Paper-based Lateral Flow Research Articles

Development of an RPA-CRISPR/Cas12a Assay for Rapid and Sensitive Diagnosis of Plant Quarantine Fungus Setophoma terrestris.

Setophoma terrestris is an important phytopathogenic fungus listed by China as a harmful fungus subject to phytosanitary import control. This pathogen is a threat to a wide range of plants, particularly as the causal agent of onion pink root rot, one of the most severe diseases of onions. In order to provide rapid identification and early warning of S. terrestris and prevent its spread, we have developed a rapid, accurate, and visually intuitive diagnostic assay for this pathogen, by utilizing recombinase polymerase amplification (RPA), coupled with CRISPR/Cas12a cleavage and fluorescence-based detection systems or paper-based lateral flow strips. The developed RPA-CRISPR/Cas12a assay exhibited remarkable specificity for the detection of S. terrestris. Moreover, this protocol can detect the pathogen at a sensitivity level of 0.01 pg/μL, which significantly outperforms the 1 pg/μL sensitivity achieved by the existing qPCR-based detection method. The entire diagnostic procedure, including DNA extraction, the RPA reaction, the Cas12a cleavage, and the result interpretation, can be accomplished in 40 min. Furthermore, the successful application of the assay in infected plant samples highlighted its potential for rapid and accurate pathogen detection in agricultural settings. In summary, this RPA-CRISPR/Cas12a diagnostic method offers a potentially valuable technological solution for quarantine and disease management.

Ultrasensitive Rapid Antigen Test by Geometric Lateral Flow Assays and Highly Doped Upconversion Nanoparticles.

The paper-based lateral flow assay (LFA) testing strips are currently the most widely used for point-of-care testing (POCT), valued for their rapid result turnaround times in a few minutes. However, their sensitivity has been limited. Upconversion nanoparticles (UCNPs), especially highly doped ones, have emerged as promising luminescent reporters to enhance the LFA sensitivity. These UCNPs exhibit a nonlinear enhancement in luminescence with excitation power density, necessitating higher power densities for higher brightness. In this study, we utilized a geometric paper strip design to minimize the immune reaction area and maximize the excitation power density, enabling ultrasensitive detection of the SARS-CoV-2 nucleoprotein antigen. This design also slowed the antigen flow on the paper strip, extending the reaction time between antigen and antibody, thereby enhancing the efficiency of the immune reaction. Through this design, our approach achieved over a 100-fold enhancement in the limit of detection (LOD) compared with the widely used LFAs, based on gold colloidal nanoparticles and europium nanoparticles. This innovation expands the scope of LFA applications that require a low LOD.

Development of a dual-component biosensor for rapid and sensitive detection of influenza H7 and H5 subtypes

The outbreak of highly pathogenic influenza virus subtypes, such as H7 and H5, presents a significant global health challenge, necessitating the development of rapid and sensitive diagnostic methods. In this study, we have developed a novel dual-component biosensor assembly, each component of which incorporates an antibody fused with a nano-luciferase subunit. Our results demonstrate the effectiveness of this biosensor in enabling the rapid and sensitive detection of influenza H7 and other subtypes. Additionally, we successfully applied the biosensor in paper-based assay and lateral flow assay formats, expanding its versatility and potential for field-deployable applications. Notably, we achieved effective detection of the H7N9 virus using this biosensor. Furthermore, we designed and optimized a dedicated biosensor to the sensitive detection of the influenza H5 subtype. Collectively, our findings underscore the significant potential of this dual-component biosensor assembly as a valuable and versatile tool for accurate and timely diagnosis of influenza virus infections, promising to advance the field of influenza diagnostics and contribute to outbreak management and surveillance efforts.

Strategic reuse of rapid antigen tests for coagulation status assessment: an integrated machine learning approach

Addressing the pressing demand for rapid and inexpensive coagulation testing in cardiovascular care, this study introduces a novel application of repurposed COVID-19 rapid antigen tests (RATs) as paper-based lateral flow assays (LFAs) combined with machine learning for coagulation status evaluation. By further developing a mobile app prototype, we present a platform that enables clinicians to perform immediate and accurate anticoagulant dosing adjustments using existing post-pandemic resources. Our proof-of-concept employs a random forest machine learning classifier to interpret image feature variations on RAT NC membrane, correlating red blood cell (RBC) wicked diffusion distance in recalcified citrated whole blood with changes in coagulative viscosity, easily interpreted. Enhanced by confocal imaging studies of paper microfluidics, our approach provides insights into the mechanisms dissecting coagulation components, achieving high classification precision, recall, and F1-scores. The inverse relationship between RBC wicked diffusion distance and enoxaparin concentration paves the way for machine learning to inform real-time dose prescription adjustments, aligning with individual patient profiles to optimize therapeutic outcomes. This study not only demonstrates the potential of leveraging surplus RATs for coagulation management but also exemplifies a cost-effective, rapid, and smart strategy to enhance clinical decision-making in the post-pandemic era.Graphical

A paper-based lateral flow immunochromatographic sensor for the detection of tricyclazole in rice

Tricyclazole is commonly used to prevent rice blast to meet the carbohydrate intake needs of half of the global population, and a large number of toxicological reports indicate that monitoring of tricyclazole is necessary. Here, we analyzed the structure of tricyclazole and designed different hapten derivatization strategies to prepare a high-performance monoclonal antibody (half inhibition concentration of 1.61 ng/mL), and then a lateral flow immunochromatographic sensor based on gold nanoparticles for the detection of tricyclazole in rice, with a limit of detection of 6.74 μg/kg and 13.58 μg/kg in polished and brown rice, respectively. The recoveries in rice were in the range of 84.6–107.4%, no complex pretreatment was required for comparison with LC-MS/MS, and the comparative analysis demonstrated that our method had good accuracy and precision. Therefore, the developed lateral flow immunochromatographic analysis was a reliable and rapid means for the on-site analysis of tricyclazole in rice.

Electrochemical Lateral Flow Immunoassay with Built-In Electrodes for Ultrasensitive and Wireless Detection of Inflammatory Biomarkers.

Paper-based lateral flow immunoassays (LFIAs) are cost-effective, portable, and simple methods for detection of diverse analytes, which however only provide qualitative or semiquantitative results and lack sufficient sensitivity. A combination of LFIA and electrochemical detection, namely, electrochemical lateral flow immunoassay (eLFIA), enables quantitative detection of analytes with high sensitivity, but the integration of external electrodes makes the system relatively expensive and unstable. Herein, the working, counter, and reference electrodes were prepared directly on the nitrocellulose membrane using screen printing, which remarkably simplified the structure of eLFIA and decreased the cost. Moreover, a horseradish peroxidase (HRP)-based electrochemical signal amplification strategy was used for further increasing the analytical sensitivity. HRP captured on the working electrode can catalyze the oxidation of tetramethylbenzidine (TMB) to form the TMB-TMBox precipitate on the electrode surface, which as an electrochemically active product can output an amplified current for quantification. We demonstrated that the eLFIA could detect low-abundant inflammatory biomarkers in human plasma samples with limits of detection of 0.17 and 0.54 pg mL-1 for interleukin-6 and C-reactive protein, respectively. Finally, a fully portable system was fabricated by integrating eLFIA with a flexible and wireless electrochemical workstation, realizing the point-of-care detection of interleukin-6.

Rapid and Amplification-free Nucleic Acid Detection with DNA Substrate-Mediated Autocatalysis of CRISPR/Cas12a.

To enable rapid and accurate point-of-care DNA detection, we have developed a single-step, amplification-free nucleic acid detection platform, a DNA substrate-mediated autocatalysis of CRISPR/Cas12a (DSAC). DSAC makes use of the trans-cleavage activity of Cas12a and target template-activated DNA substrate for dual signal amplifications. DSAC employs two distinct DNA substrate types: one that enhances signal amplification and the other that negatively modulates fluorescent signals. The positive inducer utilizes nicked- or loop-based DNA substrates to activate CRISPR/Cas12a, initiating trans-cleavage activity in a positive feedback loop, ultimately amplifying the fluorescent signals. The negative modulator, which involves competitor-based DNA substrates, competes with the probes for trans-cleaving, resulting in a signal decline in the presence of target DNA. These DNA substrate-based DSAC systems were adapted to fluorescence-based and paper-based lateral flow strip detection platforms. Our DSAC system accurately detected African swine fever virus (ASFV) in swine's blood samples at femtomolar sensitivity within 20 min. In contrast to the existing amplification-free CRISPR/Dx platforms, DSAC offers a cost-effective and straightforward detection method, requiring only the addition of a rationally designed DNA oligonucleotide. Notably, a common ASFV sequence-encoded DNA substrate can be directly applied to detect human nucleic acids through a dual crRNA targeting system. Consequently, our single-step DSAC system presents an alternative point-of-care diagnostic tool for the sensitive, accurate, and timely diagnosis of viral infections with potential applicability to human disease detection.

A Microfluidic Paper-Based Lateral Flow Device for Quantitative ELISA

This study presents an innovative lateral flow microfluidic paper-based analytical device (μPAD) designed for conducting quantitative paper-based enzyme-linked immunosorbent assays (p-ELISA), seamlessly executing conventional ELISA steps in a paper-based format. The p-ELISA device utilizes a passive fluidic circuit with functional elements such as a multi-bi-material cantilever (B-MaC) assembly, delay channels, and a buffer zone, all enclosed within housing for autonomous, sequential loading of critical reagents onto the detection zone. This novel approach not only demonstrates a rapid assay completion time of under 30 min, but also boasts reduced reagent requirements, minimal equipment needs, and broad applicability across clinical diagnostics and environmental surveillance. Through detailed descriptions of the design, materials, and fabrication methods for the multi-directional flow assay (MDFA), this manuscript highlights the device’s potential for complex biochemical analyses in a user-friendly and versatile format. Analytical performance evaluation, including a limit of detection (LOD) of 8.4 pM for Rabbit IgG, benchmarks the device’s efficacy compared to existing p-ELISA methodologies. This pioneering work lays the groundwork for future advancements in autonomous diagnostics, aiming to enhance global health outcomes through accessible and reliable testing solutions.

Carbon nanoparticle-based lateral flow assay for the detection of specific double-tagged DNA amplicons of Paracoccidioides spp.

To overcome the limitations of current methods for diagnosing paracoccidioidomycosis (PCM), it is critical to develop novel diagnostic strategies that can be implemented in low-resource settings and dramatically improve turnaround times. This study focused on the development of a portable molecular test to screen for Paracoccidioides spp. The proposed approach integrated double-tagging polymerase chain reaction (PCR) and a paper-based lateral flow assay (LFA) for readout, using carbon nanoparticles as a signal generation system. Primers tagged with biotin and digoxigenin were employed to conduct the double-tagging PCR, which can be conveniently carried out on portable thermocyclers. This method can generate billions of tagged DNA copies from a single target molecule, which can be rapidly detected by the LFA platform, providing results within minutes. Avidin-modified carbon nanoparticles served as a signal generation system, enabling detection in the immunochromatographic assay. The LFA demonstrated the capability to detect double-tagged amplicons as low as 0.21ng or 0.10ng, depending on whether the results were assessed visually or with a smartphone equipped with an image processor. These findings suggest that the proposed approach holds great promise as a point-of-care diagnostic tool for the early and accurate detection of PCM in low-resource settings. The diagnostic test is rapid and inexpensive, requires minimal handling and can be easily introduced into the general practitioner's armoury for ambulatory screening of infection. This innovative approach has the potential to make a substantial contribution to PCM diagnosis, ultimately reducing morbidity and mortality associated with this disease.

Integrating Paper-Based Microfluidics and Lateral Flow Strip into Nucleic Acid Amplification Device toward Rapid, Low-Cost, and Visual Diagnosis of Multiple Mycobacteria.

Efficient diagnosis of mycobacterial infections can effectively manage and prevent the transmission of infectiousdiseases. Unfortunately, existing diagnostic strategies are challenged by long assay times, high costs, and highly specialized expertise to distinguish between pulmonary tuberculosis (PTB) and nontuberculous mycobacterial pulmonary diseases (NTM-PDs). Herein, in this study, an optimized 3D paper-based analytical device (µPAD) is incorporated with a closed lateral flow (LF) strip into a loop-mediated isothermal amplification (LAMP)device (3D-µPAD-LF-LAMP) for rapid, low-cost, and visual detection of pathogenic mycobacteria. The platform's microfluidic featureenhanced the nucleic acid amplification, thereby reducing the costs and time as compared to boiling, easyMAG, and QIAGEN techniques. Moreover, the LF unitis specifically designed to minimize aerosol contamination for a user-friendly and visual readout. 3D-µPAD-LF-LAMP is optimized and assessed using standard strains, demonstrating a limit of detection (LOD) down to 10 fgreaction-1 . In a cohort of 815 patients, 3D-µPAD-LF-LAMP displays significantly better sensitivity, specificity, negative predictive value (NPV), positive predictive value (PPV), and diagnostic accuracy than conventional bacterial culture and Xpert techniques. Collectively, 3D-µPAD-LF-LAMP demonstrates enhanced accessibility, efficiency, and practicality for the diagnosis of multiple pathogenic mycobacteria, which can be applied across diverse clinical settings, thereby ultimately improving public health outcomes.

Real-Time On-Site Monitoring of Viruses in Wastewater Using Nanotrap® Particles and RICCA Technologies.

Wastewater-based epidemiology (WBE) is an effective and efficient tool for the early detection of infectious disease outbreaks in a community. However, currently available methods are laborious, costly, and time-consuming due to the low concentration of viruses and the presence of matrix chemicals in wastewater that may interfere with molecular analyses. In the present study, we designed a highly sensitive "Quick Poop (wastewater with fecal waste) Sensor" (termed, QPsor) using a joint approach of Nanotrap microbiome particles and RICCA (RNA Isothermal Co-Assisted and Coupled Amplification). Using QPsor, the WBE study showed a strong correlation with standard PEG concentrations and the qPCR technique. Using a closed format for a paper-based lateral flow assay, we were able to demonstrate the potential of our assay as a real-time, point-of-care test by detecting the heat-inactivated SARS-CoV-2 virus in wastewater at concentrations of 100 copies/mL and within one hour. As a proof-of-concept demonstration, we analyzed the presence of viral RNA of the SARS-CoV-2 virus and PMMoV in raw wastewater samples from wastewater treatment plants on-site and within 60 min. The results show that the QPsor method can be an effective tool for disease outbreak detection by combining an AI-enabled case detection model with real-time on-site viral RNA extraction and amplification, especially in the absence of intensive clinical laboratory facilities. The lab-free, lab-quality test capabilities of QPsor for viral prevalence and transmission in the community can contribute to the efficient management of pandemic situations.

Inhalable point-of-care urinary diagnostic platform.

Although low-dose computed tomography screening improves lung cancer survival in at-risk groups, inequality remains in lung cancer diagnosis due to limited access to and high costs of medical imaging infrastructure. We designed a needleless and imaging-free platform, termed PATROL (point-of-care aerosolizable nanosensors with tumor-responsive oligonucleotide barcodes), to reduce resource disparities for early detection of lung cancer. PATROL formulates a set of DNA-barcoded, activity-based nanosensors (ABNs) into an inhalable format. Lung cancer-associated proteases selectively cleave the ABNs, releasing synthetic DNA reporters that are eventually excreted via the urine. The urinary signatures of barcoded nanosensors are quantified within 20 min at room temperature using a multiplexable paper-based lateral flow assay. PATROL detects early-stage tumors in an autochthonous lung adenocarcinoma mouse model with high sensitivity and specificity. Tailoring the library of ABNs may enable not only the modular PATROL platform to lower the resource threshold for lung cancer early detection tools but also the rapid detection of chronic pulmonary disorders and infections.

Electrokinetic focusing of SARS-CoV-2 spike protein via ion concentration polarization in a paper-based lateral flow assay.

The COVID-19 pandemic highlighted the importance of designing sensitive and selective point-of-care (POC) diagnostic sensors for early and rapid detection of infection. Paper-based lateral flow assays (LFAs) are easy to use, inexpensive, and rapid, but they lack sensitivity. Preconcentration techniques can improve the sensitivity of LFAs by increasing the local concentration of the analyte before detection. Here, ion concentration polarization (ICP) is used to focus the analyte, SARS-CoV-2 Spike protein (S-protein), directly over a test line composed of angiotensin converting enzyme 2 (ACE2) capture probes. ICP is the enrichment and depletion of electrolyte ions at opposing ends of an ion-selective membrane under a voltage bias. The ion depleted zone (IDZ) establishes a steep gradient in electric field strength along its boundary. Enrichment of charged species (such as a biomolecule analyte) occurs at an axial location along this electric field gradient in the presence of a fluid flow that counteracts migration of those species - a phenomenon called ICP focusing. In this paper, running buffer composition and pretreatment solutions for ICP focusing in a paper-based LFA are evaluated, and the method of voltage application for ICP-enrichment is optimized. With a power consumption of 1.8 mW, S-protein is concentrated by a factor of 21-fold, leading to a 2.9-fold increase in the signal from the LFA compared to a LFA without ICP-enrichment. The described ICP-enhanced LFA is significant because the preconcentration strategy is amenable to POC applications and can be applied to existing LFAs for improvement in sensitivity.

Magnetic Separation of Micro Beads and Cells on a Paper-Based Lateral Flow System

Paper based lateral flow systems are widely used biosensor platforms to detect biomolecules in a liquid sample. Proteins, bacteria, oligonucleotides, and nanoparticles were investigated in the literature. In this work we designed a magnetic platform including dual magnets and tested the flow of micron size immunomagnetic particles alone and when loaded with cells on two different types of papers. The prewetting conditions of the paper and the applied external magnetic field are the two dominant factors affecting the particle and cell transport in paper. The images recorded with a cell phone, or with a bright field optical microscope were analyzed to measure the flow of particles and cells. The effect of prewetting conditions and magnetic force were measured, and it was shown that in the worst case, minimum 90% of the introduced cells reached to the edge of the paper. The paper based magnetophoretic lateral flow systems can be used for cell assays.

Mechanically Compressed Barriers Improve Paper-Based Lateral Flow Assay Sensitivity for COVID-19 Nucleic Acid Detection

Mechanically Compressed Barriers Improve Paper-Based Lateral Flow Assay Sensitivity for COVID-19 Nucleic Acid Detection

One-step paper-based SlipChip for the sensitive detection of C-reactive protein with porous platinum nanozyme-assisted signal amplification

The development of efficient and sensitive point-of-care testing is crucial for preparedness in the post-pandemic era. Although paper-based lateral flow assays have attracted attention and have various advantages for rapid, on-site diagnosis, they have low sensitivity. To overcome the limitations of the existing assays, in this study, we aimed to develop a new, one-step, nanozyme-amplified SlipChip for the sensitive detection of C-reactive protein (CRP). The SlipChip was constructed by combining wax-printed paper with different channel designs. The three-dimensional (3D) fluidic configuration of the SlipChip allowed for the sequential delivery of reagents, enabling mixing and signal amplification with a one-step sliding operation. As a signal-amplifying reagent, peroxidase-mimicking porous platinum nanozyme (pPtNZ) was synthesized using a simple wet chemical method. The pPtNZ conjugated on the test line catalyzes the oxidation of diaminobenzidine (DAB) in the presence of hydrogen peroxide, increasing the color intensity. The immunoassay results of the SlipChip were easily interpreted within 20 min, and the color intensity was visually enhanced by DAB precipitation over time, resulting in up to 6-fold signal amplification. The proposed pPtNZ-SlipChip exhibited high analytical performances for the one-step detection of serum and salivary CRP from 0.1 to 1000 ng/mL, with a limit of detection of 0.03 ng/mL. These results revealed the potential and applicability of the pPtNZ-SlipChip, with the advantages of simplicity, sensitivity, low cost, and portability for on-site detection and point-of-care testing.

Absolute Quantification of Serum Exosomes in Patients with an SERS-Lateral Flow Strip Biosensor for Noninvasive Clinical Cancer Diagnosis.

Exosomes (exos) widely existing in body fluids show great potential for noninvasive cancer diagnosis. Quantitative analysis of exos is traditionally performed by targeting specific exosomal surface proteins, but it is often imprecise due to the common expression of exosomal proteins and subtle expression differences between different cancer subtypes. Herein, we report quantitative surface-enhanced Raman spectroscopy (SERS) of serum exos through a combination of a paper-based lateral flow strip (LFS) biosensor with multivariate spectral unmixing analysis rather than simply quantifying exosomal proteins. Our SERS-LFS biosensor enables absolute quantification of two different serum exos with a limit of detection down to ∼106 particles/mL for both exos. We further exemplify the application of this strategy in quantitative dual-plex detection of serum exos from breast cancer patients. We find that human epidermal growth factor receptor 2+ (HER2+) and luminal A breast cancer patients undergoing no surgery are enriched in serum exos derived from SKBR-3 cells and MCF-7 cells (denoted as SKBR and MCF exos), respectively. The surgical treatment of these breast cancer patients accompanies an obvious decrease of either SKBR or MCF exos in the serum. These results suggest the great potential of the combination of the SERS-LFS biosensor and multivariate spectral unmixing for breast cancer subtyping and therapeutic surveillance with the powerful quantitative capability of exos in clinical samples.

Paper-based lateral flow assay for the point-of-care detection of neurofilament light chain.

Neurofilament light chain (NF-L) is a protein found in neurons of the nervous system and is widely used as a biomarker for neurological disorders. However, the current methods for detecting NF-L levels are complicated, expensive, and require specialized equipment, making it challenging to implement in a point-of-care (POC) setting. In this study, we developed a gold nanoshell (AuNS)-assisted lateral flow assay (LFA) based test strip for the POC detection of NF-L at a low ng/mL level (8 ng/mL = 117.65 pM). The test strip is a simple, rapid, and cost-effective method for detecting NF-L, making it suitable for use in a POC setting for the diagnosis and treatment of various neurological disorders. With its ease of use and reliability, the paper-based LFA is a valuable tool for the diagnosis and management of neurological conditions.Clinical Relevance- The AuNS-assisted LFA test strip developed in this study offers a rapid, cost-effective, and simple method for detecting NF-L levels, making it of great interest to practicing clinicians for the diagnosis of various neurological diseases such as HIV-associated dementia (HID), Amyotrophic Lateral Sclerosis (ALS), and Creutzfeldt-Jakob disease (CJD).

Rapid and Sensitive Detection of Verticillium dahliae from Complex Samples Using CRISPR/Cas12a Technology Combined with RPA.

Verticillium wilt is primarily caused by the fungus Verticillium dahliae and represents one of the most important worldwide soilborne plant diseases. The causal agent can be spread by microsclerotia and conidia attached to seeds during national/international trade or in soil between fields. Consequently, accurate, sensitive, and rapid detection of V. dahliae from complex samples is critical for restricting entry of the pathogen to a new region/environment and enforcing early management of Verticillium wilt. Based on CRISPR/Cas12a and recombinant polymerase amplification (RPA) technologies, we developed an accurate, sensitive, and rapid detection method for V. dahliae with paper-based lateral flow strips. A highly efficient and specific CRISPR RNA (crRNA) was designed for the GAPDH gene of V. dahliae and was validated using several closely related Verticillium spp. Excluding the time required for the DNA extraction from the complex samples, a minimum of 40 min was required for the detection time. The RPA-CRISPR/Cas12a detection system had a lower detection limit of ∼10 copies of genomic DNA per reaction and was able to successfully detect as little as one microsclerotium per gram of soil. In addition, field samples displaying symptoms suggestive of V. dahliae were able to be positively identified for the presence of V. dahliae. Taken together, this study broadens the applications of CRISPR/Cas12a nucleic acid detection to soilborne crop diseases and will contribute to the future development of field-deployable diagnostic tools.

Sharp Branched Gold Nanostar-Based Lateral-Flow Immunoassay for Detection of Yersinia pestis

Over the past few decades, colorimetric paper-based lateral flow immunoassay (LFIA) has emerged as a versatile analytical tool for rapid point-of-care detection of infectious diseases with high simplicity and flexibility. The LFIA sensitivity is based on color visualization of the antibody-labeled nanoparticles bound with the target analytes at the test line. Therefore, the nanoparticle design is crucial for LFIA sensitivity. The traditional LFIA is based on spherical gold nanoparticles, which usually suffer from poor sensitivity because of very low optical contrast at the test line. To improve the LFIA sensitivity, we have developed an LFIA based on gold nanostars (GNSs) with different branch lengths and sharpness (GNS-1, GNS-2, and GNS-3), which possess higher optical contrast than conventional gold nanospheres (GNSPs). We have selected the bacterium Yersinia pestis as a model analyte system. The effective affinity of GNSPs and GNSs with the Y. pestis fraction 1 (F1) protein was quantitively investigated by colorimetric and optical density measurements of the test line. The results show that GNS-3, which has maximum spike length and branch sharpness, exhibits the highest analytical sensitivity based on the limit of detection of the LFIA readout compared to other GNSs and GNSPs. The detection limit of the Y. pestis F1 antigen was achieved up to 0.1 ng/mL for GNS-3, which is 100 times lower than that for the GNSP at a 1 pmol/L concentration and 10 times lower than that for the reported procedure based on traditional gold nanoparticles. Overall, our prototype LFIA platform based on a highly spiked GNS (GNS-3) exhibits high analytical sensitivity, indicating it to be a promising candidate for routine LFIA application to detect infectious diseases.