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CN-122029428-A - Nanoenzyme-based lateral flow chromatography and device

CN122029428ACN 122029428 ACN122029428 ACN 122029428ACN-122029428-A

Abstract

The invention provides a kit, a device and in-vitro detection based on nano enzyme-based lateral flow chromatography, wherein p-phenylenediamine with optimal concentration of 0.1 mM to 10 mM is used as a chromogenic substrate of aptamer-coupled gold nano enzyme with catalytic activity, so that the sensitivity is improved. The chromogenic substrate produces a stable brown color upon oxidation by the catalytically active nanomaterial. The brown color remains stable for several weeks and forms a high contrast with the background. This overcomes the limitation that detection of analytes present at critical levels below micrograms/milliliter by conventional LFAs typically results in false negative results. This provides a versatile and highly sensitive point-of-care diagnostic technique for rapid screening of infectious disease antigens and detection of biomarkers of disease (e.g., cancer, heart disease, and neurodegenerative diseases).

Inventors

  • J. Al Ace
  • S. K. Elangowan

Assignees

  • 斯里·奇特拉·蒂鲁纳尔医学科学与技术研究所

Dates

Publication Date
20260512
Application Date
20240821
Priority Date
20230822

Claims (10)

  1. 1. A lateral flow chromatography device for detecting an analyte of interest (13) in a sample (14), the device consisting of a test strip (1) having a top surface (11) and a bottom surface (12); wherein the test strip (1) comprises: a) -a sample pad (2), said sample pad (2) being arranged on a top surface (11) of said test strip (1) adapted to receive said sample (14); b) -a detection zone (17), said detection zone (17) being provided on the top surface (11) of the test strip (1), downstream of the sample pad (2), comprising: (i) A binding pad (3), on the surface of which binding pad (3) a nanoenzyme (15) for capturing the target analyte (13) is placed, the nanoenzyme (15) comprising a first binding molecule (5) coupled to a detectable reporter molecule (4); (ii) A chromatographic membrane (6), the chromatographic membrane (6) comprising a detection point (8), the detection point (8) having immobilized thereon a second binding molecule (7); c) An absorbent pad (9), said absorbent pad (9) being located on the top surface (11) of the test strip (1) and downstream of the test zone (17) and being adapted to absorb excess fluid; wherein a flow path (10) extending from the sample pad (2) to the absorbent pad (9) is defined on the top surface (11) of the test strip (1); Wherein the target analyte (13) binds to the nanoenzyme (15) forming a first analyte-nanoenzyme complex which is further captured by the second binding molecule, thereby forming a second analyte-nanoenzyme complex at the detection point (8); Wherein the second nanoenzyme complex reacts with p-phenylenediamine and an activator (16) to produce a dark brown product at the detection site (8) confirming the presence of the target analyte (13).
  2. 2. The lateral flow chromatography device of claim 1, wherein the activator is hydrogen peroxide; wherein the chromatographic membrane (6) is made of nitrocellulose; wherein the sample pad (2) and the absorbent pad (9) are both made of cellulose fibers; Wherein the bonding pad (3) is made of glass fibers.
  3. 3. The lateral flow chromatography device of claim 1, wherein the first binding molecule (5) and the second binding molecule (7) are each independently selected from the group consisting of an aptamer, an antibody, a peptide, an enzyme, a nucleic acid, a carbohydrate, a lipid, a hormone, and a protein; wherein the detectable reporter (4) is selected from the group consisting of gold nanoparticles, carbon nanoparticles, composite nanoparticles, palladium nanoparticles, cellulose beads and quantum dots.
  4. 4. The lateral flow chromatography device of claim 1, wherein the target analyte (13) is selected from the group consisting of an antigen, a nucleic acid, a protein, a bacterial cell, a viral particle, a fungal cell, a lipid and a carbohydrate; Wherein the sample (14) is selected from the group consisting of blood, serum, plasma, urine, saliva, tears, sweat, cerebrospinal fluid, synovial fluid, stool, water, soil, air filters, food extracts, nasopharyngeal swabs, pharyngeal swabs, wound swabs, tissue, tumor cells, vaginal fluid and semen.
  5. 5. The lateral flow chromatography device of claim 1, wherein the device has a detection limit of 0.2 nanograms of the target analyte (13) per 100 microliters of sample (14).
  6. 6. An in vitro method for detecting an analyte of interest (13) in a sample (14), the method comprising the steps of: i. Adding the sample (14) to a sample pad (2) of a lateral flow chromatography device according to claim 1; allowing the sample to move along a flow path (10) of the device; Adding a chromogenic substrate together with an activator (16) to the spot (8), and Iv, observing the color intensity change at the detection point (8) after 5-10 minutes; Wherein the presence of a dark brown color at the detection point (8) indicates the presence of the target analyte (13) in the sample (14); wherein the chromogenic substrate is p-phenylenediamine at a concentration in the range of 0.1 mM to 10 mM; wherein the activator is hydrogen peroxide at a concentration in the range of 10 mM to 100 mM.
  7. 7. The in vitro method for detecting an analyte of interest (13) according to claim 6, wherein the analyte of interest (13) is selected from the group consisting of an antigen, a nucleic acid, a protein, a bacterial cell, a viral particle, a fungal cell, a lipid and a carbohydrate; Wherein the sample (14) is selected from the group consisting of blood, serum, plasma, urine, saliva, tears, sweat, cerebrospinal fluid, synovial fluid, stool, water, soil, air filters, food extracts, nasopharyngeal swabs, pharyngeal swabs, wound swabs, tissue, tumor cells, vaginal fluid and semen.
  8. 8. The in vitro method for detecting an analyte of interest (13) according to claim 6, wherein the method has a limit of detection of 0.2 nanograms of the analyte of interest (13) per 100 microliters of sample (14).
  9. 9. A lateral flow chromatography kit for detecting an analyte of interest in a sample, comprising: a. the lateral flow chromatography device of claim 1; b. P-phenylenediamine; c. an activator, and D. an operating manual.
  10. 10. The lateral flow chromatography kit of claim 9, wherein the activator is hydrogen peroxide; wherein the target analyte is selected from the group consisting of an antigen, a nucleic acid, a protein, a bacterial cell, a viral particle, a fungal cell, a lipid, and a carbohydrate; Wherein the sample is selected from the group consisting of blood, serum, plasma, urine, saliva, tears, sweat, cerebrospinal fluid, synovial fluid, stool, water, soil, air filters, food extracts, nasopharyngeal swabs, pharyngeal swabs, wound swabs, tissue, tumor cells, vaginal fluid, and semen.

Description

Nanoenzyme-based lateral flow chromatography and device Technical Field The present invention relates to the field of point-of-CARE TESTS. In particular, the present invention relates to a lateral flow device for detecting an analyte of interest in a sample, a method for detecting an analyte of interest in a sample, and a kit therefor, which lateral flow device, method and kit are capable of detecting an analyte of interest in very low concentrations and have the ability to quantify the analyte and thereby confirm the presence of a pathogen or illness/disease. Background For effective patient management and timely disease control, screening for disease antigens and detection of biomarkers for various diseases (e.g., cancer, infectious disease, heart disease, and neurodegenerative disease) needs to be accomplished in a timely manner. One diagnostic technique used to rapidly screen for infectious disease antigens is lateral flow chromatography (Lateral Flow Assay, LFA). LFA is a common point-of-care diagnostic technique used in the medical diagnostic field, but LFA is primarily regarded as a primary screening test, requiring a secondary confirmatory laboratory test, such as an enzyme-linked immunosorbent assay (ELISA) or Polymerase Chain Reaction (PCR), to verify disease. The disadvantage of LFAs is the detection of analytes present at critical levels below micrograms/milliliter, which typically results in false negative results. Further, LFA is considered to be limited in clinical transformation due to its lower specificity compared to standard laboratory techniques. Quantitative detection of analytes in biological samples is a key parameter for patient detection and clinical care. The weak signal output of LFA makes it less sensitive and impossible to quantify, further limiting its application in clinical practice. To overcome this problem and improve sensitivity and signal output, various signal amplification techniques have been integrated with LFA techniques. One effective method is a nanoenzyme-based LFA that exploits the catalytic properties inherent in nanomaterials to produce a quantifiable strong output signal. Thus, nanoenzyme-based LFAs have had a significant impact on developing on-the-fly diagnostic techniques for detecting various biological analytes present in very low concentrations (nano-levels) in clinical samples. Integration of signal amplification techniques (e.g., nanoenzyme-based LFAs) can significantly enhance LFA sensitivity and signal output, making it a promising detection tool for detection of various diseases or analytes at an early stage. Further research and development in this area may lead to widespread use of LFAs in clinical practice, thereby improving patient outcome and disease control. Nanoenzyme-based lateral flow chromatography uses catalytically active nanoparticles and chromogenic substrates to enhance the colorimetric signal and sensitivity of the detection line. Conventionally, TMB (3, 3', 5' -tetramethylbenzidine) and DAB (diaminobenzidine) are used as common chromogenic substrates that produce colored products upon oxidation by peroxidases, such as horseradish peroxidase (HRP) and catalytically active nanoparticles. EP3080272B1 discloses the use of aptamer-coupled mesoporous silica particles as catalytically active nanomaterials in LFAs, which utilize TMB as chromogenic substrate. However, TMB has limitations as a chromogenic substrate for catalytically active nanomaterials in LFA applications because of its low color contrast on the detection line, degradation of color over time, and the need for slightly acidic conditions and dark environments to produce maximum amounts of product. Some of the most advanced techniques in this field include research papers and issued patents on catalytically active nanomaterials for developing LFA kits with higher sensitivity. For example, kidwell et al (US 20180052153 A1) developed colloidal palladium nanoparticles as catalytically active nanomaterials for LFA applications. Veli et al (EP 3080272B 1) studied aptamer-coupled and signal molecule-loaded silica particles for use as nanoenzymes to increase sensitivity of lateral flow chromatography. In another study, cheng et al ,[Nanozyme-Mediated Dual Immunoassay Integrated with Smartphone for Use in Simultaneous Detection of Pathogens., Nan Cheng, Yang Song, Mohamed M. A. Zeinhom, Yu-Chung Chang, Lina Sheng, Haolin Li, Dan Du, Lei Li, Mei-Jun Zhu, Yunbo Luo, Wentao Xu, and Yuehe Lin, ACS Appl. Mater. Interfaces 2017, 9, 46, 40671–40680] used palladium and platinum core-shell nanostructures as signal enhancing nanoenzymes in LFA for accurate detection of pathogens. However, only very limited studies have been directed to chromogenic substrates that produce the greatest sensitivity in LFAs. Previous research in the field has focused on the introduction of new catalytically active nanomaterials. Thus, there is a need in the art to develop more sensitive and accurate LFA kits for det