"Cave Effect" Induces Self-Assembled Bimetallic Hollow Structure for Three-in-One Lateral Flow Immunoassay.

Bimetallic hollow structures have attracted much attention due to their unique properties, but they still face the problems of nonuniform alloys and excessive etching leading to structural collapse. Here, uniform bimetallic hollow nanospheres are constructed by pore engineering and then highly loaded with hemin (Hemin@MOF). Interestingly, in the presence of polydopamine (PDA), the competitive coordination between anionic polymer (γ-PGA) and dimethylimidazole does not lead to the collapse of the external framework but self-assembly into a hollow structure. By constructing the Hemin@MOF immune platform and using E. coli O157:H7 as the detection object, we find that the visual detection limits can reach 10, 3, and 3 CFU/mL in colorimetric, photothermal, and catalytic modes, which is 4 orders of magnitude lower than the traditional gold standard. This study provides a new idea for the morphological modification of the metal-organic skeleton and multifunctional immunochromatography detection.

Bimetallic Nanozyme: A Credible Tag for In Situ-Catalyzed Reporter Deposition in the Lateral Flow Immunoassay for Ultrasensitive Cancer Diagnosis.

The lateral flow immunoassay (LFIA) is a sought-after point-of-care testing platform, yet the insufficient sensitivity of the LFIA limits its application in the detection of tumor biomarkers. Here, a colorimetric signal amplification method, bimetallic nanozyme-mediated in situ-catalyzed reporter deposition (BN-ISCRD), was designed for ultrasensitive cancer diagnosis. The bimetallic nanozyme used, palladium@iridium core-shell nanoparticles (Pd@Ir NPs), had ultrahigh enzyme-like activity, which was further explained by the electron transfer of Pd@Ir NPs and the change in the Gibbs free energy during catalysis through density functional theory calculations. With gastric cancer biomarkers pepsinogen I and pepsinogen II as model targets, this assay could achieve a cutoff value of 10 pg/mL, which was 200-fold lower than that without signal enhancement. The assay was applied to correctly identify 8 positive and 28 negative clinical samples. Overall, this BN-ISCRD-based LFIA showed great merits and potential in the application of ultrasensitive disease diagnosis.

Efficient Photothermal Sensor Based on Coral-Like Hollow Gold Nanospheres for the Sensitive Detection of Sulfonamides.

Gold nanoparticles (AuNPs), universally regarded as colorimetric signal reporters, are widely employed in lateral flow immunoassays (LFIAs). However, it is difficult for AuNPs-LFIA to achieve a wide range and sensitive detection. Herein, novel coral-like hollow gold nanospheres (CHGNPs) are synthesized. The growth of gold nanospheres can be regulated to obtain a multibranched and hollow construction. The obtained CHGNPs possess intense broadband absorption across the visible to near-infrared region, exhibiting a high molar extinction coefficient of 14.65 × 1011 M-1 cm-1 and a photothermal conversion efficiency of 79.75%. Thus, the photothermal/colorimetric dual-readout LFIA is developed based on CHGNPs (CHGNPs-PT-LFIA and CHGNPs-CM-LFIA) to effectively improve the detection sensitivity and broaden the detection range in regard to sulfonamides (SAs). The limits of detection of the CHGNPs-PT-LFIA and CHGNPs-CM-LFIA reached 1.9 and 2.8 pg mL-1 for the quantitative detection of sulfaquinoxaline, respectively, which are 6.3-fold and 4.3-fold lower than that of the AuNPs-LFIA. Meanwhile, the CHGNPs-PT-LFIA broadened the detection range to three orders of magnitude, which ranged from 2.5 to 5000 pg mL-1. The synthesized photothermal CHGNPs have been proven effective in improving the performance of the LFIA and provide a potential option for the construction of sensing platforms.

Kiwi-Inspired Rational Nanoarchitecture with Intensified and Discrete Magneto-Fluorescent Functionalities for Ultrasensitive Point-of-Care Immunoassay.

Fluorescent lateral flow immunoassays (FLFIA) is a well-established rapid detection technique for quantitative analysis. However, achieving accurate analysis of biomarkers at the pg mL-1 level using FLFIA still poses challenges. Herein, an ultrasensitive FLFIA platform is reported utilizing a kiwi-type magneto-fluorescent silica nanohybrid (designated as MFS) that serves as both a target-enrichment substrate and an optical signal enhancement label. The spatially-layered architecture comprises a Fe3O4 core, an endocarp-fibers like dendritic mesoporous silica, seed-like quantum dots, and a kiwi-flesh like silica matrix. The MFS demonstrates heightened fluorescence brightness, swift magnetic response, excellent size uniformity, and dispersibility in water. Through liquid-phase capturing and fluorescence-enhanced signal amplification, as well as magnetic-enrichment sample amplification and magnetic-separation noise reduction, the MFS-based FLFIA is successfully applied to the detection of cardiac troponin I that achieved a limit of detection at 8.4 pg mL-1, tens of times lower than those of previously published fluorescent and colorimetric lateral flow immunoassays. This work offers insights into the strategic design of magneto-fluorescent synergetic signal amplification on LFIA platform and underscores their prospects in high-sensitive rapid and on-site diagnosis of biomarkers.

"Four-In-One" Multifunctional Dandelion-Like Gold@platinum Nanoparticles-Driven Multimodal Lateral Flow Immunoassay.

The traditional lateral flow immunoassay (LFIA) with a single signal output mode may encounter challenges such as low sensitivity, poor detection range, and susceptibility to external interferences. These limitations hinder its ability to meet the growing demand for advanced LFIA. To address these issues, the rational development of multifunctional labels for multimodal LFIA emerges as a promising strategy. Herein, this study reports a multimodal LFIA using "four-in-one" multifunctional dandelion-like gold@platinum nanoparticles (MDGP). The inherent properties of MDGP, such as the broad absorption spectrum, porous dandelion-like nanostructure, and bimetallic composition with gold and platinum, endow them with capacities in dual spectral-overlapped fluorescence quenching, optical readout, catalytic activity, and photothermal effect. Benefiting from their multifunctional properties, the MDGP-LFIA enables multimodal outputs including fluorescent, colorimetric, and photothermal signals. This multimodal MDGP-LFIA allows for the detection of acetamiprid at a range of 0.01-50 ng mL-1, with the lowest qualitative and quantitative detection results of 0.5 and 0.008 ng mL-1, respectively, significantly better than the traditional gold nanoparticles-based LFIA. The diversity, complementarity, and synergistic effect of integrated output signals in this multimodal MDGP-LFIA improve the flexibility, practicability, and accuracy of detection, holding great promise as a point-of-care testing platform in versatile application scenarios.

Investigating bottom phase extraction from aqueous two-phase systems for detecting bacteria using the lateral-flow immunoassay.

Lateral-flow immunoassays (LFAs) can be used to diagnose urinary tract infections caused by Escherichia coli (E. coli) at the point of care. Unfortunately, urine samples containing dilute concentrations of E. coli can yield false negative results on LFAs. Our laboratory was first to implement aqueous two-phase systems (ATPSs) to preconcentrate samples into smaller volumes prior to their application on LFAs. This is achieved by manipulating the ratio of the volume of the top phase to that of the bottom phase (volume ratio; VR) and concentrating biomarkers in the bottom phase which, when applied to LFAs in fixed volumes, leads to corresponding improvements in sensitivity. This work is the first demonstration that the same LOD can be achieved irrespective of the VR when the entire bottom phase is added to LFAs. A custom 3D-printed device was also developed to decrease liquid handling steps. Across different VRs expected from patient urine variability, this diagnostic workflow successfully detected E. coli concentrations down to 2 × 105 colony-forming units (cfu) mL-1 in synthetic urine, demonstrating consistent 10-fold improvements in sensitivity compared to trials conducted without ATPS preconcentration. This method successfully addresses the variability of patient samples while remaining easy to use at the point of care.

Clinical performance of a new lateral flow immunoassay for xylazine detection.

OBJECTIVES: Xylazine is a potent sedative used in veterinary medicine. Recently, recreational drugs such as fentanyl have been found to contain xylazine, increasing the risk of respiratory depression and death. Despite a similar presentation to opioid overdose, patients who ingest xylazine do not respond to treatment with Narcan. Therefore, rapid detection of xylazine could improve patient management and prevent adverse outcomes. METHODS: We evaluated the XYL500 one-step xylazine drug of abuse test for its ability to detect xylazine in 152 urine samples from patients on chronic opioid therapy for pain management or in treatment for substance use disorder. Results were compared to LC-MS/MS as the reference method. Precision, cross-reactivity, interference and stability studies were performed. RESULTS: Pooled patient samples were consistently negative or positive when tested five times on the same day and over three days of testing. The diagnostic sensitivity, specificity and accuracy of the XYL500 assay were 74, 98, and 82 % respectively, as compared with LC-MS/MS. XYL500 detected 77 of the 104 LC-MS/MS positive samples identified in our initial evaluation, including some that contained low levels of xylazine (n=8), <10 ng/mL. Minimal cross-reactivity with other opioid analgesics and commonly encountered drugs was seen with only one false positive result. Interferences by common urine contaminants were negligible. Specimens were stable up to 160 days refrigerated and up to 80 days at room temperature. CONCLUSIONS: XYL500 allows for rapid detection of xylazine, illustrating its utility in monitoring patients who ingested recreational drugs containing the additive, xylazine, and its potential to improve patient management.

A novel carboxyl polymer-modified upconversion luminescent nanoprobe for detection of prostate-specific antigen in the clinical gray zonebase by flow immunoassay strip.

Prostate specific antigen (PSA) is a widely-used biomarker for the diagnosis, screening, and prognosis of prostate cancer (PCa). It is critical to develop a rapid and convenient method to accurately detect PSA levels, especially when the PSA levels are in the clinical gray area of 4-10 ng/mL. We developed a novel upconversion nanoparticle (UCNP)-based fluorescence lateral flow test strip for qualitatively and quantitatively detecting PSA. The carboxyl group-modified UCNPs (UCNP-COOH) were labeled with anti-PSA antibodies via 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) as labeling probes to recognize PSA. The fluorescence intensity of the UCNP-probe was then measured with a laser fluorescence scanner. A total of 1397 serum and 20 fingertip blood samples were collected to validate the UCNP strip. A reliable correlation between the area ratio (TC), reflecting the fluorescence intensity of the test/control line, and the PSA concentration was observed (r = 0.9986). The dose-dependent luminescence enhancement showed good linearity in the PSA concentration range from 0.1 to 100.0 ng/mL with a detection limit of 0.1 ng/mL. Our UCNP POCT strip demonstrated excellent accuracy, anti-interference and stability in the gray zone (4-10 ng/mL) of PSA clinical application and outperformed other PSA test strips. The UCNP strip showed good consistency with the Roche chemiluminescence assay in 1397 serum samples. It also showed good performance for PSA detection using fingertip blood samples. This novel UCNP-based test strip could be a sensitive and reliable POCT assay to detect PSA, facilitating the diagnosis and surveillance of PCa.

Development of a prototypic, field-usable diagnostic tool for the detection of gram-positive cocci-induced mastitis in cattle.

BACKGROUND: Bovine mastitis is one of the most widespread diseases affecting cattle, leading to significant losses for the dairy industry. Currently, the so-called gold standard in mastitis diagnosis involves determining the somatic cell count (SCC). Apart from a number of advantages, this method has one serious flaw: It does not identify the etiological factor causing a particular infection, making it impossible to introduce targeted antimicrobial therapy. This can contribute to multidrug-resistance in bacterial species. The diagnostic market lacks a test that has the advantages of SCC and also recognizes the species of pathogen causing the inflammation. Therefore, the aim of our study was to develop a lateral flow immunoassay (LFIA) based on elongation factor Tu for identifying most prevalent Gram-positive cocci responsible for causing mastitis including Streptococcus uberis, Streptococcus agalactiae and Staphylococcus aureus. RESULTS: As a result, we showed that the assay for S. uberis detection demonstrated a specificity of 89.02%, a sensitivity of 43.59%, and an accuracy of 80.3%. In turn, the second variant - assay for Gram-positive cocci reached a specificity of 95.59%, a sensitivity of 43.28%, and an accuracy of 78.33%. CONCLUSIONS: Our study shows that EF-Tu is a promising target for LFIA and we have delivered evidence that further evaluation could improve test parameters and fill the gap in the mastitis diagnostics market.

Development and evaluation of a lateral flow-based portable optical system for determination of the pregnancy status of dairy cows.

Our objectives were to develop and evaluate an integrated system consisting of a lateral flow immunoassay (LFIA) and an electronic portable imaging device for determination of pregnancy status of cows based on plasma concentrations of pregnancy-specific protein B (PSPB). Experiment 1 was conducted to test the performance of the LFIA for PSPB (PSPB-LFIA), whereas experiment 2 was conducted to evaluate the performance of the integrated system, including both the LFIA and imaging device. The PSPB-LFIA strips were made of nitrocellulose membrane with polystreptavidin, anti-mouse antibody, europium anti-PSPB conjugates, and biotin PSPB. After adding buffer and plasma in a 96-well plate, strips were dipped to initiate flow and were read in a fluorescence microscope to estimate PSPB concentrations based on the test-to-control line signal (T/C ratio). The T/C ratio of standards was linearly associated with PSPB (R2 = 0.99 in both experiments) concentrations. To test the ability to identify pregnant cows of the PSPB-LFIA only or the integrated system, plasma samples were collected and transrectal ultrasonography (TUS) was conducted 29 to 35 d after AI in lactating Holstein cows (experiment 1: n = 83; experiment 2: n = 205). A cow was considered pregnant (Preg) if concentrations of PSPB in plasma obtained by ELISA were ≥2 ng/mL or if an embryo was visible by TUS. In experiment 1, the accuracy of the PSPB-LFIA compared with ELISA was 92.7% (91.2% sensitivity [Se]; 96.1% specificity [Sp]; 98.1% positive predictive value [PPV]; 83.3% negative predictive value [NPV]) and compared with TUS was 90.4% (100% Se; 78.9% Sp; 84.9% PPV; 100% NPV). The agreement between LFIA and ELISA (kappa = 0.84; 95% CI 0.71-0.96) or LFIA and TUS (kappa = 0.80; 95% CI 0.67-0.93) as methods to classify cows as Preg or nonpregnant (Non-Preg) was high. In experiment 2, the accuracy of the PSPB-LFIA compared with ELISA was 96.1% (93.8% Se; 100% Sp; 100% PPV; 90.5% NPV) and compared with TUS was 92.2% (99.0% Se; 84.7% Sp; 87.6% PPV; 98.8% NPV). The agreement between LFIA and ELISA (kappa = 0.92; 95% CI 0.86-0.97) or LFIA and TUS (kappa = 0.84; 95% CI 0.77-0.92) as methods to classify cows as Preg or Non-Preg was high. We conclude that a system integrating a fluorescence-based LFIA and an optical reader was effective for classifying cows as pregnant or not pregnant based on estimations of plasma concentrations of PSPB. This novel system serves as a platform for further development of on-farm pregnancy testing tools based on measurement of biomarkers of pregnancy in bodily fluids of cattle.

Hollow versatile Ag@Pt alloy nanoparticles with nanozyme activity for detection and photothermal sterilization of Helicobacter pylori.

In view of a large number of people infected with Helicobacter pylori (H. pylori) with great harm followed, there is an urgent need to develop a non-invasive, easy-to-operate, and rapid detection method, and to identify effective sterilization strategies. In this study, highly specific nanoprobes with nanozyme activity, Ag@Pt nanoparticles (NPs) with the antibody, were utilized as a novel lateral flow immunoassay (LFIA). The optical label (Ag@Pt NPs) was enhanced by the introduction of the chromogenic substrate 3,3',5,5'-tetramethylbenzidine (TMB) and compared with a gold nanoparticles (Au NPs) optical label. Under the optimal condition, Ag@Pt-LFIA and TMB-enhanced Ag@Pt-LFIA for H. pylori were successfully established, two of which were over twofold and 100-fold more sensitive than conventional visual Au NP-based LFIA, respectively. Furthermore, Ag@Pt NPs with the antibody irradiated with NIR laser (808 nm) at a power intensity of 550 mW/cm2 for 5 min exhibited a remarkable antibacterial effect. The nanoprobes could close to bacteria through effective interactions between antibodies and bacteria, thereby benefiting photothermal sterilization. Overall, Ag@Pt NPs provide promising applications in pathogen detection and therapeutic applications.

Simultaneous rapid detection of glycyrrhizin and sennoside A in Daiokanzoto samples by lateral flow immunoassay.

INTRODUCTION: Glycyrrhizin (GLY) and sennoside A (SA) are characteristic bioactive marker compounds of the Kampo medicine Daiokanzoto. Their accurate detection in blends of Rhei rhizoma and Glycyrrhizae radix of several species (4:1 or 4:2) is essential for quality control and to ensure therapeutic efficacy. A rapid, efficient assay can significantly facilitate their detection. OBJECTIVE: To establish a rapid qualitative assay for GLY and SA detection, a lateral flow immunoassay (LFA) was developed using specific monoclonal antibody (mAb) nanoparticles. METHODOLOGY: This assay harnesses the competitive binding of mAb nanoparticles to the immobilized analytes on test strips and free analytes in the samples. Two conjugates for detecting GLY and SA, GLY-bovine serum albumin and SA-human serum albumin, were separately immobilized on the test zones of LFA strips. The detection mechanism is reliant on the visual detection of color changes in the test zones. RESULTS: When GLY and SA were present in samples, they contended with the immobilized conjugates on the strip to bind with the mAb nanoparticles and produced distinct color patterns in the test zones. The limits of detection of the assay for GLY and SA were both 3.13 µg/mL. The capability of the LFA was substantiated using plant samples and Daiokanzoto, and its alignment with indirect competitive ELISA results was confirmed. CONCLUSION: The introduced LFA is a groundbreaking procedure that offers a rapid, straightforward, and sensitive method for simultaneously detecting GLY and SA in Daiokanzoto samples. It is instrumental in ensuring product quality.

Development and characterisation of highly specific monoclonal antibody-based immunoassays for the detection and quantification of genistein-7-O-[α-rhamnopyranosyl-(1→6)]-ß-glucopyranoside in Derris scandens (Roxb.) Benth.

INTRODUCTION: The stem of the plant species Derris scandens (Roxb.) Benth. (DS) contains genistein-7-O-[α-rhamnopyranosyl-(1→6)]-ß-glucopyranoside (GTG), which is a unique marker. Previous analyses of GTG using antibody-based immunoassays were compromised because of their high cross-reactivity with structurally related compounds of DS, thereby limiting their applicability in DS quality control. OBJECTIVE: Conjugation of GTG with carrier proteins was achieved using the Mannich reaction to produce a highly specific monoclonal antibody (mAb) targeting GTG (anti-GTG mAb). METHODS: The anti-GTG mAb was generated using hybridoma technology and characterised using an indirect competitive enzyme-linked immunosorbent assay (icELISA). Both lateral-flow immunoassay (LFIA) and icELISA were developed to detect and quantify GTG in DS raw materials and associated products. RESULTS: icELISA using the anti-GTG mAb showed 100% specificity for GTG, with only 1.77% cross-reactivity with genistin and less than 0.01% cross-reactivity with other compounds. icELISA demonstrated a linear range for GTG determination between 62.5 and 2000 ng/mL. The limits of detection (LOD) and quantification were 49.68 and 62.50 ng/mL for GTG, respectively. The precision of the analysis ranged from 1.28% to 4.20% for repeatability and from 1.03% to 7.05% for reproducibility. The accuracy of the analysis ranged from 101.97% to 104.01% for GTG recovery. GTG levels determined via icELISA were consistent with those confirmed via high-performance liquid chromatography (HPLC) (R2 = 0.9903). Moreover, the LOD of LFIA for GTG was 500 ng/mL. CONCLUSION: Immunoassays utilising specific anti-GTG mAbs were successfully developed, including LFIA for rapid GTG detection and icELISA for GTG quantification.

Research on the Flow Characteristics and Reaction Mechanisms of Lateral Flow Immunoassay under Non-Uniform Flow.

Lateral flow immunoassay (LFIA) is extensively utilized for point-of-care testing due to its ease of operation, cost-effectiveness, and swift results. This study investigates the flow dynamics and reaction mechanisms in LFIA by developing a three-dimensional model using the Richards equation and porous media transport, and employing numerical simulations through the finite element method. The study delves into the transport and diffusion behaviors of each reaction component in both sandwich LFIA and competitive LFIA under non-uniform flow conditions. Additionally, the impact of various parameters (such as reporter particle concentration, initial capture probe concentrations for the T-line and C-line, and reaction rate constants) on LFIA performance is analyzed. The findings reveal that, in sandwich LFIA, optimizing parameters like increasing reporter particle concentration and initial capture probe concentration for the T-line, as well as adjusting reaction rate constants, can effectively enhance detection sensitivity and broaden the working range. Conversely, in competitive LFIA, the effects are inverse. This model offers valuable insights for the design and enhancement of LFIA assays.

Au Nanoshell-Based Lateral Flow Immunoassay for Colorimetric and Photothermal Dual-Mode Detection of Interleukin-6.

Interleukin-6 (IL-6) detection and monitoring are of great significance for evaluating the progression of many diseases and their therapeutic efficacy. Lateral flow immunoassay (LFIA) is one of the most promising point-of-care testing (POCT) methods, yet suffers from low sensitivity and poor quantitative ability, which greatly limits its application in IL-6 detection. Hence, in this work, we integrated Aushell nanoparticles (NPs) as new LFIA reporters and achieved the colorimetric and photothermal dual-mode detection of IL-6. Aushell NPs were conveniently prepared using a galvanic exchange process. By controlling the shell thickness, their localized surface plasmon resonance (LSPR) peak was easily tuned to near-infrared (NIR) range, which matched well with the NIR irradiation light. Thus, the Aushell NPs were endowed with good photothermal effect. Aushell NPs were then modified with IL-6 detection antibody to construct Aushell probes. In the LFIA detection, the Aushell probes were combined with IL-6, which were further captured by the capture IL-6 antibody on the test line of the strip, forming a colored band. By observation with naked eyes, the colorimetric qualitative detection of IL-6 was achieved with limit of 5 ng/mL. By measuring the temperature rise of the test line with a portable infrared thermal camera, the photothermal quantitative detection of IL-6 was performed from 1~1000 ng/mL. The photothermal detection limit reached 0.3 ng/mL, which was reduced by nearly 20 times compared with naked-eye detection. Therefore, this Aushell-based LFIA efficiently improved the sensitivity and quantitative ability of commercial colloidal gold LFIA. Furthermore, this method showed good specificity, and kept the advantages of convenience, speed, cost-effectiveness, and portability. Therefore, this Aushell-based LFIA exhibits practical application potential in IL-6 POCT detection.

Impact of Sample Type on D-Dimer Screening.

Background: The quality of laboratory test results depends on various factors, including sample type selection. Blood samples, such as whole blood, plasma and serum are commonly used for most clinical laboratory examinations. D-dimer parameters are frequently analysed in haematology laboratories and serve as biomarkers for coagulation activation and fibrinolysis. This study aimed to assess the impact of using different sample types on the quality of D-dimer test results. Method: An observational analytical method was used. D-dimer examination was performed using the fluorescent lateral flow immunoassay method. The study sample consisted of 26 participants aged between 18 years old and 22 years old who had no blood disorders. Whole blood and ethylenediaminetetraacetic acid (EDTA) plasma samples were used for the examination of D-dimer levels. Results: D-dimer levels in 26 participants using whole blood samples had a mean value of 0.23 mg/L (230 ng/mL), while plasma samples yielded a mean value of 0.14 mg/L (140 ng/mL). D-dimer levels obtained from whole blood samples were higher than plasma samples but remained within the normal range of 0 mg/L-0.5 mg/L (0 ng/mL-500 ng/mL). Conclusion: The results showed that whole blood samples were more practical than plasma samples. Nevertheless, plasma samples gave results within the normal range of D-dimer values.

Validity of Self-testing at Home With Rapid Severe Acute Respiratory Syndrome Coronavirus 2 Antibody Detection by Lateral Flow Immunoassay.

BACKGROUND: We explore severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody lateral flow immunoassay (LFIA) performance under field conditions compared to laboratory-based electrochemiluminescence immunoassay (ECLIA) and live virus neutralization. METHODS: In July 2021, 3758 participants performed, at home, a self-administered Fortress LFIA on finger-prick blood, reported and submitted a photograph of the result, and provided a self-collected capillary blood sample for assessment of immunoglobulin G (IgG) antibodies using the Roche Elecsys® Anti-SARS-CoV-2 ECLIA. We compared the self-reported LFIA result to the quantitative ECLIA and checked the reading of the LFIA result with an automated image analysis (ALFA). In a subsample of 250 participants, we compared the results to live virus neutralization. RESULTS: Almost all participants (3593/3758, 95.6%) had been vaccinated or reported prior infection. Overall, 2777/3758 (73.9%) were positive on self-reported LFIA, 2811/3457 (81.3%) positive by LFIA when ALFA-reported, and 3622/3758 (96.4%) positive on ECLIA (using the manufacturer reference standard threshold for positivity of 0.8 U mL-1). Live virus neutralization was detected in 169 of 250 randomly selected samples (67.6%); 133/169 were positive with self-reported LFIA (sensitivity 78.7%; 95% confidence interval [CI]: 71.8, 84.6), 142/155 (91.6%; 95% CI: 86.1, 95.5) with ALFA, and 169 (100%; 95% CI: 97.8, 100.0) with ECLIA. There were 81 samples with no detectable virus neutralization; 47/81 were negative with self-reported LFIA (specificity 58.0%; 95% CI: 46.5, 68.9), 34/75 (45.3%; 95% CI: 33.8, 57.3) with ALFA, and 0/81 (0%; 95% CI: 0, 4.5) with ECLIA. CONCLUSIONS: Self-administered LFIA is less sensitive than a quantitative antibody test, but the positivity in LFIA correlates better than the quantitative ECLIA with virus neutralization.

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 Ab of 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 Ag in 100 clinical samples with 94.3% sensitivity and 90.9% specificity in 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.

Laboratory evaluation of the miniature direct-on-blood PCR nucleic acid lateral flow immunoassay (mini-dbPCR-NALFIA), a simplified molecular diagnostic test for Plasmodium.

BACKGROUND: Point-of-care diagnosis of malaria is currently based on microscopy and rapid diagnostic tests. However, both techniques have their constraints, including poor sensitivity for low parasitaemias. Hence, more accurate diagnostic tests for field use and routine clinical settings are warranted. The miniature direct-on-blood PCR nucleic acid lateral flow immunoassay (mini-dbPCR-NALFIA) is an innovative, easy-to-use molecular assay for diagnosis of malaria in resource-limited settings. Unlike traditional molecular methods, mini-dbPCR-NALFIA does not require DNA extraction and makes use of a handheld, portable thermal cycler that can run on a solar-charged power pack. Result read-out is done using a rapid lateral flow strip enabling differentiation of Plasmodium falciparum and non-falciparum malaria infections. A laboratory evaluation was performed to assess the performance of the mini-dbPCR-NALFIA for diagnosis of pan-Plasmodium and P. falciparum infections in whole blood. METHODS: Diagnostic accuracy of the mini-dbPCR-NALFIA was determined by testing a set of Plasmodium-positive blood samples from returned travellers (n = 29), and Plasmodium-negative blood samples from travellers with suspected malaria (n = 23), the Dutch Blood Bank (n = 19) and intensive care patients at the Amsterdam University Medical Centers (n = 16). Alethia Malaria (LAMP) with microscopy for species differentiation were used as reference. Limit of detection for P. falciparum was determined by 23 measurements of a dilution series of a P. falciparum culture. A fixed sample set was tested three times by the same operator to evaluate the repeatability, and once by five different operators to assess the reproducibility. RESULTS: Overall sensitivity and specificity of the mini-dbPCR-NALFIA were 96.6% (95% CI, 82.2%-99.9%) and 98.3% (95% CI, 90.8%-100%). Limit of detection for P. falciparum was 10 parasites per microlitre of blood. The repeatability of the assay was 93.7% (95% CI, 89.5%-97.8%) and reproducibility was 84.6% (95% CI, 79.5%-89.6%). CONCLUSIONS: Mini-dbPCR-NALFIA is a sensitive, specific and robust method for molecular diagnosis of Plasmodium infections in whole blood and differentiation of P. falciparum. Incorporation of a miniature thermal cycler makes the assay well-adapted to resource-limited settings. A phase-3 field trial is currently being conducted to evaluate the potential implementation of this tool in different malaria transmission areas.

A colorimetric lateral flow immunoassay based on oriented antibody immobilization for sensitive detection of SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19). The high human-to-human transmission and rapid evolution of SARS-CoV-2 have resulted in a worldwide pandemic. To contain SARS-CoV-2, it is essential to efficiently control the transmission of the virus through the early diagnosis of infected individuals, including asymptomatic people. Therefore, a rapid and accurate assay is vital for the early diagnosis of SARS-CoV-2 in suspected individuals. In this study, we developed a colorimetric lateral flow immunoassay (LFIA) in which a CBP31-BC linker was used to immobilize antibodies on a cellulose membrane in an oriented manner. The developed LFIA enabled sensitive detection of cultured SARS-CoV-2 in 15 min with a detection limit of 5 × 104 copies/mL. The clinical performance of the LFIA for detecting SARS-CoV-2 was evaluated using 19 clinical samples validated by reverse transcription-polymerase chain reaction (RT-PCR). The LFIA detected all the positive and negative samples accurately, corresponding to 100% accuracy. Importantly, patient samples with low viral loads were accurately identified. Thus, the proposed method can provide a useful platform for rapid and accurate point-of-care testing of SARS-CoV-2 in infected individuals to efficiently control the COVID-19 pandemic.