Vertical flow assays (VFAs) or flow-through assays have emerged as an alternate type of paper-based assay due to their faster detection time, larger sample volume capacity, and significantly higher multiplexing capabilities. They have been successfully employed to detect several different targets (polysaccharides, protein, and nucleic acids), although in a limited number of samples (serum, whole blood, plasma) compared to the more commonly known lateral flow assays (LFAs). The operation of a VFA relies mainly on gravity, coupled with capillary action or external force to help the sample flow through layers of stacked pads. With recent developments in this field, multiple layers of pads and signal readers have been optimized for more user-friendly operation, and VFAs have achieved a lower limit of detection for various analytes than the gold-standard methods. Thus, compared to the more widely used LFA, the VFA demonstrates certain advantages and is becoming an increasingly popular platform for obtaining qualitative and quantitative results in low-resource settings. Considering the wide application of gold nanoparticles (GNPs) in VFAs, we will mostly discuss (1) the design of GNP-based VFA along with its associated advantages/disadvantages, (2) fabrication and optimization of GNP-based VFAs for applications, and (3) the future outlook of flow-based assays for point-of-care testing (POCT) diagnostics.

The inflammation biomarker Interleukin 6 (IL-6) exhibits a concentration of less than 7 pg/mL in healthy serum but increases 10–100-fold when inflammation occurs. Increased serum IL-6 has been reported in chronic diseases such as rheumatoid arthritis (RA), as well as in life-threatening acute illnesses such as sepsis and cytokine release syndrome (CRS). This work seeks to meet the demand for rapid detection of serum IL-6 both for rapid monitoring of chronic diseases and for triaging patients with acute illnesses. Following the optimization of several types of gold nanoparticles, membrane pore sizes, and buffer systems, an ultra-sensitive vertical flow assay (VFA) was engineered, allowing the detection of recombinant IL-6 in spiked buffer with a limit of detection (LoD) of 10 pg/mL and a reportable range of 10–10,000 pg/mL with a 15-min assay time. The detection of IL-6 in spiked pooled healthy serum exhibited an LoD of 3.2 pg/mL and a reportable range of 10–10,000 pg/mL. The VFA’s stability was demonstrated over 1-day, two-week, four-week, and six-week storage durations at room temperature. The inter-operator CV and intra-operator CV were determined to be 14.3% and 15.2%, respectively. Three reference zones, high, low, and blank, were introduced into the cartridge to facilitate on-site semi-quantitative measurements across a 6-point semi-quantitative range. Finally, the performance of the IL-6 VFA was validated using 20 RA and 20 healthy control (HC) clinical serum samples, using ELISA as the gold standard platform. The ultra-sensitive, rapid IL-6 VFA could potentially be used to triage patients for intensive care, treatment adjustments, or for monitoring disease activity in inflammatory conditions.