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Lateral Flow Tests Involving Fluorescent Dyes

IP.com Disclosure Number: IPCOM000242165D
Publication Date: 2015-Jun-22

Publishing Venue

The IP.com Prior Art Database

Abstract

Here we describe a simple/highly sensitive detection technology, method and device for point-of-care testing using a novel lateral-flow system. The detection scheme is based upon fluorescent dyes (for example, but not limited to cyanine dyes) in combination with specific analyte binding moieties e.g. antigens, recombinant proteins, antibodies, affimers or aptamers. The system provides a universal detection technology for lateral-flow devices for infectious agents such as, but not limited to, influenza, Ebola Haemorrhagic fever or HIV. The lateral flow device described consists of a membrane to carry the sample from the application pad, through a conjugate release pad up to an absorbent pad. The latter is located at the apex of the device. The membrane is composed of a polymeric material which is attached to a base layer (e.g. polyester, polyvinyl or polystyrene self-adhesive backing foil) to facilitate handling. In addition, device strength/robustness is achieved by a plastic holder, where only the sample application and reading windows are exposed. The sample application and conjugate release pads are fabricated from cellulose or cross-linked silica. The application pad connects the membrane via a close contact with the conjugate release pad. Mobile recognition elements such as analyte-specific antibodies are labelled with a fluorescent dye such as Cy3B, Cy5B (encapsulated in a polystyrene or latex bead) other bright dye and/or a quencher dye. These will be dried on the conjugate release pad. Addition of a sample, followed by specific binding interactions, and the resultant analyte/antibody complex will travel along the membrane via capillary action. A schematic illustrating general aspects of the invention is shown in Figure 1. The labelled recognition antibodies may be dried inside the sample collection tube. The preferred method for the marketplace includes the labelled antibody in the conjugate release pad in a lyophilised format. In order achieve broad applicability; the technology described here includes a description of the labelled reagents in the sample collection tube. Detection will occur upon the mobility of the dye encapsulated in a latex or polystyrene particle, dyes linked to antibodies or antigen labelled through a sugar backbone, or, a dye linked to antigen and an antibody labelled with a quencher dye. Detection of analyte will occur at the reading window. Detection would be by means of a fluorescence end-point with a simple fluorimeter fitted with appropriate filter sets. The approaches described here result in a highly sensitive assay, with a low background. The detection technologies illustrated here have not been described previously involving membrane fluidics and lateral flow devices. Capturing/concentrating the analyte in a specific region of the membrane is a key advantage, greatly increasing the sensitivity of the test. Lateral flow strips were prepared from nitrocellulose or a fused silica material, supplied by either: Whatman, Pierce, Advanced Microdevices, Millipore or Pall. A relatively large pore size (up to 12µM) is required. Polyethylene is an alternative. The device and method described here is highly advantageous as when coupled with a simple fluorimeter. Very low concentrations of analyte may be rapidly detected, with little sample preparation. A number of configurations and assay architectures are described in this disclosure. Thus, here we describe a portable fluorescence biosensor device with rapid and ultrasensitive response for protein biomarkers fabricated from fluorescent dyes and a lateral flow test strip. The superior signal brightness and high photostability of the dyes are combined with the clear advantages of a lateral flow test strip, resulting in high sensitivity, selectivity and speed for protein detection. The device may be used for the rapid detection of infectious material, such as Ebola Haemorrhagic Fever influenza and HIV. The device has great potential for point-of-care and in field analysis of other protein biomarkers. Examples of organic fluorophores or fluorescent dyes that may be used with the invention described herein belong to following major chemical families: • Xanthene derivatives: fluorescein, rhodamine, Oregon green, eosin, and Texas red • Cyanine derivatives: cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, and merocyanine • Squaraine derivatives and ring-substituted squaraines, including Seta, SeTau, and Square dyes • Naphthalene derivatives (dansyl and prodan derivatives) • Coumarin derivatives • Oxadiazole derivatives: pyridyloxazole, nitrobenzoxadiazole and benzoxadiazole • Anthracene derivatives: anthraquinones, including DRAQ5, DRAQ7 and CyTRAK Orange • Pyrene derivatives: cascade blue etc. • Oxazine derivatives: Nile red, Nile blue, cresyl violet, oxazine 170 etc. • Acridine derivatives: proflavin, acridine orange, acridine yellow etc. • Arylmethine derivatives: auramine, crystal violet, malachite green • Tetrapyrrole derivatives: porphin, phthalocyanine, bilirubin

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Lateral Flow Tests Involving Fluorescent Dyes

Introduction

Point-of-care testing (POCT) is defined as diagnostic testing at or near the site of a patient or subject. Results are usually generated within 10-20 minutes. The driving force behind POCT is to bring the results immediately to the patient, clinician or veterinarian. The major benefit is that results can be generated almost immediately thereby facilitating (especially when combined with goal-directed therapies) a reduction in morbidity and mortality.

Many POCT systems are based upon lateral flow devices, usually developed from membrane-based test strips, enclosed in a plastic test cassette. This increases the likelihood that the patient, physician, and care team will receive the results quicker, which allows for immediate clinical management decisions to be made.

POCT includes: blood glucose testing, blood gas and electrolytes analysis, rapid coagulation testing (PT/INR, Alere, Microvisk Ltd), rapid cardiac markers diagnostics (TRIAGE, Alere), drugs of abuse screening, urine strips testing, pregnancy testing, faecal occult blood analysis, food pathogens screening, haemoglobin diagnostics (HemoCue), infectious disease testing and cholesterol screening.

POCT is often accomplished through the use of transportable, portable, and handheld devices and test kits (e.g., CRP, HBA1C, Homocystein, HIV salivary assay, etc.). As stated above, the goal is to collect the specimen and obtain the results in a very short period of time at or near the location of the patient so that the treatment plan can be adjusted as necessary before the patient leaves the clinic or a new treatment is commenced. Cheaper, faster, and smarter POCT devices have increased the use of POCT approaches by making these devices cost-effective for many diseases, such as diabetes, carpal tunnel syndrome and acute coronary syndrome. However there remains a need for improved sensitivity of POCT. This can be achieved with novel fluorescence detection technologies.

Thus, the invention described here describes a rapid diagnostic test or POCT test that is a medical diagnostic test, which is quick and easy to perform and uses lateral flow (or “immunochromatography”). The technology described here is suitable for preliminary or emergency medical screening and for use in medical facilities with limited resources, such as in developing countries. This invention provides same-day results within two hours, typically in approximately 20 minutes.

Some applications of the invention are listed below:

·         Rapid serological HIV testing

·         Rapid serological Ebola testing

·         Rapid influenza diagnostic testing

·         Rapid malaria testing

·         Rapid plasma regain testing

·         Rapid strep testing for Group A Streptococci

·         Rapid urease testing

The lateral flow assays that are described here may be used for qualitative, and to some extent, quantitative monitoring in non-laboratory environments.

The system described here has excellent sensitivity and a wid...