Lateral Flow Immunoassay Strip Research Articles

A nanostructured lateral flow immunoassay strip combined with Au@SiO2 SERS nanotags for multiplex biomarker detection

A nanostructured lateral flow immunoassay strip with Au@SiO2 SERS nanotags was constructed. This is the first time nanostructured T line and SERS-LFA has been combined, which has great potential for early non-invasive detection of AD biomarkers in blood.

Lateral Flow Immunoassay Strip Based on Confocal Raman Imaging for Ultrasensitive and Rapid Detection of COVID-19 and Bacterial Biomarkers

Lateral Flow Immunoassay Strip Based on Confocal Raman Imaging for Ultrasensitive and Rapid Detection of COVID-19 and Bacterial Biomarkers

Gold nanoparticle conjugate-based lateral flow immunoassay (LFIA) for rapid detection of RBD antigen of SARS-CoV-2 in clinical samples using a smartphone-based application.

The coronavirus disease 2019 (COVID-19) pandemic has emphasized the need for development of a rapid diagnostic device for the effective treatment and its mitigation. Lateral flow immunoassay (LFIA) belongs to a class of diagnostic devices, which has the benefit of providing quick results, easy to handle, low cost, and on-site applicable. So far, several LFIA has been developed for the detection of infectious severe acute respiratory syndrome-coronavirus-2(SARS-CoV-2), however, only a few of them are antigen (Ag)-based. Here, we describe an antibody (Ab)-labeled gold nanoparticles (AuNPs)-based LFIA(AuNPs-LFIA) for the detection of Receptor-Binding Domain (RBD) of SARS-CoV-2. For this, RBD Abof SARS-CoV-2 was conjugated with the AuNPs, which served as a detecting probe. The fabricated LFIA strip was optimized for different parameters such as membrane pore size, blocking conditions, Ab coating concentration, and conjugate incubation. The optimized LFIA strips were validated in spiked buffer samples and the optimal limit of detection was found to be 1 ng/ml, which was confirmed by a smartphone-based application. Moreover, the developed AuNPs-LFIA strips effectively detected RBD Agin 100 clinical samples with 94.3% sensitivity and 90.9% specificityin clinical samples when compared with the gold standard (RT-PCR). The fabricated LFIAs are reported to have storage stability of up to 21 days at 4°C and room temperature (RT).Hence, the developed LFIA can be used as a portable, cost-effective diagnostic device for rapid detection of SARS-CoV-2.

Quantum Dot Nanobeads-Labelled Lateral Flow Immunoassay Strip for Rapid and Sensitive Detection of Salmonella Typhimurium Based on Strand Displacement Loop-Mediated Isothermal Amplification

• We developed a novel method for pathogen detection in foods. • It combined QBs-labelled LFIAS and SD-LAMP to achieve quantitative detection. • The combined test rapidly and sensitively detected Salmonella Thyphimurium. • Current approach does not require almost any equipment allowing point-of-care use. • The efficacy of the current test was confirmed in different food samples. Rapid, sensitive, point-of-care detection of pathogenic bacteria is important for food safety. In this study, we developed a novel quantum dot nanobeads-labelled lateral flow immunoassay strip (QBs-labelled LFIAS) combined with strand displacement loop-mediated isothermal amplification (SD-LAMP) for quantitative Salmonella Typhimurium (ST) detection. Quantum dot nanobeads (QBs) served as fluorescence reporters, providing good detection efficiency. The customizable strand displacement (SD) probe was used in LAMP to improve the specificity of the method and prevent by-product capture. Detection was based on a sandwich immunoassay. A fluorescence strip reader measured the fluorescence intensity (FI) of the test (T) line and control (C) line. The linear detection range of the strip was 10 2 –10 8 CFU·mL −1 . The visual limit of detection was 10 3 CFU·mL −1 , indicating that the system was 10-fold more sensitive than AuNPs-labelled test strips. ST specificity was analyzed in accordance with agarose gel outputs of PCR and SD-LAMP. We detected ST in foods with an acceptable recovery of 85%–110%. The method is rapid, simple, almost equipment-free, and suitable for bacterial detection in foods and for clinical diagnosis.

Simple and rapid detection of ractopamine in pork with comparison of LSPR and LFIA sensors.

This study developed a simple and rapid strategic technique to detect ractopamine (chemical growth-promoting agent) in pork. Two highly sensitive and specific gold nanoparticle-based portable sensors, i.e., localized surface plasmon resonance (LSPR) sensors, and lateral flow immunoassay (LFIA) strips were developed to detect veterinary drug residues in food products, that have detrimental effects on humans. Optimization studies were conducted on several sensor devices to improve sensitivity. Each sensor comprised functionalized gold nanoparticles conjugated with ractopamine antibodies. The LSPR sensor chip achieved excellent detection sensitivity = 1.19 fg/mL and was advantageous for quantitative analysis due to its wide dynamic range. On the other hand, LFIA strips provided visual test confirmation and achieved 2.27 ng/mL detection sensitivity, significantly less sensitive than LSPR. The complementary sensors help overcome each other's shortcomings with both the techniques offering ease of use, affordability and rapid diagnosis. Thus, these sensors can be applied on-site for routine screening of harmful drug residues in pork meat. They also provide useful direction for advanced technologies to enhance assay performance for detecting various other food drug contaminants.

Triplex Immunostrip Assay for Rapid Diagnosis of Tobacco Mosaic Virus, Tobacco Vein Banding Mosaic Virus, and Potato Virus Y.

Mixed virus infection has increasingly become a problem in the production of Solanaceae crops in recent years; therefore, a fast and accurate detection method is needed. In this study, a novel triplex immunostrip assay was developed for the simultaneous detection of tobacco mosaic virus (TMV), tobacco vein banding mosaic virus (TVBMV), and potato virus Y (PVY). The limits of detection of this novel immunostrip reached 200 ppb (ng/ml), 1 ppm (µg/ml), and 2 ppm for TMV, PVY, and TVBMV particles, respectively. Importantly, no cross-reactivity was observed among TMV, TVBMV, and PVY or to a nontarget virus. When the assay was applied to suspected virus-infected tobacco, tomato, and potato samples collected from fields in Southwest China, samples of single or mixed virus infection were successfully identified. In conclusion, the triplex immunostrip assay provides a fast and easy to use on-site detection method for field epidemiological studies of TMV, TVBMV, and PVY, and for managing diseases that are caused by them.

Lateral Flow Immunoassays for Detecting Viral Infectious Antigens and Antibodies.

Abundant immunological assays currently exist for detecting pathogens and identifying infected individuals, making detection of diseases at early stages integral to preventing their spread, together with the consequent emergence of global health crises. Lateral flow immunoassay (LFIA) is a test characterized by simplicity, low cost, and quick results. Furthermore, LFIA testing does not need well-trained individuals or laboratory settings. Therefore, it has been serving as an attractive tool that has been extensively used during the ongoing COVID-19 pandemic. Here, the LFIA strip's available formats, reporter systems, components, and preparation are discussed. Moreover, this review provides an overview of the current LFIAs in detecting infectious viral antigens and humoral responses to viral infections.

A Bedside Test to Detect the Presence of Embryonic or Fetal Tissue in Vaginal Blood.

To evaluate a rapid bedside test that detects alpha-fetoprotein (AFP) and insulin-like growth factor-binding protein 1 (IGFBP-1) to identify the presence of embryonic or fetal tissue in vaginal blood. This was a prospective cohort study. Reproductive-aged individuals were recruited into three groups: a negative control group consisting of nonpregnant individuals undergoing dilation and curettage (D&C) or experiencing vaginal bleeding; a positive control group of individuals with confirmed intrauterine pregnancy undergoing D&C; and the study group of pregnant individuals with first-trimester bleeding. Lateral flow immunoassay strips capable of detecting both AFP and IGFBP-1 were used to test vaginal blood for the presence of embryonic or fetal tissue. Ninety individuals were recruited: 31 in the positive control group, 23 in the negative control group, and 36 in the study group, including 12 individuals with ectopic pregnancies, 16 with active miscarriages, four with threatened miscarriages, and four with complete miscarriages. Vaginal blood from 14 of the 16 individuals with active miscarriages was correctly positive for embryonic or fetal tissue. Vaginal blood from all individuals with ectopic pregnancies, threatened miscarriages, and complete miscarriages was negative for embryonic or fetal tissue. Overall, 45 of 47 individuals with confirmed embryonic or fetal tissue in vaginal blood correctly tested positive using the test strips, a test sensitivity of 95.7% (95% CI 85.5-99.5%). Of the 43 individuals with confirmed absence of embryonic or fetal tissue in their vaginal blood, 42 were correctly negative, a test specificity of 97.7% (95% CI 87.7-99.9%). A rapid test strip detecting both AFP and IGFBP-1 can accurately identify the presence of embryonic or fetal tissue in vaginal blood. When positive, this could aid in diagnosing miscarriage and ruling out ectopic pregnancy at the bedside.

Detection of Ureaplasma urealyticum by Catalytic Hairpin Assembly Combined with a Lateral Flow Immunoassay Strip.

Ureaplasma urealyticum is a common genital mycoplasma in men and women, which can cause reproductive tract infection and infertility, and is also related to adverse pregnancy outcomes and neonatal diseases. Pathogen culture and polymerase chain reaction (PCR) are the main methods for the diagnosis of U. urealyticum. However, pathogen culture takes too long, and PCR requires professional personnel and sophisticated instruments. Here, we report a simple, convenient, sensitive, and specific detection method, which combines catalytic hairpin assembly with a lateral flow immunoassay strip. Only a water bath and a fluorescence reader are needed to detect the results in 30 min. We can realize the point-of-care testing of U. urealyticum by this method. To verify this method, we selected 10 clinical samples for testing, and the test results were exactly the same as the clinical report.

Biosensing Chlorpyrifos in Environmental Water Samples by a Newly Developed Carbon Nanoparticle-Based Indirect Lateral Flow Assay.

Pesticides are used in agriculture to prevent pests. Chlorpyrifos (CHLP) is an insecticide with potentially detrimental effects on humans, bees, and the aquatic environment. Its effects have led to a total ban by the European Union (EU), but outside the EU, CHLP is still produced and used. An indirect lateral flow immunoassay (LFIA) for the detection of CHLP was developed and integrated into a cassette to create a lateral flow device (LFD). Species-specific reporter antibodies were coupled to carbon nanoparticles to create a detector conjugate. Water samples were mixed with a specific CHLP monoclonal antibody and detector conjugate and applied to the LFD. Dose-response curves elicited the detection of low concentrations of CHLP (<1 µg/L). This sensitivity was recorded through a rapid handheld digital imaging device but also visually by naked eye. The CHLP LFD was applied to a range of European surface water samples, fortified with CHLP, revealing a sensitivity in these matrices of 2 µg/L, both by digital and visual analysis. To improve the simplicity of the CHLP LFIA, the assay reagents were dried in tubes, enabling to carry out the test by simply adding water samples and inserting the LFIA strips. This CHLP LFIA is thus suited for the on-site screening of surface waters.

Fabrication of lateral flow immunoassay strip for rapid detection of acute hepatopancreatic necrosis disease.

Acute hepatopancreatic necrosis disease (AHPND) is a contagious disease for the shrimp cultivation, thus early detection of disease is an unmet need. This present study documented for the first time a simple lateral flow immunoassay (LFIA) strip using polyclonal antibodies was created for the rapid detection both of PirAvpand PirBvpprotein simultaneously. LFIA method based on the principle of sandwich format. The label is the colloidal gold. The polyclonal antibody was conjugated with the colloidal gold acting as biorecognition element and coated onto the conjugate pad. The rabbit anti-Pirvp, anti-PirBvpantibodies, and goat anti-rabbit IgG antibody were separately sprayed onto a nitrocellulose membrane to form two test lines and one control line, respectively. The appearance of red bands at the control line and the test line indicated a positive result. A single coloured band at control area indicated a negative result. The limit of detection of LFIA was found to be 125ng, which could be visually detected by naked eye within 15min. Therewasno cross-reactivity observed with VPnon-AHPND. Furthermore, the sensitivity and specificity of LFIA were 94.0% and 98.0%, respectively. The developed test strip could be a game changer for early andin situdiagnosis of AHPND.

Lateral flow immunoassay strips based on europium(III) chelate microparticle for the rapid and sensitive detection of Trichinella spiralis infection in whole blood samples of pigs.

Trichinellosis is a major food-borne parasitosis caused by ingesting raw or semi-raw meat products from pigs infected with Trichinella spiralis (T. spiralis). Although China is the largest consumer of pork in the world, the current diagnostic method of T. spiralis is exclusively performed in a laboratory setting, due to its complexity and laborious procedure. Here, in order to solve the detection problems in the pig breeding industry, a rapid, sensitive, and on-site serological diagnosis method was developed. The novel lateral flow immunoassay strip (ICS) is based on europium(III) chelate microparticle (ECM) to detect T. spiralis-specific IgG antibody in the serum and whole blood samples from pigs. The structure of the blood-filtering pad and the conjugate pad was added to the ICS, allowing for whole blood samples to be detected and enabling on-site deployment. By comparing the detection results of the serum samples and the whole blood samples, the detection limit of this method was evaluated. Thereafter, this method was used to investigate Trichinella infection in Chongqing, Sichuan, Inner Mongolia, Guangxi, and Liaoning provinces of China, and the results were almost consistent with the standard method of artificial digestion. Taking advantage of its user-friendly procedure, short detection time (3 min), and sensitivity, the ECM-ICS could be employed for monitoring the epidemic of Trichinella infection and ensuring meat safety.

Detection of trh+ Vibrio parahaemolyticus in seafood by lac dye coloration-based label-free lateral flow immunoassay strip.

Vibrio parahaemolyticus (V.parahaemolyticus) is an important foodborne pathogen known to cause severe enteric disease. Thus, timely detection of V.parahaemolyticus in seafood is crucial to prevent food poisoning and reduce economic losses. Traditional lateral flow immunoassay strips (LFIS) required good labelling materials and pairing of two antibodies, which made them costly and difficult to manufacture. In this study, a label-free and lac dye coloration-based LFIS (LD-LFIS) to detect trh+ V.parahaemolyticus was developed for the first time. Lac dye was used to stain V.parahaemolyticus, and LFIS was used to detect stained bacteria. Dimethyl sulphoxide (DMSO) and simultaneous mordanting were chosen as the best solvent and the best staining method for lac dye. In addition, three mordants [KAl(SO4 )2 ·12H2 O, NH4 Fe(SO4 )2 ·12H2 O, and AlCl3 ·6H2 O] were selected to improve dyeing efficiency. The detection limit of LD-LFIS was 3.9 × 105 CFU/ml when NH4 Fe(SO4 )2 ·12H2 O was used as mordant. Feasibility of the LD-LFIS method was verified by detecting trh+ V.parahaemolyticus in true and spiked seafood samples. The method developed in this study is expected to reduce restrictions on labelling materials and pairing of two antibodies on LFIS, and proposes a novel idea for the rapid detection of V.parahaemolyticus in seafood.

Hue Recognition Competitive Fluorescent Lateral Flow Immunoassay for Aflatoxin M1 Detection with Improved Visual and Quantitative Performance.

Immunological detection of small molecules in a point-of-care (POC) format is of great significance yet remains challenging for accurate visual discrimination and quantitative analysis. Here, we report a novel hue recognition competitive fluorescent lateral flow immunoassay (HCLFIA) strip that allows both visual and quantitative detection of aflatoxin M1 (AFM1). The HCLFIA strip works on the basis of the ratiometric change of emission, arising from the overlap of fluorescence signals of two nanocomposites tagged with probe antibodies and coated antigens. A visually discernible orange-red-to-green fluorescence color change allows the naked eye semiquantitative readout of AFM1 around the threshold concentration (0.05 ng mL-1), yielding a visible detection limit of 0.02 ng mL-1. Moreover, using a custom smartphone-based device and color chart analysis, ultrasensitive quantitative detection of AFM1 can be achieved with a low limit of detection at 0.0012 ng mL-1, which is considerably better than those of the previously reported colorimetric and fluorescent strips. The accuracy performed in spiked milk samples ranged from 97.91 to 113.12% with a coefficient of variation below 7.8%, showing good consistency with the results from isotope dilution liquid chromatography-tandem mass spectrometry. Thanks to the unique hue recognition scheme, the HCLFIA strip holds great potential for POC detection of small molecules.

Ultrasensitive and multiplex detection of four pathogenic bacteria on a bi-channel lateral flow immunoassay strip with three-dimensional membrane-like SERS nanostickers

A lateral flow immunoassay (LFA) technique for sensitive and multiplexed on-site detection of bacteria remains a challenge. Here, we develop a bi-channel surface-enhanced Raman scattering (SERS)-based LFA by using three-dimensional membrane-like SERS tags as nanostickers (named GO@Au/Ag) for direct and ultrasensitive analysis of multiple pathogens in a single test. The grafting of numerous Ag satellites onto nanosticker significantly increased the relative surface area for bacteria binding and generated efficient SERS hotspots over large area to improve the sensing sensitivity. Antibody-labeled GO@Au/Ag nanostickers can rapidly stick onto the target bacteria and generate superior SERS signals and fluidity on the paper strip, thus conquering the adverse effect of bacteria size and improving the multiplex analysis ability of LFA. The integration of two different Raman reporter molecules into nanostickers allows simultaneous detection of four pathogens on two test lines, which significantly simplifies the reading process of SERS signals. The proposed biosensor can quantitatively detect four different bacteria in real clinical samples with low detection limit (9 cells mL−1 level), short assay time (20 min), high accuracy and excellent stability, indicating its great application potential for on-site detection of pathogens.

Development of a smartphone-based quantum dot lateral flow immunoassay strip for ultrasensitive detection of anti-SARS-CoV-2 IgG and neutralizing antibodies

BackgroundAs several vaccines for SARS-CoV-2 have been developed, a large proportion of individuals have been vaccinated worldwide so far. The rapid and accurate immunoassays are urgently needed for detecting the specific virus-neutralizing antibody (NAb), which reflect the protective effect of the vaccines among different populations.MethodsIn this study, we designed a quantum dot lateral flow immunoassay strip (QD-LFIA) for smartphones for the detection of specific IgG or neutralizing antibodies in SARS-CoV-2 in human serum or whole blood samples. The recombinant receptor binding domain of the SARS-CoV-2 spike protein was used as the antigen to combine with NAb or angiotensin-converting enzyme 2.ResultsAmong 81 patients who recovered from COVID-19 who were diagnosed using the nucleic acid test initially, 98.8% (80/81) were positive for IgG and 88.9% (72/81) were positive for NAb by QD-LFIA. Among 64 individuals inoculated with inactivated vaccines and six subunit vaccines, 90% (63/70) were positive for IgG and 82.9% (58/70) were positive for NAb by QD-LFIA, whereas no cross-reaction was found in 150 healthy blood donors, two patients with influenza B, and three patients with common cold.ConclusionThe established platform could achieve a rapid and accurate detection of NAb specific to SARS-CoV-2, which could be used for detecting the protective effect of the vaccines in areas of world that currently affected by the pandemic.

Gold-based strip sensor for the rapid and sensitive detection of butralin in tomatoes and peppers

Butralin is a widely used dinitroaniline herbicide. Butralin residues in vegetables or fruits represent a threat to human health. In this study, we developed a rapid and sensitive gold-based lateral flow immunoassay (LFIA) for butralin detection in tomato and green pepper samples based on a screened monoclonal antibody (mAb) against butralin. The mAb possessed a half-maximal inhibitory concentration (IC50) of 12.7 ng/mL, with no cross-reactivity toward other dinitroaniline herbicides. The established LFIA strip had a visible limit of detection (LOD) of 50 ng/g and a cut-off value of 2000 ng/g in tomato and green pepper samples. According to the calibration curves for quantitative analysis, the calculated LODs of the LFIA strip were 4.7 ng/g and 4.3 ng/g in tomato and green pepper, respectively. The results were obtained within 10 min. The average recoveries ranged between 95.4% and 109.6% with a coefficient of variation (CV) of 4.3% to 7.1% in tomato samples and between 94.8% and 109.1% with a CV of 3.9% to 6.1% in green pepper samples. These data suggested that our proposed LFIA is a sensitive, specific, and reliable method for the rapid detection of butralin in real samples.

Deeply-dyed nanobead system for rapid lateral flow assay testing of drugs at point-of-care

Point-of-care test (POCT) allows for rapid and sensitive screening of drugs of abuse. However, traditional gold nanoparticle-based lateral flow immunoassay strip is not sensitive enough for detection of trace drugs in hair samples. Herein, we aimed to develop a more specific system using a composite polymer nanobead that is deeply dyed with oil soluble dyes, and termed as deeply dyed nanobead (DDNB). The prepared composite nanobeads displayed a clear core-shell structure and the core/shell ratios were readily controlled by polymer/dye feeding ratios. The absorbance stable nanobeads capped with carboxyl groups were covalently conjugated with antibodies, and were employed for preparation of lateral flow immunoassay strips for sensitive detection of drugs in hair with naked eye. The developed drug strips allow the detection of drugs such as morphine and methamphetamine in hair samples within 13 min (including hair sample processing ~5 min). The cut-off value of DDNB strip for methamphetamine detection with naked eye is down to concentration of 8.0 ng/mL, which is about 3.1 times more sensitive than the traditional gold nanoparticles based lateral flow immunoassay. Moreover, the colorful DDNB system has the potential for multiplexing detection of analytes at point-of-care settings and low cost.

Design of multiplexing lateral flow immunoassay for detection and typing of foot-and-mouth disease virus using pan-reactive and serotype-specific monoclonal antibodies: Evidence of a new hook effect.

The foot-and-mouth disease (FMD) is the most important transboundary viral disease of livestock in the international context, because of its extreme contagiousness, widespread diffusion, and severe impact on animal trade and animal productions. The rapid and on-field detection of the virus responsible for the FMD represents an urgent demand to efficiently control the diffusion of the infection, especially in low resource setting where the FMD is endemic. Colorimetric lateral flow immunoassay (LFIA) is largely used for the development of rapid tests, due to the extreme simplicity, cost-effectiveness, and on-field operation. In this work, two multiplex LFIA devices were designed for the diagnosis of FMD and the simultaneous identification of major circulating serotypes of the FMD virus. The LFIAs relied on the sandwich-type immunoassay and combined a set of well-characterised monoclonal antibodies (mAb) pairs. One LFIA aimed at detecting and identifying O, A and Asia-1 serotypes, the second device enabled the detection and differentiation of the SAT 1 and SAT 2 serotypes. Both devices also incorporated a broad-specific test line reporting on infection from FMDV, regardless the strain and the serotype involved. Accordingly, five and four reactive zones were arranged in the two devices to achieve a total of six simultaneous analyses. The development of the two multiplex systems highlighted for the first time the relevance of the mAb positioning along the LFIA strip in connection with the use of the same or different mAb as capture and detector ligands. In fact, the excess of detector mAb typically employed for increasing the sensitivity of sandwich immunoassay induced a new type of hook effect when combined with the same ligand used as the capture. This effect strongly impacted assay sensitivity, which could be improved by an intelligent alignment of the mAb pairs along the LFIA strip. The analytical and diagnostic performances of the two LFIAs were studied by testing reference FMDV strains grown in cell cultures and some representative field samples (epithelium homogenates). Almost equivalent sensitivity and specificity to those of a reference Ag-ELISA kit were shown, except for the serotype SAT 2. These simple devices are suitable in endemic regions for in-field diagnosis of FMD accompanied by virus serotyping and, moreover, could be deployed and used for rapid confirmation of secondary outbreaks after FMD incursions in free-areas, thus contributing to promptly implement control measures.

Thermometric lateral flow immunoassay with colored latex beads as reporters for COVID-19 testing

Temperature sensing is a promising method of enhancing the detection sensitivity of lateral flow immunoassay (LFIA) for point-of-care testing. A temperature increase of more than 100 °C can be readily achieved by photoexcitation of reporters like gold nanoparticles (GNPs) or colored latex beads (CLBs) on LFIA strips with a laser power below 100 mW. Despite its promise, processes involved in the photothermal detection have not yet been well-characterized. Here, we provide a fundamental understanding of this thermometric assay using non-fluorescent CLBs as the reporters deposited on nitrocellulose membrane. From a measurement for the dependence of temperature rises on the number density of membrane-bound CLBs, we found a 1.3-fold (and 3.2-fold) enhancement of the light absorption by red (and black) latex beads at 520 nm. The enhancement was attributed to the multiple scattering of light in this highly porous medium, a mechanism that could make a significant impact on the sensitivity improvement of LFIA. The limit of detection was measured to be 1 × 105 particles/mm2. In line with previous studies using GNPs as the reporters, the CLB-based thermometric assay provides a 10× higher sensitivity than color visualization. We demonstrated a practical use of this thermometric immunoassay with rapid antigen tests for COVID-19.