Conventional Lateral Flow Assay Research Articles

Highly sensitive detection of high-risk bacterial pathogens using SERS-based lateral flow assay strips

Bacterial pathogens such as Yersinia pestis, Francisella tularensis, and Bacillus anthracis are classified into the highest rank of potential bioterrorism agents. Colorimetric lateral flow assay (LFA) strips are commercially available but these conventional strips have drawbacks in terms of low sensitivity and limit of quantitative analysis. Therefore, there is an urgent need for a new sensing platform to detect these pathogens in the early contamination stage. In this study, a novel surface-enhanced Raman scattering (SERS)-based LFA strip was developed for sensitive detection of bacterial pathogens. Target-specific SERS nanotags (Raman reporter-labeled gold nanoparticles) were used as an alternative to the gold nanoparticles in conventional LFA strips. Using these SERS nanotags the presence of bacteria could be identified through a simple color change in the test line. Additionally, highly sensitive and accurate quantitative analysis could be performed by monitoring the characteristic Raman peak intensity of SERS nanotags that were captured in the test line. This highly sensitive method required a short assay time (15 min) and a tiny volume of pathogen sample (40 μL). We believe that the proposed SERS-based LFA technique has great potential as a valuable tool in the early detection of specific bacterial pathogens in the field due to its excellent analytical sensitivity.

Improved LFIAs for highly sensitive detection of BNP at point-of-care.

Heart failure (HF) has become a major cause of morbidity and mortality with a significant global economic burden. Although well-established clinical tests could provide early diagnosis, access to these tests is limited in developing countries, where a relatively higher incidence of HF is present. This has prompted an urgent need for developing a cost-effective, rapid and robust diagnostic tool for point-of-care (POC) detection of HF. Lateral flow immunoassay (LFIA) has found widespread applications in POC diagnostics. However, the low sensitivity of LFIA limits its ability to detect important HF biomarkers (e.g., brain natriuretic peptide [BNP]) that are normally present in low concentration in blood. To address this issue, we developed an improved LFIA by optimizing the gold nanoparticle (GNP)–antibody conjugate conditions (e.g., the conjugate pH and the amount of added antibody), the diameter of GNP and the concentration of antibody embedded on the test line and modifying the structure of test strip. Through these improvements, the proposed test strip enabled the detection of BNP down to 0.1 ng/mL within 10–15 min, presenting ~15-fold sensitivity enhancement over conventional lateral flow assay. We also successfully applied our LFIA in the analysis of BNP in human serum samples, highlighting its potential use for clinical assessment of HF. The developed LFIA for BNP could rapidly rule out HF with the naked eye, offering tremendous potential for POC test and personalized medicine.

Multiple test zones for improved detection performance in lateral flow assays

Lateral flow assays (LFAs) have found applications in clinical diagnostics, veterinary medicine, food safety, environmental monitoring, bio-defence, drug abuse, etc. However, conventional LFAs with single test zone for one analyte (sLFA) show qualitative, positive/negative results (that are usually not so accurate for decision-making) and suffer from the hook effect (that easily induces false negative results). In this study, a modified LFA with multiple test zones for one analyte (mLFA) is developed for detection of nucleic acid showing two methods to interpret assay results. One is the sum of detection signals of test zones, which exhibits a stable and broader detection range compared with sLFA. The other is the pattern of test zones that is presented is correlated to the concentration of analyte, facilitating semi-quantitative detection without the aid of a reader or analyzer.

A three-line lateral flow assay strip for the measurement of C-reactive protein covering a broad physiological concentration range in human sera

The lateral flow assay (LFA) strip sensor possesses many advantages as a diagnostic device, including the capabilities of rapid, one-step assay performance, and high throughput production. A major limitation of the sensor, however, is its difficulty in measuring a broad concentration range of target proteins, including C-reactive protein (CRP), due to the “hook effect.” In this study, we report the use of a three-line LFA strip sensor, adding an antigen line to the conventional two-line LFA sensor, for detecting CRP within a broad concentration range in human sera. We introduced an antigen line between test and control lines in the LFA sensor. The antigen line was formed by dispensing a CRP antibody solution followed by a CRP solution in nitrocellulose membrane. All other conditions were identical to those applied to the conventional LFA strip sensor. The CRP level in test samples was generated by data processing from the intensities of three lines. The strip sensor measured a linear detection range of CRP concentration from 1ng/mL to 500μg/mL within 10min, with a calculated detection range of 0.69ng/mL–1.02mg/mL. Using the developed three-line LFA sensor, 50 clinical samples were measured at a detection range of 0.4–84.7μg/mL. This novel and easy-to-use CRP sensor can be a useful tool for rapid, sensitive, and cost-effective detection of a broad physiological concentration range of CRP capabilities that are vital for various diagnostic applications.

Comparison of conventional lateral-flow assays and a new fluorescent immunoassay to detect influenza viruses

Sofia, a novel, fluorescent lateral-flow immunoassay was compared with two conventional colorimetric assays, Quickvue Influenza A+B and Directigen FLU A+B, to identify influenza viral antigen from patient nasopharyngeal specimens. A total of 118 frozen original influenza-positive specimens and 57 prospective specimens were examined. Using rt-PCR as a referee assay, sensitivity values (%) for influenza A/B of 80.0/74.8, 73.3/59.3 and 73.3/40.7 were obtained using the Sofia, Quickvue and Directigen assays, respectively. All assays demonstrated reduced sensitivity for influenza B as compared with influenza A virus. With respect to the Sofia assay, the sensitivity of influenza B for the Directigen assay was significantly diminished.False positive results were not observed in the Sofia and Directigen assays. The Quickvue assay produced 3 false-positive results (2 influenza A and 1 influenza B) resulting in a specificity (%) of 96 and 98 for influenza A and B, respectively. Cross-reactivity to other respiratory viruses was not observed among immunoassays. A sensitivity rank (highest to low) of rt-PCR>culture>Sofia>Quickvue>Directigen was established using dilutions of influenza A and B. Sofia provides enhanced sensitivity and objective result interpretation over conventional colorimetric immunoassays.

Enhanced Sensitivity of Lateral Flow Tests Using a Two-Dimensional Paper Network Format

Point-of-care diagnostic assays that are rapid, easy-to-use, and low-cost are needed for use in low-resource settings; the lateral flow test has become the standard bioassay format in such settings because it meets those criteria. However, for a number of analytes, conventional lateral flow tests lack the sensitivity needed to have clinical utility. To address this limitation, we are developing a paper network platform that extends the conventional lateral flow test to two dimensions. The two-dimensional structures allow incorporation of multistep processes for improved sensitivity, while still retaining the positive aspects of conventional lateral flow tests. Here we create an easy-to-use, signal-amplified immunoassay based on a modified commercial strip test for human chorionic gonadotropin, the hormone used to detect pregnancy, and demonstrate an improved limit of detection compared to a conventional lateral flow assay. These results highlight the potential of the paper network platform to enhance access to high-quality diagnostic capabilities in low-resource settings in the developed and developing worlds.

A dual gold nanoparticle conjugate-based lateral flow assay (LFA) method for the analysis of troponin I

For signal amplification without an additional operation step in a gold nanoparticle (AuNP)-based lateral flow assay (LFA), a new and simple method utilizing two AuNP-antibody conjugates was developed. The 1st conjugate was the AuNP immobilized with an anti-troponin I antibody and blocked with bovine serum albumin (BSA), and the 2nd conjugate was the AuNP immobilized with an anti-BSA antibody and blocked with human serum albumin. The two conjugates were encapsulated in different pads, respectively. A scheme of the LFA system is described in the part A of first figure. The size of the two conjugates was very critical in the detection sensitivity of troponin I. When 10 nm for the 1st and 40 nm for the 2nd were used, the detection sensitivity increased about a 100-fold compared to the conventional LFA. We could detect as low as 0.01 ng/mL troponin I in 10 min using the dual AuNP conjugate-based LFA, which was successfully applied in the analysis of serum samples of patients with myocardial infarction.