Optimization of flow path parameters for enhanced sensitivity lateral flow devices.

Lateral flow devices (LFDs) or lateral flow tests (LFTs) are one of the most widely used biosensor platforms for point-of-care (POC) diagnostics. The basic LFD design has remained largely unchanged since its first appearance, and this has limited LFD use in clinical applications due to a general lack of analytical sensitivity. We report here a comprehensive study of the use of laser-patterned geometric control barriers that influence the flow dynamics within an LFD, with the specific aim of enhancing LFD sensitivity and lowering the limit of detection (LOD). This control of sample flow produces an increase in the time available for optimizing the binding kinetics of the implemented assay. The geometric modification to the flow path is in the form of a constriction that is produced by depositing a photo-sensitive polymer onto the nitrocellulose membrane which when polymerized, creates impermeable barrier walls through the depth of the membrane. Both the position of the constriction within the flow path and the number of constrictions allow for an increase in the sensitivity because of a slower overall flow rate within the test and a larger volume of sample per unit width of the test line. For these high sensitivity LFDs (HS-LFD), through optimization of the constriction position and addition of a second constriction we attained a 62% increase in test line color intensity for the detection of procalcitonin (PCT) and were also able to lower the LOD from 10 ng/mL to 1 ng/mL. In addition, of relevance for future commercial exploitation, this also significantly decreases the antibody consumption per device leading to reduced costs for test production. We have further tested our HS-LFD with contrived human samples, validating its application for future clinical use.

Semi-quantitative detection of inflammatory biomarkers using a laser-patterned multiplexed lateral flow device.

Inflammatory markers including C-reactive protein (CRP) and procalcitonin (PCT) have been shown to be useful biomarkers to improve triage speed and prevent the inappropriate use of antibiotics for infections such as pneumonia. Here, we present a novel and exciting solution to guide the administration of antibiotic treatment via rapid, semi-quantitative and multiplexed detection of CRP and PCT using an advanced lateral flow device (LFD) designed to have multiple parallel flow-paths, produced via the precise laser-based partitioning of the single flow-path of a standard LFD. Each flow-path within this multiplexed LFD has a unique detection capability which permits tailored detection of CRP within a predefined cut-off range (20 µg/mL - 100 µg/mL) and PCT above a pre-defined threshold (0.5 ng/mL). We demonstrate the use of this LFD in the successful detection of CRP and PCT semi-quantitatively within spiked human serum samples. This multiplexed near-patient assay has potential for development into a rapid triage and treatment of patients with suspected pneumonia.

Capillary-based reverse transcriptase loop-mediated isothermal amplification for cost-effective and rapid point-of-care COVID-19 testing.

As the SARS-CoV-2 pandemic continues to spread, the necessity for rapid, easy diagnostic capabilities could never have been more crucial. With this aim in mind, we have developed a cost-effective and time-saving testing methodology/strategy that implements a sensitive reverse transcriptase loop-mediated amplification (RT-LAMP) assay within narrow, commercially available and cheap, glass capillaries for detection of the SARS-CoV-2 viral RNA. The methodology is compatible with widely used laboratory-based molecular testing protocols and currently available infrastructure. It employs a simple rapid extraction protocol that lyses the virus, releasing sufficient genetic material for amplification. This extracted viral RNA is then amplified using a SARS-CoV-2 RT-LAMP kit, at a constant temperature and the resulting amplified product produces a colour change which can be visually interpreted. This testing protocol, in conjunction with the RT-LAMP assay, has a sensitivity of ∼100 viral copies per reaction of a sample and provides results in a little over 30 min. As the assay is carried out in a water bath, commonly available within most testing laboratories, it eliminates the need for specialised instruments and associated skills. In addition, our testing pathway requires a significantly reduced quantity of reagents per test while providing comparable sensitivity and specificity to the RT-LAMP kit used in this study. While the conventional technique requires 25 µl of reagent, our test only utilises less than half the quantity (10 µl). Thus, with its minimalistic approach, this capillary-based assay could be a promising alternative to the conventional testing, owing to the fact that it can be performed in resource-limited settings, using readily available apparatus, and has the potential of increasing the overall testing capacity, while also reducing the burden on supply chains for mass testing.