Lateral Flow Immunoassay Research Articles

Educational Models in Analytical Chemistry Lab: The Story behind Lateral Flow Immunoassays in the Context of COVID-19

Educational Models in Analytical Chemistry Lab: The Story behind Lateral Flow Immunoassays in the Context of COVID-19

Strategy for Accurate Detection of Six Tropane Alkaloids in Honey Using Lateral Flow Immunosensors

Honey, a widely consumed food, is susceptible to contamination by various toxic substances during production. Tropane alkaloids, with their potent neurotoxicity, are frequently found in honey. Hence, there is an acute need for rapid and effective detection methods to monitor these alkaloids. Lateral flow immunoassay (LFIA), known for its simple operation, low cost, and reliable results, holds great promise. In this study, we developed an efficient and user-friendly analytical method for the simultaneous detection of six tropane alkaloids (atropine, L-hyoscyamine, scopolamine, anisodamine, homatropine, and apoatropine) in honey based on an AuNPs lateral flow immunoassay (AuNPs-LFIA) with broad-spectrum antibodies. Under optimal conditions, the calculated detection limits were 0.22, 0.29, 0.51, 6.34, 0.30, and 0.94 ng/mL, respectively. By diluting the honey sample five times, the contaminants can be readily detected using LFIA. Semi-quantitative and quantitative analyses can be completed within 17 min. This innovative method fills the void in LFIA for detecting tropane alkaloids and serves as a valuable reference for LFIA detection of honey samples, providing a crucial strategy for the accurate detection of these important compounds.

Smartcard: an integrated approach for contaminant monitoring, from field to laboratory.

Effective food safety monitoring requires a multi-step approach from farm to fork, involving different methods, ranging from convenient screening devices to sophisticated laboratory confirmatory testing. However, sample transportation to routine laboratories is time-consuming and expensive. Simplified on-site sampling followed by laboratory analysis offers a potential solution. Dried blood spot (DBS) cards ensure stability and ease of sample transportation and are used in clinical testing. However, the applicability of such an approach could be broader and include the storage of dried extract from more complex (solid) matrices. Therefore, a simplified approach is presented here, using DBS cards for on-site sampling and subsequent laboratory confirmation for food contaminants. To achieve this, an analytical tool (Smartcard) was designed using 3D-printing technology. As a proof of concept, the approach was applied to detect the pesticide fipronil, which is widely used in ornamental flower production to limit pests and on poultry farms. The Smartcard can securely store the sample extracts on a DBS card (dried extract spot (DExS) card), incorporate the lateral flow immunoassay (LFIA) and immediately provide an estimate of contamination levels. After simplified in-syringe extraction of the sample, the LFIA allows direct screening of fipronil (half maximum inhibitory concentration of 6.5µg/l with calibration standards), and the same sample extract can be directly applied to the DExS card for storage and transport to the laboratory, where analyte re-extraction and instrumental analysis is performed using ultra high-pressure liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) detecting fipronil down to 0.8µg/kg.

A fluorescence-based lateral flow immunoassay using AIEgen-encapsulated nanoparticles to rapidly and sensitively detect pro-gastrin-releasing peptide.

Afluorescence lateral flow immunoassay (F-LFIA) is presentedusing nanoparticles with encapsulated molecules whose emission is caused by aggregation as the fluorescent label to quantitatively detect gastrin-releasing peptide precursor(proGRP) in serum. The detection system was optimized to achieve a broad linear detection range of 10 ~ 5000 pg/mL and a detection limit of 6 pg/mL for F-LFIA. The proposed method exhibited good sensitivity, specificity, and reproducibility. The performance and applicability of the F-LFIA were evaluated in the analysisof 183 human serum samples, and the results were strongly correlated with those of the electrochemiluminescence immunoassay. The proposed F-LFIA can serve as a precise and rapid detection method to detect proGRP and has potential for the early diagnosis of small-cell lung cancer (SCLC).

Au@Pt@Pd nanozymes based lateral flow immunoassay for quantitative detection of SARS-CoV-2 nucleocapsid protein in nasal swab samples.

Three-metal-core-shell nanoparticles (Au@Pt@PdNPs) providing excellent peroxidase-like activity were applied in lateral flow immunoassay (LFIA), designated as Au@Pt@Pd-LFIA, for detecting the nucleocapsid protein (NP) of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2). An Au@Pt@Pd-LFIA was developed for quantitatively testing of SARS-CoV-2 NP with a range 0.12-31.25ng/mL. The limit of detection (LOD) of Au@Pt@Pd-LFIA strip was 0.06ng/mL, which was 16-fold or eightfold more sensitive than that of the gold lateral flow immunoassay (Au-LFIA) and the gold flower flow immunoassay (AF-LFIA) strips, respectively. For detection of clinical samples from nasal swabs using teststrips, Au@Pt@Pd-LFIA had 84.09% sensitivity, 100% specificity, and 92.55% accuracy. In terms of detection time, the testing of Au@Pt@Pd-LFIA strip was 16min similar to Au-LFIA (15min) and AF-LFIA (10min), but much shorter than ELISA (2h). In conclusion, Au@Pt@Pd-LFIA isa sensitive, rapid, and simple test for quantitative detection of SARS-CoV-2 NP in nasal swab samples.

Traffic signal-inspired fluorescence lateral flow immunoassay utilizing self-assembled AIENP@Ni/EC for simultaneous multi-pesticide residue detection

The presence of multiple pesticide residues in fruits and vegetables poses a serious threat to human health. Conventional gold nanoparticles (AuNPs) used in multiplex lateral flow immunoassays (M−LFIA) often exhibit poor sensitivity and potential for misidentification. This study introduces a traffic signal-inspired fluorescent LFIA (T-FLFIA) designed for the simultaneous quantitative detection of chlorothalonil (CTN), paclobutrazol (PBZ), and fipronil (FIP) in cowpea and apple samples. Utilizing the hydrophobic interactions of aggregation-induced emission luminogens (AIEgens) and the coordination of epicatechin (EC) with Ni2+, we developed a novel nanostructure-aggregation-induced emission nanoparticle (AIENP) coated with a metal-polyphenol network of Ni/EC (AIENP@Ni/EC) via a self-assembly method and employed it as fluorescence probe in T-FLFIA. Three distinct colors of AIENP@Ni/EC featuring green, yellow, and red fluorescence were successfully synthesized by coating various AIEgens. This multicolor fluorescence capability facilitates more precise and expedited discrimination among multiple detection targets. High-performance AIE fluorescence probes were fabricated by directly coupling AIENP@Ni/EC with different antibodies through electrostatic interactions, leveraging the exceptional biocompatibility of Ni/EC. The T-FLFIA exhibited sensitive detection of CTN, PBZ, and FIP at concentrations as low as 0.038 ng/mL, 0.025 ng/mL, and 0.046 ng/mL, respectively, with high qualitative accuracy (92.5 %) within a rapid timeframe (5 s). This exceptional performance can be attributed to its outstanding optical properties and distinguishable colors. This approach demonstrated enhanced sensitivity and accuracy, reduced consumption of antibodies and immunoprobes, and a broader detection range compared to single-color AuNPs-M−LFIA. This study introduces a novel, practical, and cost-effective tool for rapid screening of diverse pesticide residues in field settings

SERS-lateral flow immunoassay based on AuNR@Ag@SiO2-AuNP assembly for ultra-sensitive detection of deoxynivalenol in grain

In view of the serious harm and widespread pollution caused by deoxynivalenol (DON), it is crucial to quantify its presence in the food safety assessment process. Here, we designed a core-shell-satellite nano assembly structure as a probe, composed of an anisotropic AuNR@Ag core, an ultra-thin SiO2 layer and a high surface coverage AuNPs satellites, which showed outstanding SERS activity and high stability. Anti-DON antibodies were modified on the surface of the prepared core-shell-satellite nano-assembly structure as SERS immunoprobe for DON specific detection using SERS-based lateral flow immunoassay (LFIA) with the advantages of simplicity, rapidity, and high sensitivity. Under optimal conditions, the detection limit for detecting DON using the SERS-LFIA method was 0.053 fg/mL, and the linear detection ranged from 0.1 fg/mL to 1 μg/mL. The SERS-LFIA strips based on AuNR@Ag@SiO2-AuNP nanostructures demonstrated the great potential for quick quantitative DON detection, providing a reference for trace detection of highly toxin mycotoxins.

Performance evaluation of Eu3+-based CRP/SAA and PCT/IL-6 lateral flow immunoassay kits and their diagnostic value in respiratory tract infections

Respiratory infections are among the most common infectious diseases, resulting in significant morbidity and mortality. C-reactive protein (CRP), serum amyloid A (SAA), procalcitonin (PCT), and interleukin-6 (IL-6) are advantageous for diagnosing respiratory tract infections. This study assessed the analytical performance and accuracy of new kits for Eu3+-based CRP/SAA and PCT/IL-6 lateral flow immunoassay and its diagnostic value in respiratory tract infections. This study evaluated the detection performance of a test kit using guidelines from the Center for Medical Device Evaluation (CMDE) and the Clinical and Laboratory Standards Institute (CLSI). The test results were compared to those of the commercial kits (CRP: Mindray; SAA: Norman; PCT: Shanghai Upper; IL-6: Wantai BioPharm). A total of 156 patients with respiratory tract infections (53 with bacterial infections (Bac group); 50 with viral infections (Vir group); and 53 with co-infections (Bac+Vir group)) were enrolled, along with 50 healthy controls (HC group). Venous blood samples were collected to measure levels of SAA, PCT, CRP, and IL-6 using both the test and commercial kits. The diagnostic value of these biomarkers was assessed using receiver operating characteristic (ROC) curves. Correlation analysis demonstrated a strong concordance between the test kits and commercial kits (CRP: r=0.9396, P<0.0001; SAA: r=0.8986, P<0.0001; PCT: r=0.9594, P<0.0001; IL-6: r=0.9009, P<0.0001). The diagnostic performance of the test kits in identifying bacterial, viral, and co-infections was highly consistent with that of the commercial kit. The Eu3+-based CRP/SAA and PCT/IL-6 lateral flow immunoassay test kits demonstrated high levels of consistency with commercial kits in terms of quantitative outcomes and diagnostic performance for respiratory tract infections.

An Ultraminiaturized Inflammation Monitoring Platform Implemented by Long Afterglow Lateral Flow Immunoassay

An Ultraminiaturized Inflammation Monitoring Platform Implemented by Long Afterglow Lateral Flow Immunoassay

A novel multiplex and glycoprotein-based immunochromatographic serologic IgM test for the rapid diagnosis of Escherichia coli O157 and O145 causing bloody diarrhea and hemolytic uremic syndrome.

Shiga toxin-producing Escherichia coli (STEC) are the main etiological agents of hemolytic uremic syndrome (HUS). Good clinical management of STEC infections and HUS depends on early, rapid, and accurate diagnosis. Here, we have developed and evaluated the first multiplex and glycoprotein-based immunochromatographic test for the detection of IgM antibodies against the O-polysaccharide of the lipopolysaccharide of E. coli O157 and O145 in human serum samples. A retrospective study was carried out resulting in a diagnostic sensitivity of the E. coli O157/O145 LFIA (lateral flow immunoassay) of 97.1% and 98.9% for O157 and O145, respectively, and 97.9% for both serogroups. The diagnostic specificity was 98.7% for O157 and O145, and the overall specificity 97.4%. In samples obtained before 3 days after the onset of diarrhea, the detection percentage was 83%, increasing to 100% from 3 days onward. Finally, the association of bloody diarrhea (BD) or HUS cases to an STEC infection increased from 22.8% to 77.2% when stool culture and stx/Stx detection were combined with serology by LFIA. Our results demonstrate that the E. coli O157/O145 LFIA is a highly accurate and serospecific test for the early and rapid diagnosis of E. coli O157 and O145 infections in BD or HUS cases. This test allows the detection of specific IgM antibodies very early in the course of the infection, making it an ideal diagnostic tool to be implemented in pediatric emergencies and, thus, avoid delays in the application of the correct supportive or specific treatment and prevent complications associated with HUS.

Dual-Fluorescent Quantum Dot Nanobead-Based Lateral Flow Immunoassay for Simultaneous Detection of C-Reactive Protein and Procalcitonin.

Simultaneous detection of C-reactive protein (CRP) and procalcitonin (PCT) at the point of care is crucial for the management of infections in patients with inflammation and in critical care settings. The challenge of detecting high concentrations of CRP alongside low concentrations of PCT in plasma from inflammatory patients has limited the clinical application of multiplexed immunoassays. Herein, we developed a lateral flow immunoassay (LFIA) that employs quantum dot nanobeads (QDNBs) of varying sizes and colors to enable the simultaneous quantification of PCT and CRP in human plasma. To extend the dynamic range of CRP detection, we combined QDNBs with smaller particle sizes with the CRP detection antibodies, thereby increasing the assay's dynamic range and reducing the hook effect. At the same time, the stronger fluorescence emitted by these larger QDNBs, in conjugation with the PCT detection antibodies, allows for the detection of PCT at the nanogram level, meeting the demand for high sensitivity. The results show that this method can detect CRP concentrations from 0.1 to 3 mg/L and PCT with a detection limit of 0.09 ng/mL, which is on par with clinically used methods. By employing this dual-color and dual-size QDNB labeling strategy, we successfully achieved simultaneous detection of CRP with a broad dynamic range and PCT with high sensitivity in a one-step point-of-care rapid test.

A Versatile catalytic and photothermal lateral flow immunoassay Based on ultrathin Fe‐MoS2 nanosheets for sensitive and accurate detection of Influenza A

A Versatile catalytic and photothermal lateral flow immunoassay Based on ultrathin Fe‐MoS₂ nanosheets for sensitive and accurate detection of Influenza A

Loop-Mediated Isothermal Amplification Assay for the Detection of Citrus Canker Causing Bacterial Variant, Xanthomonas citri pv. citri Aw Strain.

Citrus canker, a highly transmissible bacterial disease, has three major types, with Asiatic canker (Canker A), caused by Xanthomonas citri pv. citri (Xcc A), being the most widespread and severe, affecting most citrus varieties. Xcc A has two mild variants, Xcc A* and Aw with a limited host range, reported in Southwest Asia and Florida, respectively. Since 2015, the canker caused by Xcc Aw has been being reported in the Rio Grande Valley of South Texas where the Texas commercial citrus industry is located. In 2016, a more severe Canker A was reported in the upper Texas gulf coast region, north of the Rio Grande Valley, posing a potential threat to the Texas citrus industry. Given that existing diagnostic methods cannot reliably distinguish Xcc Aw from Xcc A, we developed a loop-mediated isothermal amplification (LAMP) assay specific to Xcc Aw (LAMP-Aw) for rapid, field-based identification of this bacterial variant. The detection limit of LAMP-Aw was ~4.52 Log10 copies of the target molecule. This study also evaluated the field applicability of the LAMP-Aw assay by coupling the LAMP-Aw assay with a lateral flow immunoassay system.

Lateral Flow Immunoassay for the Rapid Detection of Thiamethoxam in Tea Based on a SERS Tag Constructed by Phenolic-Mediated Coating Engineering.

Sensitive and accurate detection of thiamethoxam in tea is significant to ensuring consumer health. In this study, surface-enhanced Raman scattering (SERS) tags were prepared by using a polyphenol-mediated coating engineering strategy. This approach involved the self-assembly of tannic acid (TA) and self-polymerization of benzene-1,4-dithiol (BDT) on the surface of gold nanoparticles, resulting in the formation of Au@pBDT-TA. The SERS tags possess high Raman signals and antibody adsorption properties, avoiding the complex decoration or label steps of SERS reporters. We discovered that Au@pBDT-TA, composed of gold nanoparticles at 40 nm and a pBDT-TA thickness of 10 nm, was optimal for the development of the SERS lateral flow immunoassay (LFIA). In comparison to the gold nanoparticle-based LFIA, the SERS-LFIA demonstrated 4-fold and 720-fold enhancements in visual and quantitative limits of detection in buffer solution. The SERS-LFIA demonstrated quantitative limits of detection of 0.06 and 0.1 ng/g for black and green tea, respectively, with a broader linear range spanning over 2 orders of magnitude and a short detection time of 20 min. The proposed SERS-LFIA not only offers a sensitive and reliable method for monitoring thiamethoxam in tea but also possesses a versatile potential that can be easily adapted for the trace detection of various other targets.

Comparative Characteristics of Immunochromatographic Test Systems for Tylosin Antibiotic in Meat Products.

Tylosin (TYL) is a macrolide antibiotic widely used in animal husbandry. Due to associated health risks, there is a demand for sensitive methods for mass screening of TYL in products of animal origin. This article describes the development of lateral flow immunoassays (LFIAs) for TYL detection using direct (anti-TYL antibodies conjugated with nanoparticles) and indirect antibody labeling (anti-species antibodies conjugated with nanoparticles and combined with native anti-TYL antibodies). The choice of LFIA conditions, such as concentrations of hapten-protein conjugates, specific antibodies, and gold nanoparticle (GNP) conjugates with antibodies, as well as incubation time of reagents and the concentration of detergent in the sample buffer, is presented. The achieved limits of TYL detection using LFIAs with indirect labeling were 0.8 ng/mL (visual) and 0.07 ng/mL (instrumental), compared to 4 ng/mL (visual) and 0.4 ng/mL (instrumental) for the case of direct labeling. The sensitivity of the LFIA using the indirect format was up to seven times higher, allowing the determination of the target analyte at low concentrations. TYL detection in ground meat using LFIA with indirect antibody labeling ranged from 76-119%.

Highly sensitive multiplexed colorimetric lateral flow immunoassay by plasmon-controlled metal-silica isoform nanocomposites: PINs.

Lateral flow assay (LFA) systems use metal nanoparticles for rapid and convenient target detection and are extensively studied for the diagnostics of various diseases. Gold nanoparticles (AuNPs) are often used as probes in LFAs, displaying a single red color. However, there is a high demand for colorimetric LFAs to detect multiple biomarkers, requiring the use of multicolored NPs. Here, we present a highly sensitive multiplexed colorimetric lateral flow immunoassay by multicolored Plasmon-controlled metal-silica Isoform Nanocomposites (PINs). We utilized the localized surface plasmon resonance effect to create multi-colored PINs by precisely adjusting the distance between the NPs on the surface of PINs through the controlled addition of reduced gold and silver precursors. Through simulations, we also confirmed that the distance between nanoparticles on the surface of PINs significantly affects the color and colorimetric signal intensity of the PINs. We achieved multicolored PINs that exhibit stronger colorimetric signals, offering a new solution for LFA detection with high sensitivity and a 33 times reduced limit of detection (LOD) while maintaining consistent size deviations within 5%. We expect that our PINs-based colorimetric LFA will facilitate the sensitive and simultaneous detection of multiple biomarkers in point-of-care testing.

Rigidifying aggregation-induced emission luminogens by metal–organic framework formation for sensitive lateral flow immunoassay

T-2 toxin, the strongest toxic of type A trichothecenes, widely exists in barley, wheat, corn, oats, and even human food, which is urgent to establish a rapid and sensitive detection method for T-2 toxin. Herein, we developed a fluorescent lateral flow immunoassay (LFIA) for sensitive detection of T-2 toxin. Specially, the monoclonal antibody (mAb) was prepared by immunizing mice and cell fusion, and exhibited a low half maximal inhibitory concentration (IC50) of 0.482 ng mL−1. In addition, an aggregation-induced emission (AIE)-based metal-organic framework (HfMOFs) with excellent fluorescence performance (QY: 72.5 %) was developed as reporter. Benefiting from these advantages, the limit of detection (LoD) of HfMOFs-LFIA was 0.0487 ng mL−1, which was 7.7-fold lower than traditional nanoparticles-based LFIA (0.375 ng mL−1). In addition, HfMOFs-LFIA was used for the detection of T-2 toxin in actual samples and showed satisfactory recoveries (71.3 %–123.7 %) with coefficient of variation (2.96 %–14.07 %). Overall, the dual-improvement in mAb and label might power the development in LFIA, achieving innovative prospects for LFIA in food safety fields.

ImageJ and smartphone app chemistry analyzer using image-based colorimetric assay for quantitative detection of melamine

Abstract A smartphone-assisted microchemistry analyzer with an image-based colorimetric assay using ImageJ and a smartphone app detection method was successfully developed for the quantitative detection of melamine (MEL). The color changes of the MEL in the colloidal gold lateral flow immunoassay strip were captured and analyzed using a smartphone-controlled analyzer with an LED light source and a smartphone camera. The smartphone camera and light source were used to read the colorimetric signal from the strip, and a smartphone app was written and installed onto the smartphone. The quantitative analysis was validated with ImageJ and smartphone app colorimetric analysis. The limits of detection (LODs) for ImageJ and the smartphone app were calculated as 0.30 and 0.07 mg/L, respectively. The highly quantitative relationships between the MEL concentrations and the optical density and gray value of the ImageJ and smartphone app detection method. The designed image-based biosensor is successfully applied to detect MEL in solution of standard MEL and commercial milk samples.

Enhanced Peroxidase-like Activity of Ruthenium-Modified Single-Atom-Thick A Layers in MAX Phases for Biomedical Applications.

Nanozymes have demonstrated significant potential as promising alternatives to natural enzymes in biomedical applications. However, their lower catalytic activity compared to that of natural enzymes has limited their practical utility. Addressing this challenge necessitates the development of innovative enzymatic systems capable of achieving specific activity levels of natural enzymes. In this study, we focus on enhancing the catalytic performance of nanozymes by introducing Ru atoms into the single-atom-thick A layer of the V2SnC MAX phase, resulting in the formation of V2(Sn0.8Ru0.2)C with Ru single-atom sites. The V2(Sn0.8Ru0.2)C MAX phase demonstrated an exceptional peroxidase-like specific activity of up to 1792.6 U mg-1, surpassing the specific activity of a previously reported horseradish peroxidase (HRP). Through X-ray photoelectron spectroscopy (XPS) and density functional theory (DFT) investigations, it has been revealed that both the V2C atom layers and single-atom-thick Sn readily accept a negative charge from Ru, leading to a reduction of the energy barrier for H2O2 adsorption. This discovery has enabled the successful application of V2(Sn0.8Ru0.2)C in the development of a lateral flow immunoassay for heart failure biomarkers, achieving a detection sensitivity of 4 pg mL-1. Additionally, V2(Sn0.8Ru0.2)C demonstrated exceptional broad-spectrum antibacterial efficacy. This study lays the groundwork for the precise design of MAX phase-based nanozymes with high specific activity, offering a viable alternative to natural enzymes for various applications.

Modular Point-of-Need Tropane Alkaloid Detection at Regulatory Levels: Combining Solid-Liquid Extraction from Buckwheat with a Paper-Immobilized Liquid-Phase Microextraction and Immuno-Detection in Interconnectable 3D-Printed Devices.

Contamination with tropane alkaloids in cereals is expected to increase globally. However, current identification tools (e.g., liquid chromatography-mass spectrometry) for tropane alkaloids are time-consuming and expensive. Furthermore, their miniaturized alternatives lack sensitivity and robustness. Therefore, there is a pressing need for inexpensive and effective screening methods. Here, an on-site applicable modular workflow for tropane alkaloid detection in buckwheat is presented. The modular workflow combines paper microfluidics and interconnectable 3D-printed sample preparation tools and was evaluated for different tropane alkaloids, including atropine and scopolamine. Furthermore, integration with an indirect competitive lateral flow immunoassay (icLFIA) for atropine detection at relevant levels was demonstrated. In the modular workflow, to minimize matrix coextraction, tropane alkaloids were extracted from the milled buckwheat cereals by a mixture of alkaline aqueous and immiscible organic solvents (extraction recoveries: 66-79%). The tropane alkaloids were subsequently concentrated with a newly developed paper-immobilized liquid-phase microextraction (PI-LPME, extraction recoveries: 34-60%, concentration factor to immobilized solution in paper: 60-108×). After the PI-LPME, with an integrated 3D-printed setup, the tropane alkaloids were directly eluted (elution recoveries: 83-93%) and detected with the icLFIA. Digital read-out of the icLFIA, by employing a hand-held reader, enabled semiquantification of atropine (IC50 = 0.56 ng mL-1 in standard solutions). The modular workflow was validated by analyzing 24 blank and spiked buckwheat cereal samples with 5 and 10 μg kg-1 atropine. A cutoff value was established with an estimated false negative rate of 1% and estimated false positive rate of 0.68%. Therefore, the modular workflow can aid in fast, inexpensive, and on-site atropine detection by nonexperts, and when integrated with a scopolamine-specific icLFIA expanded toward scopolamine detection. Moreover, the developed sample extraction and concentration method (PI-LPME) is suitable for the analysis of many other compounds with pH-dependent polarity.