An ASIC based strip reader controller in 0.18 μm CMOS technology for lateral flow immunoassay

As the COVID-19 pandemic moves towards endemic status, testing strategies are being de-escalated. A rapid and effective point of care test (POCT) assessment of SARS-CoV-2 immune responses can inform clinical decision-making and epidemiological monitoring of the disease. This cross-sectional seroprevalence study of anti-SARS-CoV-2 antibodies in Irish healthcare workers assessed how rapid anti-SARS-CoV-2 antibody testing can be compared to a standard laboratory assay, discusses its effectiveness in neutralisation assessment and its uses into the future of the pandemic. A point of care lateral flow immunoassay (LFA) detecting anti-SARS-CoV-2 spike (S)-receptor binding domain (RBD) neutralising antibodies (Healgen SARS-CoV-2 neutralising Antibody Rapid Test Cassette) was compared to the Roche Elecsys/-S anti-SARS-CoV-2 antibody assays and an in vitro surrogate neutralisation assay. A correlation between anti-spike (S), anti-nucleocapsid (N) titres, and in vitro neutralisation was also assessed. 1,777 serology samples were tested using Roche Elecsys/-S anti-SARS-CoV-2 assays to detect total anti-N/S antibodies. 1,562 samples were tested using the POC LFA (including 50 negative controls), and 90 samples were tested using an in vitro ACE2-RBD binding inhibition surrogate neutralisation assay. The POCT demonstrated 97.7% sensitivity, 100% specificity, a positive predictive value (PPV) of 100%, and a negative predictive value (NPV) of 61% in comparison to the commercial assay. Anti-S antibody titres determined by the Roche assay stratified by the POC LFA result groups demonstrated statistically significant differences between the "Positive" and "Negative" LFA groups (p < 0.0001) and the "Weak Positive" and "Positive" LFA groups (p < 0.0001). No statistically significant difference in ACE2-RBD binding inhibition was demonstrated when stratified by the LFA POC results. A positive, statistically significant correlation was demonstrated between the in vitro pseudo-neutralisation assay results and anti-S antibody titres (rho 0.423, p < 0.001) and anti-N antibody titres (rho = 0.55, p < 0.0001). High sensitivity, specificity, and PPV were demonstrated for the POC LFA for the detection of anti-S-RBD antibodies in comparison to the commercial assay. The LFA was not a reliable determinant of the neutralisation capacity of identified antibodies. POC LFA are useful tools in sero-epidemiology settings, pandemic preparedness and may act as supportive tools in treatment decisions through the rapid identification of anti-Spike antibodies.

A rapid and sensitive lateral flow immunoassay (LFIA) test for the on-site detection of banana bract mosaic virus in banana plants

To date, a specific point-of-care test (POCT) for 3,4-methylenedioxymethamphetamine (MDMA, ecstasy, 'E') in latent fingerprints (LFPs) has not been explored. Other POCTs identify MDMA in sweat by detecting the drug as a cross-reactant rather than target analyte, thus decreasing the test's sensitivity. The study's aim was to design a sensitive POCT for the detection of MDMA in LFPs using surface plasmon resonance (SPR) and lateral flow immunoassay (LFA) technology. A high-affinity antibody binding pair was identified using the former technique, deeming the pair suitable for a LFA. Titrations of fluorescently labelled antibody and antigen concentrations were tested to identify a sharp drop-in signal upon the addition of MDMA to allow a clear distinction between negative and positive outcomes. We trialled the LFA by producing dose response curves with MDMA and a group of drugs that share a similar chemical structure to MDMA. These were generated through spiking the LFA with increasing levels of drug (0-400 pg/10μl of MDMA; 0-10,000 pg/10μl of cross-reactant). Fluorescent test signals were measured using a cartridge reader. The cut-off (threshold) 60 pg/10μl calculated better cartridge performance (1.00 sensitivity, 0.95 specificity and 0.98 accuracy), when compared with 40 pg/10μl. The biggest cross-reactant was PMMA (250%), followed by MDEA (183%), MBDB (167%), MDA (16%) and methamphetamine (16%). A sensitive LFP screening tool requiring no sample preparation was successfully designed.

The lateral flow immunoassay (LFIA) is the most successful point-of-care testing (POCT) method to date. In the case of clinical biomarkers that require quantification, it remains a challenge to quantitate those biomarkers using the lateral flow immunoassay remains a challenge due to the cost of the reader and possibly the type of marker used. In the present work, a new concept of a platform LFIA device configuration is proposed in which different, aligned membrane components, some already existing in the classical lateral flow immunoassay, and the others created with special new functions in the present device. As the sample containing the target analyte passes through the aforementioned membranes, the target analyte will initially interact with a target-specific antibody-conjugated to horseradish peroxidase (HRP). Thereafter, the newly formed immunocomplex will diffuse through a proprietary capture membrane (that ensures that the nontarget-bound antibodies do not continue further and thus remain “captured” to that specific area). This is done by having the target molecules (or components thereof) immobilized onto the said capture layer. The target-bound immunocomplexes will then be allowed by the system configuration to continue further to the last layer, where the signal will be generated and quantified. Thus, in the absence of the target analyte in the sample, the free antibodies will be filtered at the capture layer by preimmobilized analyte molecules, thus preventing a false positive signal to occur. We validated the concept in the detection of dengue NS1 protein in view of making a triage test. The sample containing NS1 will first meet HRP-conjugated NS1-specific antibodies and become attached, thus producing an NS1-specific antibody–HRP immunocomplex. The sample then flows through the blocking layer, where the immunocomplex is unchallenged and thus allowed to reach the last “absorbent” pad, incorporating the substrate for the HRP marker. In the case of a positive test, a signal is generated, that is proportional to the amount of immunocomplexes (and therefore the NS1 concentration), and then analyzed and measured at the absorbent pad. Any unbound anti-NS1 antibody will be stopped at the blocking matrix by preimmobilized NS1, so there will be no false positive. As this study is the initial study of a novel configuration, much of the work comprised of optimization steps, such as determining the required NS1 membrane-immobilization concentration and the required target-specific capture antibody concentration. Our immunoassay was tested with spiked buffer and serum samples to mimic the clinical conditions, with a range of NS1 concentrations, and was found, at this time, to be fivefold more sensitive than a gold standard enzyme-linked immunosorbent assay (ELISA) test (5 ng mL–1) performed in our laboratory. This method shows another form of LFIA that has the potential to be quantitative (at least semiquantitative), albeit not solving the reader cost; however, unlike the regular LFIA, we do not use nanobeads but instead enzymes, allowing, in theory, greater sensitivity, while retaining the one-step procedure. The test is accurate and has low production costs.

Nanoengineered plasmonics-enhanced photothermal tags for sensitive detection of cardiac biomarker troponin I using lateral flow immunoassay

Lateral flow immunoassay (LFIA) is a vital point-of-care testing (POCT) technique that is widely used for on-site detection and in vitro diagnosis. Many diseases, particularly cardiovascular diseases (CVDs), require the quantitative detection of multiple biomarkers across a broad dynamic range. Recurrent viral infections like COVID-19 elevate CVDs risk, necessitating early warning and prognostic monitoring, especially based on POCT of multiple blood biomarkers of D-dimer, NT-proBNP, and cTnI across a broad range from pg/mL to μg/mL. Traditional LFIAs lack ultrasensitivity and broad-range quantification. We design the surface chemistry-mediated template-free assembly of metal-aggregation-induced emission luminogens (AIEgen) framework@gold nanoparticles (MAF@AuNPs) for developing ultrasensitive and dynamic dual-quantitative LFIA (ddLFIA). The template-free self-assembled MAF@AuNPs have strong fluorescent and plasmonic properties, avoiding complicated synthesis, chemical modifications, and covalent conjugations, thus leading to highly convenient and versatile visual POCT. MAF@AuNPs ddLFIA achieves a normal detection at ng/mL using chromogenic AuNPs and ultrasensitive detection at pg/mL using fluorogenic MAFs, for which the fluorescence signal is 102-103 times more sensitive than the color signal. MAFs and AuNPs are functionally complementary to form a dynamic, broad-range quantification system covering over 5 orders of magnitude, at the same time having a naked eye sensitivity near 1 pg/mL. The visual POCT using MAF@AuNPs ddLFIA aligns well with clinical chemiluminescent assays. MAF@AuNPs ddLFIA perfectly matches ultrasensitive and broad-range multiple biomarker detections for clinical CVDs diagnosis.

Currently, the global trend of several hundred thousand new confirmed COVID-19 patients per day has not abated significantly. Serological antibody detection has become an important tool for the self-screening of people. While the most commonly used colorimetric lateral flow immunoassay (LFIA) methods for the detection of COVID-19 antibodies are limited by low sensitivity and a lack of quantification ability. This leads to poor accuracy in the screening of early COVID-19 patients. Therefore, it is necessary to develop an accurate and sensitive autonomous antibody detection technique that will effectively reduce the COVID-19 infection rate. Here, we developed a three-line LFIA immunoassay based on polydopamine (PDA) nanoparticles for COVID-19 IgG and IgM antibodies detection to determine the degree of infection. The PDA-based three-line LFIA has a detection limit of 1.51 and 2.34 ng/mL for IgM and IgG, respectively. This assay reveals a good linearity for both IgM and IgG antibodies detection and is also able to achieve quantitative detection by measuring the optical density of test lines. In comparison, the commercial AuNP-based LFIA showed worse quantification results than the developed PDA-based LFIA for low-concentration COVID-19 antibody samples, making it difficult to distinguish between negative and positive samples. Therefore, the developed PDA-based three-line LFIA platform has the accurate quantitative capability and high sensitivity, which could be a powerful tool for the large-scale self-screening of people.

Long afterglow nanoprobes labeled image enhancement using deep learning in rapid and sensitive lateral flow immunoassay

Anon-site detection strategy is reportedbased on dual-color SiO2@quantum dot (QD)-integrated lateral flow immunoassay (LFA) strip to realize the quantitative and simultaneous detection of C-reactive protein (CRP) and procalcitonin (PCT) in serum. The dual-color SiO2@QD nanotags with monodispersity and excellent luminescence were synthesized using polyethyleneimine-mediated electrostatic adsorption of dense red CdSe/ZnS-COOH (excitation/emission 365/625nm) or green CdSe/ZnS-COOH (excitation/emission 365/525nm) QDs on the surface of 180nm SiO2 spheres and were conjugated with anti-PCT and anti-CRP monoclonal antibodies, as stable and fluorescent-enhanced QD nanotags in the LFA system. The use of SiO2@QDs with two different fluorescent signals causedthe sensitivity and specificity of themultiplex LFA system. As a result, the proposed assay provided a wide logarithmic determinationrange with a CRP quantitative range of 0.5-103ng/mL and PCT quantitative range of 0.05-103ng/mL. The limits of detection (LODs) of CRP and PCT reached 0.5 and 0.05ng/mL, respectively. The SiO2@QD-based LFA showed great potential as rapid detection tool for the simultaneous monitoring of CRP and PCT in serum sample.

Dual recombinase polymerase amplification system combined with lateral flow immunoassay for simultaneous detection of Staphylococcus aureus and Vibrio parahaemolyticus

MAAN-eLFA: A portable electrical lateral flow assay enabled by cooperative MXene@Au-Ag nanowires networks for H1N1 detection.

Performance enhancement of the lateral flow immunoassay by use of composite nanoparticles as signal labels

Lateral flow immunoassay (LFIA) is an important detection method in vitro diagnosis, which has been widely used in medical industry. It is difficult to analyze all peak shapes through classical methods due to the complexity of LFIA. Classical methods are generally some peak-finding methods, which cannot distinguish the difference between normal peak and interference or noise peak, and it is also difficult for them to find the weak peak. Here, a novel method based on deep learning was proposed, which can effectively solve these problems. The method had two steps. The first was to classify the data by a classification model and screen out double-peaks data, and second was to realize segmentation of the integral regions through an improved U-Net segmentation model. After training, the accuracy of the classification model for validation set was 99.59%, and using combined loss function (WBCE + DSC), intersection over union (IoU) value of segmentation model for validation set was 0.9680. This method was used in a hand-held fluorescence immunochromatography analyzer designed independently by our team. A Ferritin standard curve was created, and the T/C value correlated well with standard concentrations in the range of 0–500 ng/ml (R2 = 0.9986). The coefficients of variation (CVs) were ≤ 1.37%. The recovery rate ranged from 96.37 to 105.07%. Interference or noise peaks are the biggest obstacle in the use of hand-held instruments, and often lead to peak-finding errors. Due to the changeable and flexible use environment of hand-held devices, it is not convenient to provide any technical support. This method greatly reduced the failure rate of peak finding, which can reduce the customer’s need for instrument technical support. This study provided a new direction for the data-processing of point-of-care testing (POCT) instruments based on LFIA.

Lateral flow immunoassay (LFIA), as a prominent point-of-care (POC) test platform, has been extensively adopted for rapid, on-site, and facile diagnosis of pathogen infections and disease biomarkers. Exploring novel structured optical labels of LFIA with amplified signal and complementary detection modes favors the sensitive and flexible POC diagnosis. Here, bimodal labels with both colorimetric and fluorescent readout were fabricated via a layered sequential assembly strategy based on affinity templates and hydrophobic metal-containing nanounits. High-quality colorimetric and fluorescent nanoparticles were densely incorporated into the colloidal supports and confined in separated regions, without interfering with each other. The hierarchical integration of gold nanoparticles and quantum dots with high loading density and good optical preservation realized dual readout and amplified signals from the assemblies of individual single nanoparticles. The "all-in-one" optical labels allowed both colorimetric and fluorescent detection of cystatin C (Cys C) after surface conjugation with antibodies. The LFIA strips revealed noninterfering dual signals for both visual inspection and quantitative detection of Cys C via the naked eye and portable devices, respectively. The limits of detection by colorimetric and fluorescent modes were 0.61 and 0.24 ng mL-1, respectively. The novel LFIA platform demonstrated sensitive, specific, and reproducible POC testing of biomarkers with flexible detection modes and was reliable for clinical diagnosis.

Highly active antiretroviral therapy (HAART) includes very potent drugs that are often characterized by high toxicity. Tenofovir (TFV) is a widely used drug prescribed mainly for pre-exposure prophylaxis (PreP) and the treatment of human immunodeficiency virus (HIV). The therapeutic range of TFV is narrow, and adverse effects occur with both underdose and overdose. The main factor contributing to therapeutic failure is the improper management of TFV, which may be caused by low compliance or patient variability. An important tool to prevent inappropriate administration is therapeutic drug monitoring (TDM) of compliance-relevant concentrations (ARCs) of TFV. TDM is performed routinely using time-consuming and expensive chromatographic methods coupled with mass spectrometry. Immunoassays, such as enzyme-linked immunosorbent assays (ELISAs) and lateral flow immunoassays (LFIAs), are based on antibody–antigen specific recognition and represent key tools for real-time quantitative and qualitative screening for point-of-care testing (POCT). Since saliva is a non-invasive and non-infectious biological sample, it is well-suited for TDM. However, saliva is expected to have a very low ARC for TFV, so tests with high sensitivity are required. Here, we have developed and validated a highly sensitive ELISA (IC50 1.2 ng/mL, dynamic range 0.4–10 ng/mL) that allows the quantification of TFV in saliva at ARCs and an extremely sensitive LFIA (visual LOD 0.5 ng/mL) that is able to distinguish between optimal and suboptimal ARCs of TFV in untreated saliva.