Conventional Lateral Flow Research Articles

Colorimetric stack pad immunoassay for bacterial identification

A new colorimetric immunoassay concept, utilizing conventional lateral flow membranes (e.g., conjugation, sample, absorption and nitrocellulose), were placed in a different configuration in a stacking manner, where the liquid sample that may contain the analyte diffuses from the bottom to the upper-most layer. The key element of this proprietary technology is a capture layer, where a nitrocellulose membrane is modified with the target analyte of interest, namely in this study target Escherichia coli. During the immunoassay operation, samples contaminated with the target bacteria will conjugate to their corresponding HRP-antibodies laying in wait and the immune-target measurand complex flows by capillarity towards the upper-most layer to generate a colorimetric signal (positive answer) through an enzymatic reaction. In target-free samples, previously immobilized target bacteria on the capture layer will prevent the HRP-labeled anti-target antibodies from migrating to the upper-most layer, where the enzymatic substrate lays in wait. After optimization, the sensitivity of this approach was found to be 1,000 folds higher than ELISAs (102cellsmL−1). The advantages of the stacked pad assay include: miniaturization, operational simplicity, fast response time (less than 5min), useful sensitivity.

Paper-based enzymatic microfluidic fuel cell: From a two-stream flow device to a single-stream lateral flow strip

Abstract This work presents a first approach towards the development of a cost-effective enzymatic paper-based glucose/O 2 microfluidic fuel cell in which fluid transport is based on capillary action. A first fuel cell configuration consists of a Y-shaped paper device with the fuel and the oxidant flowing in parallel over carbon paper electrodes modified with bioelectrocatalytic enzymes. The anode consists of a ferrocenium-based polyethyleneimine polymer linked to glucose oxidase (GOx/Fc-C 6 -LPEI), while the cathode contains a mixture of laccase, anthracene-modified multiwall carbon nanotubes, and tetrabutylammonium bromide-modified Nafion (MWCNTs/laccase/TBAB-Nafion). Subsequently, the Y-shaped configuration is improved to use a single solution containing both, the anolyte and the catholyte. Thus, the electrolytes pHs of the fuel and the oxidant solutions are adapted to an intermediate pH of 5.5. Finally, the fuel cell is run with this single solution obtaining a maximum open circuit of 0.55 ± 0.04 V and a maximum current and power density of 225 ± 17 μA cm −2 and 24 ± 5 μW cm −2 , respectively. Hence, a power source closer to a commercial application (similar to conventional lateral flow test strips) is developed and successfully operated. This system can be used to supply the energy required to power microelectronics demanding low power consumption.

Investigation of Reagent Delivery Formats in a Multivalent Malaria Sandwich Immunoassay and Implications for Assay Performance.

Conventional lateral flow tests (LFTs), the current standard bioassay format used in low-resource point-of-care (POC) settings, have limitations that have held back their application in the testing of low concentration analytes requiring high sensitivity and low limits of detection. LFTs use a premix format for a rapid one-step delivery of premixed sample and labeled antibody to the detection region. We have compared the signal characteristics of two types of reagent delivery formats in a model system of a sandwich immunoassay for malarial protein detection. The premix format produced a uniform binding profile within the detection region. In contrast, decoupling the delivery of sample and labeled antibody to the detection region in a sequential format produced a nonuniform binding profile in which the majority of the signal was localized to the upstream edge of the detection region. The assay response was characterized in both the sequential and premix formats. The sequential format had a 4- to 10-fold lower limit of detection than the premix format, depending on assay conjugate concentration. A mathematical model of the assay quantitatively reproduced the experimental binding profiles for a set of rate constants that were consistent with surface plasmon resonance measurements and absorbance measurements of the experimental multivalent malaria system.

Development of a Dual Path Platform (DPP®) immunoassay for rapid detection of Candida albicans in human whole blood and serum

Candida albicans is an opportunistic pathogen which can lead to Candidiasis and blood-stream infections, resulting in a mortality rate near 40%. Given its high fatality and emerging pathogenicity, there is a strong need for the development of a rapid C. albicans diagnostic assay. Point-of-care devices, specifically lateral flow assays, are an attractive and often employed diagnostic modality for C. albicans detection. However, they lack the required performance characteristics needed for accurate pathogen detection and subsequent treatment options. Thus, we describe herein the utility of the Dual Path Platform (DPP®) device as an immunochromatographic Point-of-care assay for C. albicans. The limit of detection for hyphal and budding C. albicans in DPP® tests are reported to be as low as 7.94×105 whole cells/mL in human serum. C. albicans cells were detected with up to a 3.9 fold increase in sensitivity on DPP® when compared to conventional lateral flow modalities.

Novel strategies to enhance lateral flow immunoassay sensitivity for detecting foodborne pathogens.

Food contaminated by foodborne pathogens causes diseases, affects individuals, and even kills those affected individuals. As such, rapid and sensitive detection methods should be developed to screen pathogens in food. One current detection method is lateral flow immunoassay, an efficient technique because of several advantages, including rapidity, simplicity, stability, portability, and sensitivity. This review presents the format and principle of lateral flow immunoassay strip and the development of conventional lateral flow immunoassay for detecting foodborne pathogens. Furthermore, novel strategies that can be applied to enhance the sensitivity of lateral flow immunoassay to detect foodborne pathogens are presented; these strategies include innovating new label application, designing new formats of lateral flow immunoassay, combining with other methods, and developing signal amplification systems. With these advancements, detection sensitivity and detection time can be greatly improved.

Use of gold nanoparticle-labeled secondary antibodies to improve the sensitivity of an immunochromatographic assay for aflatoxin B1

We describe a sensitive method for the immunochromatographic determination of aflatoxin B1. It is based on the following steps: 1) Competitive interaction between non-labeled specific primary antibodies and target antigens in a sample and in the test zone of a membrane; 2) detection of the immune complexes on the membrane by using a secondary antibodies labeled with gold nanoparticles. The method enables precise adjustment of the required quantities of specific antibodies and the colloidal (gold) marker. It was applied in a lateral flow format to the detection of aflatoxin B1 and exhibits a limit of detection (LOD) of 160 pg · mL−1 if detected visually, and of 30 pg · mL−1 via instrumental detection. This is significantly lower than the LOD of 2 ng · mL−1 achieved by conventional lateral flow analysis using the same reagents.

Smartphone based bacterial detection using biofunctionalized fluorescent nanoparticles

We are describing immunochromatographic test strips with smart phone-based fluorescence readout. They are intended for use in the detection of the foodborne bacterial pathogens Salmonella spp. and Escherichia coli O157. Silica nanoparticles (SiNPs) were doped with FITC and Ru(bpy), conjugated to the respective antibodies, and then used in a conventional lateral flow immunoassay (LFIA). Fluorescence was recorded by inserting the nitrocellulose strip into a smart phone-based fluorimeter consisting of a light weight (40 g) optical module containing an LED light source, a fluorescence filter set and a lens attached to the integrated camera of the cell phone in order to acquire high-resolution fluorescence images. The images were analysed by exploiting the quick image processing application of the cell phone and enable the detection of pathogens within few minutes. This LFIA is capable of detecting pathogens in concentrations as low as 105 cfu mL−1 directly from test samples without pre-enrichment. The detection is one order of magnitude better compared to gold nanoparticle-based LFIAs under similar condition. The successful combination of fluorescent nanoparticle-based pathogen detection by LFIAs with a smart phone-based detection platform has resulted in a portable device with improved diagnosis features and having potential application in diagnostics and environmental monitoring.

Capillary-driven surface-enhanced Raman scattering (SERS)-based microfluidic chip for abrin detection

Herein, we firstly demonstrate the design and the proof-of-concept use of a capillary-driven surface-enhanced Raman scattering (SERS)-based microfluidic chip for abrin detection. The micropillar array substrate was etched and coated with a gold film by microelectromechanical systems (MEMS) process to integrate into a lateral flow test strip. The detection of abrin solutions of various concentrations was performed by the as-prepared microfluidic chip. It was shown that the correlation between the abrin concentration and SERS signal was found to be linear within the range of 0.1 ng/mL to 1 μg/mL with a limit of detection of 0.1 ng/mL. Our microfluidic chip design enhanced the operability of SERS-based immunodiagnostic techniques, significantly reducing the complication and cost of preparation as compared to previous SERS-based works. Meanwhile, this design proved the superiority to conventional lateral flow test strips in respect of both sensitivity and quantitation and showed great potential in the diagnosis and treatment for abrin poisoning as well as on-site screening of abrin-spiked materials.

Paper-based analytical devices for point-of-care infectious disease testing

Paper-based devices provide an alternative technology for simple, low-cost, portable, and disposable or recyclable diagnostic tools for many applications, including clinical diagnosis, food quality control, and environmental monitoring. The present review focuses on new paper-based tests for point-of-care (POC) infectious disease testing. This review provides a brief presentation of the fabrication techniques and the main sample preparation procedures. Recent immunological and molecular testing formats based on new paper-based solutions which go beyond conventional lateral flow formats are also added. Emphasis is placed on how paper systems could be used for detecting whole and viable bacteria associated to infectious diseases. Paper has recently become attractive, since it is a ubiquitous and extremely cheap material. It is easy to store, easy to use, and is compatible with many (bio)chemical and (bio)medical applications. Paper absorbs and transports liquids by capillary force without additional mechanical assistance. Hence, paper-based analytical devices are promising and possibly game-changing, even if they still suffer from limitations, including accuracy and sensitivity. It is anticipated that, in the near future, with advances in fabrication procedures and associated analytical techniques, there will be a continuous flow of innovative paper-based diagnostics kits.

A highly sensitive europium nanoparticle-based lateral flow immunoassay for detection of chloramphenicol residue

A europium nanoparticle-based lateral flow immunoassay for highly sensitive detection of chloramphenicol residue was developed. The detection result could be either qualitatively resolved with naked eye or quantitatively analyzed with the assistance of a digital camera. In the qualitative mode, the limit of detection (LOD) was found to be 0.25ng/mL. In the quantitative mode, the half-maximal inhibition concentration (IC50) was determined to be 0.45ng/mL and the LOD can reach an ultralow level of 0.03ng/mL, which is ~100 times lower than that of the conventional colloidal gold-based lateral flow immunoassay. Potential application of the established method was demonstrated by analyzing representative cow milk samples.

Lateral Flow Immunoassay with the Signal Enhanced by Gold Nanoparticle Aggregates Based on Polyamidoamine Dendrimer

In order to amplify the signal in a gold nanoparticle-based lateral flow immunoassay, a simple and sensitive method utilizing gold nanoparticle aggregates as a colored reagent formed with a polyamidoamine dendrimer was developed. The results were compared with that achieved by employing the individual nanoparticles used in the conventional lateral flow immunoassay. Under the optimized experimental conditions, a detection limit of 0.1 ng mL⁻¹ for rabbit immunoglobulin G was achieved, which is almost 20-fold lower than that of the traditional method using individual gold nanoparticles. We believe that this simple, practical bioassay platform will be of interest for use in areas such as disease diagnostics, pathogen detection, and quality monitoring of food and water.

Functionalized viral nanoparticles as ultrasensitive reporters in lateral-flow assays

Two types of viral nanoparticles were functionalized with target-specific antibodies and multiple copies of an enzymatic reporter (horseradish peroxidase). The particles were successfully integrated into an immunochromatographic assay detecting MS2 bacteriophage, a model for viral pathogens. The sensitivity of the assay was greatly superior to conventional gold nanoparticle lateral flow assays, and results could easily be evaluated, even without advanced lab instruments.

Two-Dimensional Paper Network Format That Enables Simple Multistep Assays for Use in Low-Resource Settings in the Context of Malaria Antigen Detection

The lateral flow test has become the standard bioassay format in low-resource settings because it is rapid, easy to use, and low in cost, uses reagents stored in dry form, and is equipment-free. However, lateral flow tests are often limited to a single chemical delivery step and not capable of the multistep processing characteristic of high performance laboratory-based assays. To address this limitation, we are developing a paper network platform that extends the conventional lateral flow test to two dimensions; this allows incorporation of multistep chemical processing, while still retaining the advantages of conventional lateral flow tests. Here, we demonstrate this format for an easy-to-use, signal-amplified sandwich format immunoassay for the malaria protein PfHRP2. The card contains reagents stored in dry form such that the user need only add sample and water. The multiple flows in the device are activated in a single user step of folding the card closed; the configuration of the paper network automatically delivers the appropriate volumes of (i) sample plus antibody conjugated to a gold particle label, (ii) a rinse buffer, and (iii) a signal amplification reagent to the capture region. 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.

Chemiluminescence-based biosensor for fumonisins quantitative detection in maize samples

A compact portable chemiluminescent biosensor for simple, rapid, and ultrasensitive on-site quantification of fumonisins (fumonisin B1 + fumonisin B2) in maize has been developed. The biosensor integrates a competitive lateral flow immunoassay based on enzyme-catalyzed chemiluminescence detection and a highly sensitive portable charge-coupled device (CCD) camera, employed in a contact imaging configuration. The use of chemiluminescence detection allowed accurate and objective analyte quantification, rather than qualitative or semi-quantitative information usually obtained employing conventional lateral flow immunoassays based on colloidal gold labeling. A limit of detection of 2.5 μg L −1 for fumonisins was achieved, with an analytical working range of 2.5–500 μg L −1 (corresponding to 25–5000 μg kg −1 in maize flour samples, according to the extraction procedure). Total assay time was 25 min, including sample preparation. A simple and convenient extraction procedure, performed by suspending the sample in a buffered solution and rapidly heating to eliminate endogenous peroxidase enzyme activity was employed for maize flour samples analysis, obtaining recoveries in the range 90–115%, when compared with LC–MS/MS analysis. The chemiluminescence immunochromatography-based biosensor is a rapid, low cost portable test suitable for point-of-use applications.

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.

Chemical signal amplification in two-dimensional paper networks

Two-dimensional paper networks (2DPNs) hold great potential for extending the utility of paper-based chemical and biochemical diagnostics at a cost and ease-of-use that are comparable to conventional lateral flow strips. 2DPNs enable the automated sequential delivery of multiple reagents to a detection region with a single user activation step, and therefore have the potential to extend the processing capabilities of inexpensive paper-based assays with comparable ease of use to conventional lateral flow tests. In this communication, we used a simple 3 inlet 2DPN to perform signal amplification of a colloidal gold label using a gold enhancement solution, thus demonstrating the capability of 2DPNs to perform processes that can improve limits of detection.

Aptamer-Functionalized Gold Nanoparticles as Probes in a Dry-Reagent Strip Biosensor for Protein Analysis

The highly specific molecular recognition properties of aptamers are combined with the unique optical properties of gold nanoparticles for the development of a dry-reagent strip biosensor that enables qualitative (visual)/quantitative detection of protein within minutes. A model system comprising thrombin as an analyte and a pair of aptamer probes is used to demonstrate the proof-of-concept on the conventional lateral flow test strip. The assay avoids the multiple incubation and washing steps performed in most current aptamer-based protein analyses. Although qualitative tests are realized by observing the color change of the test zone, quantitative data are obtained by recording the optical responses of the test zone with a portable "strip reader". The response of the biosensor is linear over the range of 5-100 nM of thrombin with a detection limit of 2.5 nM (S/N = 3). By comparing the analytical performances of the aptamer-based strip biosensor with the antibody-based strip sensor, we can demonstrate that aptamers are equivalent or superior to antibodies in terms of specificity and sensitivity, respectively. The sensor was used successfully for detection of thrombin in human plasma samples. It shows great promise for use of aptamer-functionalized gold nanoparticle probes in dry-reagent strip biosensors for point-of-care or in-field detection of proteins.

Improving the Sensitivity of Immunoassays by Tuning Gold Nanoparticles to the Tipping Point

Conventional lateral flow immunoassays are based on labeled antibodies. In this paper we describe an alternative design based on gold nanoparticles labeled with haptens. The haptens are conjugated to gold nanoparticles by a method that allows the number per particle to be tuned to the point of maximum sensitivity. This leads to improvements compared with conventional lateral flow devices without relinquishing any of their advantages. In parallel assays for the environmental pollutant 2,4-dinitrophenol the alternative devices were 50% more sensitive.