Conventional Lateral Flow Assay Research Articles

Compact Digital Immunoassay Platform Integrating ELISA with a Lateral Flow Strip

Background/Objectives: On-site diagnosis of infection in their early stages requires assays with high sensitivities that are compact and easy to operate out of the laboratory and hospital environments. However, current assay technologies fall short of these requirements and require highly skilled technicians to set up, operate, and interpret the results. Methods: To address these challenges, we developed and evaluated a Point-of-Care-Testing (PoCT) immunoassay platform called the D-strip. The D-strip platform combines the capabilities of a digital enzyme-linked immunoassay (ELISA) with a lateral flow assay (LFA). The D-strip sample flow cell is composed of the same components found in conventional LFAs, and its high sensitivity is due to its efficient implementation of ELISA. The fully integrated platform is simple and requires minimal user intervention to operate. Results: The D-strip exhibited a sample-to-result time of 15 min with a limit of detection (LOD) of 1.7 × 103 copies/mL for severe acute respiratory syndrome coronavirus 2 (SARS-2-CoV) antigen. The LOD of the D-strip is 488-fold higher than that for conventional LFAs and is comparable to a clinical laboratory test. Conclusions: The D-strip is a compact and highly sensitive immunoassay platform with a strong potential for application as a confirmatory assay outside the clinical laboratory.

Wheat Germ Agglutinin-Modified "Three-in-One" Multifunctional Probe Driven Broad-Spectrum and Flexible Immunochromatographic Diagnosis of viruses With High Sensitivity.

The conventional lateral flow assay (LFA) fails to the demands for the accurate screening of viruses as a result of its low sensitivity of colorimetric signal output and poor universality limited by antibody pairs. Here, a magnetically assisted dual-signal output LFA platform is developed for the ultrasensitive, universal, and flexible detection of viruses. A "three-in-one" multifunctional probe (MAuDQD) is prepared using a 180nm Fe3O4 core to load numerous Au nanoparticles (NPs) and two layers of QDs, which can substantially improve the sensitivity of LFA through coupling with the effects of magnetic enrichment and colorimetric/fluorescent enhancement. Wheat germ agglutinin (WGA)-modified MAuDQD attained the broad-spectrum capture viral membrane proteins and the colorimetric/fluorescent dual-mode detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and monkeypox virus (MPXV) on the LFA strip. In the colorimetric mode, the target viruses detected directly, with the visual sensitivity reaching 0.1-0.5ngmL-1 and the fluorescent mode supported quantitative analysis of SARS-CoV-2/MPXV with limits of detection decreasing to pgmL-1 level. Practicability of the MAuDQD@WGA-LFA is verified through the detection of 33 real clinical samples, showing the proposed assay has a great potential to become a sensitive, accurate, and universal tool for on-site monitoring of viral infections.

Dual-mode photothermal/chemiluminescence vertical flow assay for sensitive point-of-care detection of carcinoembryonic antigen using Cu2-xAgxS@liposome on a filter membrane

Conventional lateral flow assays based on colorimetry and fluorescence still have shortages in sensitivity and selectivity due to the severe background interference from complex human fluid sample matrices. In this work, Cu2-xAgxS nanocrystals with high photothermal conversion efficiency and good peroxidase-like activity were synthesized and applied in the construction of a dual-mode near-infrared-photothermal/chemiluminescence (CL) vertical flow assay of carcinoembryonic antigen (CEA). These two-mode principles showed nearly zero background and the synthesized Cu2-xAgxS exhibited a high photothermal conversion efficiency of 75.23%, enabling the luminol-H2O2 CL system to have over 4 min of chemiluminescence. By combining filter membrane enrichment, Cu2-xAgxS@liposome encapsulation amplification, and nanozyme catalysis, a dual-mode photothermal/CL portable assay was constructed for sensitive and accurate detection of CEA in serum, with linear ranges of 0.02–40 and 0.001–30 ng mL−1, and detection limits of 0.0023 and 0.00029 ng mL−1, respectively. Furthermore, a smartphone application and a 3D printing device were combined for point-of-care testing. This assay can be completed within 20 min, with simple operation and no need for large instruments. It exhibited good sensitivity, selectivity, and stability, and is expected to be used in early diagnosis and prevention of relevant diseases in resource-limited areas.

NT-proBNP detection with a one-step magnetic lateral flow channel assay

Point-of-care sensors targeting blood marker analysis must be designed to function with very small volumes since acquiring a blood sample through a simple, mostly pain-free finger prick dramatically limits the sample size and comforts the patient. Therefore, we explored the potential of converting a conventional lateral flow assay (LFA) for a significant biomarker into a self-contained and compact polymer channel-based LFA to minimize the sample volume while maintaining the analytical merits. Our primary objective was to eliminate the use of sample-absorbing fleece and membrane materials commonly present in LFAs. Simultaneously, we concentrated on developing a ready-to-deploy one-step LFA format, characterized by dried reagents, facilitating automation and precise sample transport through a pump control system. We targeted the detection of the heart failure biomarker NT-proBNP in only 15 µL human whole blood and therefore implemented strategies that ensure highly sensitive detection. The biosensor combines streptavidin-functionalized magnetic beads (MNPs) as a 3D detection zone and fluorescence nanoparticles as signal labels in a sandwich-based immunoassay. Compared to the currently commercialized LFA, our biosensor demonstrates comparable analytical performance with only a tenth of the sample volume. With a detection limit of 43.1 pg∙mL−1 and a mean error of 18% (n ≥ 3), the biosensor offers high sensitivity and accuracy. The integration of all-dried long-term stable reagents further enhances the convenience and stability of the biosensor. This lateral flow channel platform represents a promising advancement in point-of-care diagnostics for heart failure biomarkers, offering a user-friendly and sensitive platform for rapid and reliable testing with low finger-prick blood sample volumes.Graphical abstract

Nanoarchitectonics of photothermal materials to enhance the sensitivity of lateral flow assays.

Lateral flow assays (LFAs) are currently the most widely used point-of-care testing technique with remarkable advantages such as simple operation, rapid analysis, portability, and low cost. Traditionally, gold nanoparticles are employed as tracer element in LFAs due to their strong localised surface plasmon resonance. However, this conventional LFA technique based on colorimetric analysis is neither useful to determine critical analytes with desired sensitivity, nor can it quantify the analytes. Various signal amplification strategies have been proposed to improve the sensitivity and the quantitative determination of analytes using LFAs. One of the promising strategies is to enhance the photothermal properties of nanomaterials to generate heat after light irradiation, followed by a temperature measurement to detect and quantify the analyte concentration. Recently, it has been observed that the nanoscale architecture of materials, including size, shape, and nanoscale composition, plays a significant role in enhancing the photothermal properties of nanomaterials. In this review, we discuss the nanoarchitectonics of nanomaterials regarding enhanced photothermal properties and their application in LFAs. Initially, we discuss various important photothermal materials and their classification along with their working principle. Then, we highlight important aspects of the nanoscale architecture (i.e., size, shape, and composition) to enable maximum light-to-heat conversion efficiency. Finally, we discuss some of the recent advances in photothermal LFAs and their application in detecting analytes.

Applications of magnetic nanomaterials in the fabrication of lateral flow assays toward increasing performance of food safety analysis: Recent advances

Up to now, the life of humans is complex, and it has been under attack by numerous factors. In other words, human beings are relationship with living in a healthy and clean environment. Remarkably, life on the planet Earth has been threatened by food contamination in all areas of the world. Over the last few years, the portability of analytical methods has received considerable attention in biosensors. Among these, lateral flow assays (LFAs) can consider one of the critical types of portable sensing platforms. The vast majority of conventional LFAs operate according to the gold nanoparticles (AuNPs) signaling. However, the presence of NaCl in real samples can decrease the performance of the AuNPs-LFA. Thereby, the substitute of AuNPs with other types of nanomaterials (NMs) or using hybrid NMs can boost the application of these sensing biodevices in different real samples. Elaborately, magnetic nanomaterials (MNMs) can play a significant role in developing sensing approaches, especially later flow assays LFAs. These NMs not only do quantitative measurements by coupling with an external reader, but they can also act as an immunomagnetic separation agent. In addition, ease of use, less coat, and size uniformity can highlight their application in food safety. Therefore, the application of MNMs in LFA with different formats can significantly reduction in the limit of detection (LOD) of the analyte without methodically complicating the analytical procedure. In this review, we attempted to highlight the recent progress and technical breakthroughs of MNMs in LFA for food safety.

Chemically Amplified Multiplex Detection of SARS-CoV-2 and Influenza A and B Viruses via Paint-Programmed Lateral Flow Assays.

Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) continues to threaten lives by evolving into new variants with greater transmissibility. Although lateral flow assays (LFAs) are widely used to self-test for coronavirus disease 2019 (COVID-19), these tests suffer from low sensitivity leading to a high rate of false negative results. In this work, a multiplexed lateral flow assay is reported for the detection of SARS-CoV-2 and influenza A and B viruses in human saliva with a built-in chemical amplification of the colorimetric signal for enhanced sensitivity. To automate the amplification process, the paper-based device is integrated with an imprinted flow controller, which coordinates the routing of different reagents and ensures their sequential and timely delivery to run an optimal amplification reaction. Using the assay, SARS-CoV-2 and influenza A and B viruses can be detected with ≈25x higher sensitivity than commercial LFAs, and the device can detect SARS-CoV-2-positive patient saliva samples missed by commercial LFAs. The technology provides an effective and practical solution to enhance the performance of conventional LFAs and will enable sensitive self-testing to prevent virus transmission and future outbreaks of new variants.

Ultrasensitive lateral-flow assays via plasmonically active antibody-conjugated fluorescent nanoparticles.

Lateral-flow assays (LFAs) are rapid and inexpensive, yet they are nearly 1,000-fold less sensitive than laboratory-based tests. Here we show that plasmonically active antibody-conjugated fluorescent gold nanorods can make conventional LFAs ultrasensitive. With sample-to-answer times within 20 min, plasmonically enhanced LFAs read out via a standard benchtop fluorescence scanner attained about 30-fold improvements in dynamic range and in detection limits over 4-h-long gold-standard enzyme-linked immunosorbent assays, and achieved 95% clinical sensitivity and 100% specificity for antibodies in plasma and for antigens in nasopharyngeal swabs from individuals with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Comparable improvements in the assay's performance can also be achieved via an inexpensive portable scanner, as we show for the detection of interleukin-6 in human serum samples and of the nucleocapsid protein of SARS-CoV-2 in nasopharyngeal samples. Plasmonically enhanced LFAs outperform standard laboratory tests in sensitivity, speed, dynamic range, ease of use and cost, and may provide advantages in point-of-care diagnostics.

High-flux smartphone-integrated lateral flow assay based on chrysanthemum-like Au@polydopamine for sensitive detection of enrofloxacin in milk.

Lateral flow assays (LFAs) are widely used in screening analysis. However, the detection flux and sensitivity of LFAs are unsatisfactory because of the 'one-to-one' read mode and the ordinary performance conventional probe. In this work, a high-flux smartphone-integrated LFA (sLFA) based on chrysanthemum-like Au@polydopamine (AuNC@PDA) for the detection of enrofloxacin (ENR) in milk was established. The smartphone-integrated device was developed and applied in the LFA, giving rise to a reading maximum of 64 samples with only two photos in less than 2 min compared to a 'one-to-one' read mode of a conventional LFA in more than 14 min with a 36.4% deviation. For ENR detection, the limits of detection of sLFA based on AuNC@PDA (33.78 pg mL-1) had 5.0-fold and 6.4-fold reduction compared with those based on AuNP (169.99 pg mL-1, conventional probe) and vvAuNP (216.47 pg mL-1, commercial probe), respectively. This study provides an effective technical means for screening ENR in large quantities of milk samples.

A Low-Cost Cellulose-Based POC Device for Detection of COVID-19

Point-of-care diagnostic (POC) is of utmost importance for the fight against pandemics which provides easy, instant results right at your place. POC can transform healthcare management and create superior infrastructure. In POC, employing lateral flow assays (LFAs) can accelerate the assessment and shorten the treatment time. LFA is a small handheld device made from paper that controllably delivers the analyte without the need of an active pump to the detection area to produce the signal, generally coloured nanoparticles (Au, carbon).Conventional LFA device is made of four components, sample pad, conjugate pad, nitrocellulose membrane and absorbent pad arranged sequentially. At the sample pad, the analyte is dispensed. From here, the analyte moves to the conjugate pad and hybridizes with recognition antibodies (placed onto nanoparticles) to form analyte-antibody-gold nanoparticles conjugate. Further movement takes this to the nitrocellulose membrane. Here, other recognizant antibodies are placed at the test and control line to immobilize by forming the sandwich assay with the analyte-antibody-gold nanoparticles. The excess liquid is absorbed in the absorbent pad. From these components of the LFA device, nitrocellulose membrane and antibodies need replacements as they increase the device's overall cost. Cellulose paper holds promise to counter nitrocellulose membrane; however, the development of fabrication methods for cellulose paper-based LFA devices is needed. In comparison to antibodies, aptamers improve batch-to-batch variability, ruggedness, and longevity of LFA devices.This work describes the fabrication of half-strip cellulose paper (Whatman® Grade 4 filter paper) based LFA device exploiting aptamers as the recognition element for antigen detection. The covalent bonding of aptamers was achieved by NanoCheck-ATH® (NC) and p-phenylene disothiocyanate (PDITC). NC provides plentiful amine functional groups and constricts the test line width by covalently bonding aptamer to the NC region only. The chitosan-based NanoCheck on paper assists in realizing sharp test and control lines by providing the controlled surface for PDITC reaction through amine groups. PDITC functions as a linker between NC and aptamers. Finally, a proof-of-concept nucleocapsid protein of SARS-CoV-2 as an antigen detection was shown with the minimal detectable concentration down to ~20 ng/mL with excellent selectivity towards casein, BSA, and albumin. We envisage that using facile modification chemistry of NC, PDITC, and aptamers for antigen detection will allow the fabrication of paper-based LFA devices in a roll-to-roll setting and, thus, decrease the overall cost. The developed method is generic and can be adapted to detect other antigens to fight future pandemics.

Mitigating hook effect in one-step quantitative sandwich lateral flow assay by timed conjugate release.

Sandwich lateral flow assay (LFA) is one of the most successfully commercialized paper-based biosensors, which offers a rapid, low-cost, one-step assay. Despite its advantages, conventional sandwich LFA is fundamentally limited by the high-dose "hook" effect-a phenomenon that occurs at very high analyte concentrations and results in false-negative results. In this paper, we present a novel strategy of automatic timed detection antibody release to mitigate the hook effect in sandwich LFA without additional manual steps. We introduced an intermediate pad treated with saturated sucrose solution to regulate the flow between the nitrocellulose membrane and the conjugate pad in order to delay the reaction between detection antibodies and analytes. Using C-reactive protein (CRP) as a representative analyte, we demonstrated that our strategy exhibited a range of detection 10 times wider than that of our conventional LFA, without sacrificing the limit of detection. Comparing to other published strategies, our work could offer a one-step, cost-effective approach that is closely unified with the benefits of the LFA.

Recent advances in sensitivity enhancement for lateral flow assay.

Conventional lateral flow assay (LFA) is typically performed by observing the color changes in the test lines by naked eyes, which achieves considerable commercial success and has a significant impact on the fields of food safety, environment monitoring, disease diagnosis, and other applications. However, this qualitative detection method is not very suitable for low levels of disease biomarkers’ detection. Although many nanomaterials are used as new labels for LFA, additional readers limit their application to some extent. Fortunately, a lot of work has been done for improving the sensitivity of LFA. In this review, currently reported LFA sensitivity enhancement methods with an objective evaluation are summarized, such as sample pretreatment, the change of flow rate, and label evolution, and future development direction and challenges of LFAs are discussed.

Rapid and Sensitive SERS-Based Lateral Flow Test for SARS-CoV2-Specific IgM/IgG Antibodies.

As an immune response to COVID-19 infection, patients develop SARS-CoV-2-specific IgM/IgG antibodies. Here, we compare the performance of a conventional lateral flow assay (LFA) with a surface-enhanced Raman scattering (SERS)-based LFA test for the detection of SARS-CoV-2-specific IgM/IgG in sera of COVID-19 patients. Sensitive detection of IgM might enable early serological diagnosis of acute infections. Rapid detection in serum using a custom-built SERS reader is at least an order of magnitude more sensitive than the conventional LFAs with naked-eye detection. For absolute quantification and the determination of the limit of detection (LOD), a set of reference measurements using purified (total) IgM in buffer was performed. In this purified system, the sensitivity of SERS detection is even 7 orders of magnitude higher: the LOD for SERS was ca. 100 fg/mL compared to ca. 1 μg/mL for the naked-eye detection. This outlines the high potential of SERS-based LFAs in point-of-care testing once the interference of serum components with the gold conjugates and the nitrocellulose membrane is minimized.

Duplex Shiny app quantification of the sepsis biomarkers C-reactive protein and interleukin-6 in a fast quantum dot labeled lateral flow assay

Fast point-of-care (POC) diagnostics represent an unmet medical need and include applications such as lateral flow assays (LFAs) for the diagnosis of sepsis and consequences of cytokine storms and for the treatment of COVID-19 and other systemic, inflammatory events not caused by infection. Because of the complex pathophysiology of sepsis, multiple biomarkers must be analyzed to compensate for the low sensitivity and specificity of single biomarker targets. Conventional LFAs, such as gold nanoparticle dyed assays, are limited to approximately five targets—the maximum number of test lines on an assay. To increase the information obtainable from each test line, we combined green and red emitting quantum dots (QDs) as labels for C-reactive protein (CRP) and interleukin-6 (IL-6) antibodies in an optical duplex immunoassay. CdSe-QDs with sharp and tunable emission bands were used to simultaneously quantify CRP and IL-6 in a single test line, by using a single UV-light source and two suitable emission filters for readout through a widely available BioImager device. For image and data processing, a customized software tool, the MultiFlow-Shiny app was used to accelerate and simplify the readout process. The app software provides advanced tools for image processing, including assisted extraction of line intensities, advanced background correction and an easy workflow for creation and handling of experimental data in quantitative LFAs. The results generated with our MultiFlow-Shiny app were superior to those generated with the popular software ImageJ and resulted in lower detection limits. Our assay is applicable for detecting clinically relevant ranges of both target proteins and therefore may serve as a powerful tool for POC diagnosis of inflammation and infectious events.

Lateral flow assay modified with time-delay wax barriers as a sensitivity and signal enhancement strategy

The ease of use, low cost and quick operation of lateral flow assays (LFA) have made them some of the most common point of care biosensors in a variety of fields. However, their generally low sensitivity has limited their use for more challenging applications, where the detection of low analytic concentrations is required. Here we propose the use of soluble wax barriers to selectively and temporarily accumulate the target and label nanoparticles on top of the test line (TL). This extended internal incubation step promotes the formation of the immune-complex, generating a 51.7-fold sensitivity enhancement, considering the limit of quantification, and up to 96% signal enhancement compared to the conventional LFA for Human IgG (H-IgG) detection.

Improvement in Detection Limit for Lateral Flow Assay of Biomacromolecules by Test-Zone Pre-enrichment

Lateral flow assay (LFA) is one of the most prevalent commercially available techniques for point-of-care tests due to its simplicity, celerity, low cost and robust operation. However, conventional colorimetric LFAs have inferior limits of detection (LODs) compared to sophisticated laboratory-based assays. Here, we report a simple strategy of test-zone pre-enrichment to improve the LOD of LFA by loading samples before the conjugate pad assembly. The developed method enables visual LODs of miR-210 mimic and human chorionic gonadotropin protein, to be improved by 10–100 fold compared with a conventional LFA setup without introducing any additional instrument and reagent except for phosphate running buffer, while no obvious difference occurred for Aflatoxin B1 (AFB1). It takes about 6–8 min to enrich every 50 μL of sample diluted with phosphate running buffer, therefore we can get visual results within 20 min. We identified a parameter by modeling the entire process, the concentration of probe-analyte conjugate at test zone when signaling unit being loaded, to be important for the improvement of visual limit of detection. In addition, the test-zone pre-enrichment did not impair the selectivity when miR-210 mimic was adopted as target. Integrated with other optimization, amplification and modification of LFAs, the developed test-zone pre-enrichment method can be applied to further improve LOD of LFAs.

Highly Sensitive Chemiluminescence-Based Lateral Flow Immunoassay for Cardiac Troponin I Detection in Human Serum.

Lateral flow assays (LFAs) have become the most common biosensing platforms for point-of-care testing due to their compliance with the ASSURED (affordable, sensitive, specific, user-friendly, rapid/robust, equipment-free, and deliverable to end-users) guidelines stipulated by the World Health Organization. However, the limited analytical sensitivity and low quantitative capability of conventional LFAs, which use gold nanoparticles (AuNPs) for colorimetric labeling, have prevented high-performance testing. Here, we report the development of a highly sensitive chemiluminescence (CL)-based LFA involving AuNPs conjugated with aldehyde-activated peroxidase and antibody molecules—i.e., AuNP-(ald)HRP-Ab—as a new conjugation scheme for high-performance testing in LFAs. When paired with the CL-based signal readout modality, the AuNP-(ald)HRP-Ab conjugate resulted in 110-fold enhanced sensitivity over the colorimetric response of a typical AuNP-Ab conjugate. To evaluate the performance of the CL-based LFA, we tested it with human cardiac troponin I (cTnI; a standard cardiac biomarker used to diagnose myocardial infarction) in standard and clinical serum samples. Testing the standard samples revealed a detection limit of 5.6 pg·mL−1 and acceptably reliable precision (with a coefficient of variation of 2.3%–8.4%), according to clinical guidelines. Moreover, testing the clinical samples revealed a high correlation (r = 0.97) with standard biochemical analyzers, demonstrating the potential clinical utility of the CL-based LFA for high-performance cTnI testing.

Challenges and perspectives in the development of paper-based lateral flow assays

Lateral flow assays (LFAs) have been introduced and developed over the last half century. This technology is widely used as a tool for diagnosis in several fields such as environment, food quality and healthcare. Point-of-care (POC) diagnosis using LFAs has been attracting attention of the research community, particularly aiming for the development of a platform that can evaluate of biological markers in bodily fluids such as saliva and urine. The existence of a disease or the pregnancy can be determined by a test device, before further investigation and medical treatment. LFAs make use of a disposable test strip, which can provide diagnosis result on the spot within minutes. Thus, LFAs is a promising alternative of preliminary diagnosis for laboratory instruments that are costly, time consuming and require trained personnel. This paper includes a brief overview of the conventional LFAs: material selection based on its roles and characteristics, working principles, fundamentals, applications, and design criteria. We mainly discuss the technical challenges in both engineering and biochemical aspects and recommends possible solutions. We identify current research trends and provide perspectives of advanced technologies for enhancing assay performance.

Nucleic acid lateral flow assay with recombinase polymerase amplification: Solutions for highly sensitive detection of RNA virus

We propose nucleic acid lateral flow assay (LFA) coupled with reverse transcription recombinase polymerase amplification (RT-RPA) resulting from step-by-step multiparametric adjustments to both RT-RPA reactions and LFA interactions. The assay was realized for RNA virus detection using the example of potato virus X (PVX), a dangerous phytopathogen. The assay stages were adjusted for sensitive detection. (1) DNA target was designed and verified. A fragment (146 bp) of coat protein gene (gp5) and biotin-/fluorescein-labeled forward/reverse primers were chosen to produce target amplicons. (2) In a test strip, the construction advantage of the realization of the highest affinity interaction (biotin–streptavidin in our research) through gold nanoparticle conjugate (streptavidin immobilized on the GNP surface) was demonstrated. (3) RPA with reverse transcription was adjusted including primer concentration, order of components’ mixing, and reaction temperature. Due to the adjustments, the assay was able to detect 0.14 ng PVX per g potato leaves at 30 min. The PVX assay was 260 times more sensitive than conventional lateral flow assay based on antibodies and demonstrated the same sensitivity as PCR detection. The proposed adjustments are applicable for ultrasensitive and rapid detection of various RNA viruses.

Lateral flow assay ruler for quantitative and rapid point-of-care testing.

Lateral flow assay (LFA) is a well-established platform for point-of-care (POC) testing due to its low cost and user friendliness. Conventional LFAs provide qualitative or semi-quantitative results and require dedicated instruments for quantitative detection. Here, we developed an "LFA ruler" for quantitative and rapid readout of LFA results, using a 3D printed strip cassette and a simple, inexpensive microfluidic chip. Platinum nanoparticles are used as signal amplification reporters, which catalyze the generation of oxygen to push ink advancement in the microfluidic channel. The concentration of the target is linearly correlated with the ink advancement distance. The entire assay can be completed within 30 minutes without external instruments and complicated operations. We demonstrated quantitative prostate specific antigen testing using the LFA ruler, with a limit of detection of 0.54 ng mL-1, linear range of 0-12 ng mL-1, and high correlation with a clinical gold standard assay. The LFA ruler achieves low cost, quantitative, sensitive and rapid detection, which has great potential in POC testing and can be extended to quantify other disease biomarkers.