COLORIMETRIC DETECTION SYSTEM FOR RAPID DETECTION OF INFECTIOUS PULMONARY DISEASES AND A FACE MASK WITH SAID COLORIMETRIC DETECTION SYSTEM

Abstract

The present invention relates to a colorimetric detection system for rapid detection of lung diseases. The detection system comprises antibodies and/or aptamers coupled to a substrate for capturing a lung disease specific antigen. The colorimetric system further comprises dyed microspheres modified with the antibodies and/or aptamers for visually detecting the lung disease specific antigen. The present invention also relates to a method of producing said detection system and a mask with such a detection system.

Claims

1. A colorimetric detection system for rapid detection of lung diseases comprising antibodies and/or aptamers coupled to a substrate for capturing a lung disease specific antigen, wherein the colorimetric system further comprises dyed microspheres modified with the antibodies and/or aptamers for visually detecting the lung disease specific antigen.

2. The colorimetric detection system according to claim 1 wherein the dyed microspheres have a mean diameter of less than 6 μm.

3. The colorimetric detection system according to claim 1, wherein the substrate comprises a protease.

4. The colorimetric detection system according to claim 1, wherein the substrate comprises discrete sections, each section being modified with antibodies and/or aptamers different from one of the other section for capturing a lung disease specific antigen different from the one captured in one of the other section.

5. The colorimetric detection system according to claim 1, wherein the antibodies and/or aptamers are influenza virus antibodies and/or aptamers; or a pneumonia or pneumonia-inducing antibodies and/or aptamers.

6. The colorimetric detection system according to claim 1, wherein the substrate is a hydrophilic material and/or oxidized cellulose.

7. A colorimetric face mask comprising a first layer with a colorimetric detection system according to claim 1.

8. The colorimetric face mask according to claim 7 comprising a second outer layer.

9. The colorimetric face mask according to claim 7, wherein the colorimetric detection system is located in the centre of the first layer.

10. The colorimetric mask according to claim 7 comprising a third inner layer for hiding the result of the visually detected lung disease.

11. The colorimetric mask according to claim 10, wherein the third inner layer is permeable to fluids.

12. The colorimetric mask according claim 7, wherein the mask comprises fixation loops.

13. A method for producing a colorimetric detection system according to claim 1, the method comprising the steps of Providing a substrate with reactive sites towards antibodies and/or aptamer, coupling antibodies and/or aptamers specific for a lung disease causing antigen to the reactive sites, blocking of non-reacted active sites, attaching dyed microspheres modified with the antibodies and/or aptamers to the substrate.

14. The method according to claim 13, wherein a protease is coupled to the substrate.

15. The method according to claim 13, wherein two or more different antibodies and/or aptamers each specific for another lung disease causing antigen are coupled to the substrate.

16. The method according claim 13, wherein the substrate is a hydrophilic material and/or oxidized cellulose.

17. The method according claim 13, wherein the non-modified dyed microspheres have a diameter less than 6 μm.

18. The mask according claim 7 produced by a method comprising: providing a first layer with a colorimetric detection system obtainable by a method including: providing a substrate with reactive sites towards antibodies and/or aptamer, coupling antibodies and/or aptamers specific for a lung disease causing antigen to the reactive sites, blocking of non-reacted active sites, and attaching dyed microspheres modified with the antibodies and/or aptamers to the substrate.

19. The method according to claim 18, wherein a second outer layer is provided.

20. (canceled)

Description

[0064] The invention will be described in more details with regard to the following figures and examples. The invention is not limited to these specific embodiments.

[0065] It shows:

[0066] FIG. 1: A detection system according to the invention.

[0067] FIG. 2A: A face mask according to the invention from the front side.

[0068] FIG. 2B: The mask of FIG. 2A from the opposite side in a perspective view.

[0069] FIG. 3: Preparation method of the colorimetric detection system.

[0070] FIG. 4: Working principle of the colorimetric detection system.

[0071] FIG. 1 shows a colorimetric detection system 1 according to the invention. The colorimetric detection system 1 comprises a substrate 11, here in form of a strip. In the centre of the strip 11 is the detection area 12 in form of a circle with discrete detection sections A, B, C. Each detection section A, B and C are able to detect another pathogen specific antigen. For example, section A can comprise influenza virus antibodies/aptamers coupled to blued-dyed microsphere. Section B can comprise pneumonia antibodies/aptamer coupled to green dyed microsphere. Section C can comprise Covid-19 antibodies/aptamer coupled to red dyed microsphere.

[0072] If a patient's sample is applied to the detection system 1 and the patient is infected with influenza virus, the detection section A will change from colorless to blue while the other sections B and C remain colorless.

[0073] If a patient's sample is applied to the detection system 1 and the patient is infected with pneumonia, the detection section B will change from colorless to green while the other sections A and C remain colorless.

[0074] If a patient's sample is applied to the detection system 1 and the patient is infected with Covid 19, the detection section C will change from colorless to red while the other sections A and B remain colorless.

[0075] FIG. 2A shows the face mask 2 according to the invention from the front with the outer layer 21 and the fixation loops 22 on each side for fixation on the ears.

[0076] FIG. 2B shows the mask 2 from the backside, meaning the side facing mouth and nose of the user. The figure shows the first layer 23 comprising the detection area 12. Thus, in this specific example, the first layer also forms the substrate of the colorimetric detection system. The detection area 12 is provided as a circle in the centre of the mask 2, being the region closest to mouth and nose when in use. The detection area 12 is divided into three different pie-shaped sections A, B, C. Each section will be coupled with another antibodies/aptamer system specific for one particular antigen thus providing the possibility of detecting three different pathogen specific antigen with one mask.

[0077] FIG. 3 shows the modification of the substrate. Firstly, a cellulose substrate 31 is provided which is oxidized with sodium periodate (step a). Secondly, capture antibodies or aptamers or both are added to the oxidized cellulose and coupled by known carbodiimide coupling techniques to the cellulose (step b). Step b can also comprise the step of coupling protease to the cellulose using horseradish peroxidase (not shown). Previously prepared dyed microsphere modified with the antibodies/aptamers (detection antibodies) 32 are attached to the substrate by immersing the cellulose substrate into a solution containing the dyed microspheres (step c). The dyed microsphere are preferably obtained by reaction of colored polystyrene microspheres with free carboxyl groups with the respective antibodies and/or aptamers by the previously mentioned carbodiimide coupling techniques known in the art.

[0078] Then, the substrate 31 is dried, attached to a first layer of a mask or being the first layer itself and mounted together with an outer layer 21 to the colorimetric mask 2 of FIG. 2.

[0079] FIG. 4 shows the working principle of the colorimetric detection system. When salvia or nasal muscus sample is applied to the substrate, the pathogen specific antigen 41 will be captured by the capture antibody/aptamer 42 (step d). If protease is present the antigen 41 may be cleaved into smaller fragments (step e), thus allowing more detection antibodies 32 to be agglutinated around the antigen 41 leading to a agglutinated network 43. The network 43 comprises sufficient detection antibodies 32 to become visible to the naked eye. Movement of the detection antibodies 32 to the antigen's 41 reactive sites is preferably achieved through capillary effects upon humidification of the substrate.

EXAMPLE

[0080] 1. Reagents

[0081] Matrix metalloproteinase-2 (MMP2) was synthesized by Pepmic Co. (Suzhou, Jiangsu, China).

[0082] Sodium phosphate and monopotassium phosphate, Tris-Base, Sodium acetate; bovine serum albumin (BSA), sodium chloride, phosphate buffered saline (PBS), ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), N-hydroxysuccinimide (NHS), ethylenediaminetetraacetic acid (EDTA) phosphate buffered were all purchased from Sigma-Aldrich (Darmstadt, Germany). Carboxylic acid functionalized microspheres of different colors (orange and blue) were obtained from Polysciences Inc. (Polysciences Inc., Warrington, Pa.).

[0083] All Aptameres of pathogen were provided from Aptamer Sciences (Aptamer Sciences Inc, Korea).

[0084] All antibody and antigen for testing the system were provided from abcam (abcam, UK), Sodium peroxide/H.sub.2SO.sub.4 were purchased from Sigma-Aldrich (Darmstadt, Germany). Pure cotton cellulose were obtained from Filmar (Egypt).

[0085] 2. Activation of Cotton Cellulose Serving as Substrate

[0086] A pure cotton cellulose cloth was activated by immersing them in sodium periodate (NaIO.sub.4) and of sulfuric acid (H.sub.2SO.sub.4) overnight at room temperature to obtained oxidized cellulose. The oxidized cotton cellulose was washed with PBS buffer, subsequently, with cold distilled water, to remove excess of oxidizing agent. Periodate oxidation of cellulose cotton primarily activates hydroxyl groups into aldehyde or carboxyl groups, which will later be coupled with free amine groups of the antibodies or aptamers.

[0087] 3. Immobilization of Protease/Antibody on the Activated Cotton

[0088] Human respiratory syncytial virus (hRSV) antibody (Ab) and MMP2 protease were coated on the cotton cellulose to later support cleavage of the pathogen. The activated cotton cellulose was incubated into a well-mixed solution of hRSV Ab and PBS and incubated overnight at 4° C. Accordingly, MMP2 containing coupling buffer was used to couple MMP2 onto the cellulose similar as to the method described for the antibody. Horseredish peroxidase was used for facilitate coupling. The cotton cellulose was washed to remove any unbounded antibody and MMP2 and thereafter 1% BSA was added to block the active sites of the cotton cellulose. A control cotton cellulose cloth was incubated with 1% BSA solution in PBS buffer for 30 min at room temperature.

[0089] 4. Conjugation of Aptamer to Blue Polymeric Microsphere

[0090] Blue dyed microsphere with diameter in the range of 300 nm were used. The microsphere contained attached carboxyl groups. hRSV aptamer was washed with distilled water several times before being reacted with a coupling mixture of EDC/NHS overnight. Subsequently, the microspheres were washed with distilled water to remove excess coupling reagent. hRSV aptamer was thus linked to activated blue-dyed microspheres. Blocking of any unbound sites was achieved using 1% BSA solution in PBS for 30 min.

[0091] 5. Attachment of Microsphere to the Cotton Cellulose

[0092] The modified cotton cellulose with the attached antibodies from step 3 were washed with PBS followed by immersion the cotton cellulose into a solution of the modified microspheres of step 4, conjugated hRSV aptamer. Reaction was allowed to occur for a couple of minutes. Subsequently, the cotton was washed with cold PBS to remove any unreacted microsphere. The cotton cellulose was dried and store at room temperature for 24 hrs.

[0093] 6. The Assay Technique for Control

[0094] After 24 hrs, the cellulose cotton with the associated microspheres and capture antibodies were exposed to a sample of a patient's salvia, infected with respiratory syncytial virus. A blue color appeared within 3 min, visually displaying the infection.