Acute myocardial infarction (AMI), a life-threatening cardiovascular disease, requires rapid and accurate diagnosis for timely intervention. Early detection of elevated cardiac troponin I (cTnI) levels in the blood is crucial for AMI diagnosis. Lateral-flow assays (LFAs) are point-of-care testing (POCT) methods offering rapid results, but current LFAs for cTnI often suffer from limitations, including slow detection times (15-30 minutes) and low sensitivity. These limitations stem from the performance of conventional chromatographic membranes, such as fiber-based nitrocellulose membranes (NC Mem), which exhibit slow liquid flow, high liquid residue, and non-specific adsorption of detectable complexes. The slow flow prolongs detection time, abundant pores trap the sample solution and detectable complexes, and residual complexes reduce signal-to-noise ratio (SNR). This research aims to overcome these limitations by developing a novel membrane technology that enhances the speed and sensitivity of cTnI detection in LFAs. The successful development of an ultra-fast and highly-sensitive LFA for cTnI could significantly improve the early diagnosis and management of AMI, leading to better patient outcomes and reduced mortality.

Existing literature highlights the importance of rapid and sensitive cTnI detection for AMI diagnosis. Several studies have explored different LFA approaches, including the use of nanomaterials for signal amplification and modifications to the chromatographic membranes to improve performance. However, achieving both ultra-fast detection (within minutes) and high sensitivity remains a challenge. Current commercially available cTnI assays, while achieving high sensitivity and specificity, typically require longer assay times (15-30 minutes). The development of novel membrane technologies aimed at improving the fluid dynamics within the LFA strip is an active area of research, with attempts made to control and direct liquid flow to enhance both speed and signal-to-noise ratio. Many studies have focused on modifying the surface properties of the membrane to optimize hydrophilicity and hydrophobicity, but achieving significantly faster flow rates while maintaining high specificity and sensitivity remains a challenge.

This study introduces a novel barbed arrow-like structure membrane (BAS Mem) designed to address the limitations of current LFA membranes. The BAS Mem features interconnected barbed arrow-like grooves, bounded by sidewalls, which drive unidirectional and fast liquid flow. The design parameters, including the angles of the short and long arcs (α and β), the shortest width between long-arc sidewalls (W), and the height of the sidewalls (H), were optimized to maximize the rectification coefficient. The fabrication process involved laser carving a microchannel pattern onto an aluminum sheet, creating a mold for polydimethylsiloxane (PDMS) casting. The PDMS master was then used for hot embossing the pattern onto a polymer membrane (e.g., high-density polyethylene, HDPE). A hydrophilic coating was applied to the surface to further enhance liquid flow. The principle of unidirectional flow was analyzed using force balance on menisci, considering forces from sidewalls and the underside of the grooves. The rectification coefficient (k = Ls/Lp, where Ls is the forward flow length and Lp is the backward flow length) was used to quantify the unidirectional flow capability. The flow behavior was also characterized using high-speed imaging and simulations. The BAS Mem was then incorporated into lateral-flow strips for cTnI detection using a nanogold-based LFA. The performance of the BAS Mem-based LFA was compared to a conventional NC Mem-based LFA in terms of detection time, signal-to-noise ratio (SNR), limit of detection (LOD), sensitivity, and specificity. Clinical samples from suspected AMI patients were used to evaluate the clinical performance of the BAS Mem-based LFA. The study also investigated the impact of different surface contact angles on the unidirectional flow behavior of the BAS Mem, using oxygen plasma treatment to modify the surface wettability. Comparisons to commercial lateral flow assays were also made, considering detection time, sensitivity, specificity, and limit of detection. The use of larger sized antibody-labeled signal amplification probes were also tested to verify the advantages of the BAS Mem in accommodating larger molecules.

The BAS Mem exhibited a significantly higher rectification coefficient (14.5) compared to NC Mem (1.02), demonstrating superior unidirectional flow capabilities. The liquid flow velocity on BAS Mem was approximately 23 times faster than on NC Mem. LFAs using the BAS Mem achieved a detection time of only 240 seconds (4 minutes), compared to the typical 15-30 minutes for NC Mem-based LFAs. The limit of detection (LOD) for cTnI using the BAS Mem-based LFA was 1.97 pg mL⁻¹. In clinical trials, the BAS Mem-based LFA demonstrated 100% specificity and 93.3% sensitivity in detecting cTnI in serum samples from 25 suspected AMI patients. The area under the curve (AUC) of the receiver operating characteristic (ROC) curve was 0.953. The BAS Mem demonstrated superior performance in terms of reduced sample volume and reduced residual sample solution compared to NC Mem. Importantly, the BAS Mem also showed an advantage when used with larger-diameter antibody-labeled signal amplification probes (500 nm and 1 µm), successfully completing the assay, while NC Mem-based assays failed or exhibited significant background noise. The BAS Mem showed significantly improved signal-to-noise ratio in these tests.

The results demonstrate the significant improvement in LFA performance achieved using the BAS Mem. The ultra-high rectification coefficient, fast flow velocity, and low residual sample solution contribute to the ultra-fast detection time and high sensitivity of the cTnI assay. The significantly reduced detection time (4 minutes) compared to conventional methods (15-30 minutes) is particularly impactful for timely AMI diagnosis and treatment. The high sensitivity and specificity of the BAS Mem-based LFA in clinical samples further validate its potential for reliable AMI diagnosis. The success of the BAS Mem with larger sized signal amplification probes significantly expands the potential applications of this technology to a wider range of analytes and detection methods. The findings could lead to the development of more rapid and sensitive diagnostic tools for other diseases and point-of-care applications. The improved SNR also translates to a more accurate and reliable assay, reducing the likelihood of false positive or false negative results.

This study successfully demonstrated the effectiveness of a novel barbed arrow-like structure membrane (BAS Mem) in significantly improving the performance of lateral-flow assays for cTnI detection. The ultra-high rectification coefficient of the BAS Mem enabled an ultra-fast and highly sensitive assay, capable of providing results within 4 minutes with a LOD of 1.97 pg mL⁻¹. The excellent clinical performance, demonstrated by high sensitivity and specificity, highlights the potential of this technology for rapid and reliable AMI diagnosis. Future research could focus on exploring further applications of the BAS Mem in other POCT platforms, optimizing the membrane design for different target analytes, and investigating the scalability and cost-effectiveness of the fabrication process.

While the BAS Mem-based LFA demonstrated exceptional performance, some limitations should be noted. The study involved a relatively small number of clinical samples, limiting the generalizability of the findings. Further validation with a larger and more diverse patient cohort is necessary. The long-term stability and durability of the BAS Mem under various storage conditions also needs further investigation. The performance of the BAS Mem may vary depending on the specific material used and fabrication parameters. Optimization of the fabrication process to ensure consistency and reproducibility across batches is important for large-scale production and commercialization.