Hydrogel-Enhanced Lateral Flow Assays: Simplified Manufacturing and Increased Sensitivity for Point-of-Care Diagnostics

Lateral flow assays (LFAs) are widely used in point-of-care diagnostics because of they provide rapid, cost-effectiveness, and simple to use. There are currently many commercial applications based on LFAs in medical diagnostics, environmental testing, food safety, and veterinary medicine. Despite their numerous advantages, the traditional design and manufacturing processes of LFAs still have significant challenges that limit their broader application and effectiveness, particularly in more demanding diagnostic scenarios. For instance, one of the primary challenges with traditional LFAs is the necessity of preprinting costly antibodies or aptamers, onto the nitrocellulose (NC) membrane at the test and control lines. This requirement increases the manufacturing complexity and cost and limits as well the scalability and accessibility of these devices. Moreover, the sensitivity of conventional LFAs is often inadequate for detecting low-abundance targets, which requires the development of more sensitive and efficient detection methods. Over the years, several approaches have been proposed to enhance the sensitivity of LFAs, including signal amplification techniques such as colorimetric enhancement and fluorescence/laser-induced signal amplification and while these methods significantly improve sensitivity, they often require specialized detection agents or complex readers which compromise the inherent simplicity and low-cost advantages of LFAs. To this end, new study published in ACS Applied Bio Materials and conducted by Tao Ma, Linlin Peng, Qinying Ran, Yan Zeng, and Professor Feng Liang from the State Key Laboratory of Refractories and Metallurgy at School of Chemistry and Chemical Engineering, Wuhan University of Science and Technology, developed a novel strategy that simplifies the manufacturing process of LFAs but in the same time enhanced sensitivity without the need for additional complex equipment or steps. The researchers’ novel approach involves the modification of LFAs with hydrogel lines (HL) and hydrogel control lines (HCL).

The research team synthesized various natural and synthetic hydrogels, including agarose, κ-carrageenan, gelatin, poly(acrylic acid) (PAA), polyacrylamide-sodium alginate (PAM-SA), and poly(vinyl alcohol)-sodium alginate (PVA-SA), and incorporated as extra lines on commercial LFA strips. The HLs were coated onto the NC membrane between the absorption pad and the control line, followed by vacuum drying. Afterward, they tested the modified LFA strips for COVID-19 antigen detection. The authors found that incorporating HLs significantly enhanced the sensitivity of the commercial LFA strips. According to the authors, the sensitivity of agarose-modified strips increased by 2-fold. Similarly, κ-carrageenan-modified strips exhibited a 2-fold increase in sensitivity, and gelatin-modified strips showed a 5-fold improvement. The authors believe these improvements are due to the hydrogels’ ability to obstruct fluid movement, which prolong the interaction time between the antigen and antibodies. Moreover, the researchers wanted to develop HCL that can replace traditional antibody-based control lines. To do this, they tested different hydrogels including agarose, gelatin, and κ-carrageenan for their ability to intercept colloidal gold nanoparticles (GNPs) and develop color as control lines. The researchers evaluated different concentrations of these hydrogels to identify the optimal formulation for HCL and assessed the performance of the HCLs by measuring the limit of detection (LOD) for COVID-19 antigens and compared it with traditional control lines. They found that HCLs effectively replaced traditional antibody-based control lines, and achieved comparable or even higher sensitivity. Furthermore, the authors tried to understand the mechanism behind the enhanced sensitivity of HCLs, and investigated the flow dynamics and hydrophilicity/hydrophobicity of hydrogel-modified NC membranes. They measured water flow rates on hydrogel-modified NC membranes by recording the time taken for fluid to traverse specific distances and also the permeation and hydrophilicity of hydrogels were assessed using scanning electron microscopy (SEM) and water contact angle measurements. The researchers also examined the internal structure of the NC membrane modified with different concentrations of hydrogels. The authors’ findings showed that hydrogel modifications significantly influenced the flow rate and wettability of the NC membrane with high-concentration hydrogels created less hydrophilic surfaces with stronger water-blocking abilities, which slowed down fluid flow and prolonged interaction times. SEM images revealed that hydrogels penetrated the NC membrane to varying degrees, with optimal concentrations effectively filling the membrane pores and delaying fluid movement. These findings explained the enhanced sensitivity observed with HCLs, as the hydrogels’ deceleration and interception effects allowed for greater capture of conjugated nanoparticles. Additionally, the researchers wanted to demonstrate their technique is versatile, therefore they applied HCL in host-guest detection systems involving macrocyclic host-guest interactions and found the HCL-based LFA successfully detected ADA-NH2, with an LOD of 5.4 nM for CB[7]-functionalized strips and 24.5 nM for β-CD-functionalized strips. The results demonstrated the applicability of HCLs in various detection systems, confirming their potential as a universal and practical strategy for LFA development.

In conclusion, Professor Feng Liang and colleagues demonstrated enhanced sensitivity of LFAs with HL  and HCL. The sensitivity improvements were substantial, with increases of 2-5 fold compared to traditional LFA strips. This enhancement is vital for detecting low-abundance antigens which can make LFAs more effective in various diagnostic applications, including early disease detection. Moreover, the authors’ method is more cost effective because it avoids the use of antibodies which are used in traditional LFA production and requires extensive screening, expression, and purification of appropriate bioreceptor pairs, which are time-consuming and expensive. The new hydrogel-based approach eliminates these steps, and reduces both production time and costs. It is significant indeed the new development of a universal HCL that can accommodate multiple LFA detection modes. This versatility allows the same LFA platform to be adapted for various types of tests, including antigen-antibody interactions, host-guest interactions, and other molecular recognition systems. This universal advantage can simplify the design and production of LFAs, making them more adaptable to different diagnostic needs. Moreover, the simplified manufacturing process using hydrogel modifications will enable scalable and rapid production of LFAs. This is most critical during public health emergencies, such as outbreaks of infectious diseases, where there is a need for mega quantities of diagnostic tests in a short period.