offer an alternative to traditional microfluidic devices that do not require external pumps to generate flow. However, µPADs are not typically used for particle analysis because most particles become trapped in the porous fiber network. Here we report the ability of newly developed fast-flow microfluidic paper-based analytical devices (ffPADs) to perform magnetophoresis. ffPADs use capillary action in a gap between stacked layers of paper and transparency sheets to drive flow at higher velocities than traditional µPADs. The multi-layer ffPADs allow particles and cells to move through the gap without being trapped in the paper layers. We first demonstrate that ffPADs enable magnetic particle separations in a µPAD with a neodymium permanent magnet and study key factors that affect performance. To demonstrate utility, E. coli was used as a model analyte and was isolated from human urine before detection with a fluorescently labeled antibody. A capture efficiency of 61.5% was then obtained of E. coli labeled magnetic beads in human urine. Future studies will look at the improvement of the capture efficiency and to make this assay completely off-chip without the need of a fluorescent label. The assay and device described here demonstrate the first example of magnetophoresis in a paper based, pump free microfluidic device.

Accessing Valuable Ligand Supports for Transition Metals: A Modified, Intermediate Scale Preparation of 1,2,3,4,5-Pentamethylcyclopentadiene.

A reliable, intermediate scale preparation of 1,2,3,4,5-pentamethylcyclopentadiene (Cp*H) is presented, based on modifications of existing protocols that derive from initial 2-bromo-2-butene lithiation followed by acid mediated dienol cyclization. The revised synthesis and purification of the ligand avoids the use of mechanical stirring while still permitting access to significant quantities (39 g) of Cp*H in good yield (58%). The procedure offers other additional benefits, including a more controlled quench of excess lithium during the production of the intermediate heptadienols and a simplified isolation of Cp*H of sufficient purity for metallation with transition metals. The ligand was subsequently used to synthesize [Cp*MCl2]2 complexes of both iridium and ruthenium to demonstrate the utility of the Cp*H prepared and purified by our method. The procedure outlined herein affords substantial quantities of a ubiquitous ancillary ligand support used in organometallic chemistry while minimizing the need for specialized laboratory equipment, thus providing a simpler and more accessible entry point into the chemistry of 1,2,3,4,5-pentamethylcyclopentadiene.