WO2021112660A1

WO2021112660A1 - Kit and cylindrical device for the detection of an infectious agent

Info

Publication number: WO2021112660A1
Application number: PCT/MX2020/000047
Authority: WO
Inventors: Samira HOSSEINI; Sergio Omar Martínez Chapa; Braulio Cárdenas Benítez; Azari PEDRAM
Original assignee: Instituto Tecnológico y de Estudios Superiores de Monterrey
Priority date: 2019-12-05
Filing date: 2020-11-23
Publication date: 2021-06-10
Also published as: MX2019014639A

Abstract

In a first aspect, the present invention relates to a device for the detection of an infectious agent, characterised in that it comprises: (i) a female piece comprising at least one indentation; and (ii) a male piece. In a second aspect, the invention relates to a kit for the detection of an infectious agent, characterised that it comprises: (a) at least one detection fibre comprising a predetermined antibody for the detection of a predetermined infectious agent; (b) at least one device for the detection of an infectious agent; and a microplate comprising a plurality of wells and a solution in each well, which potentially comprises a biomarker related to a predetermined infectious agent, wherein each device is coupled to one of the wells of the plurality of wells, allowing the detection fibre to be submerged in the solution.

Description

KIT AND CYLINDRICAL DEVICE FOR THE DETECTION OF AN INFECTIOUS AGENT.

FIELD OF THE INVENTION

The present invention is related to the detection of an infectious agent from probes with fibers or paper, and more particularly it relates to a kit and cylindrical device for the detection of an infectious agent.

BACKGROUND OF THE INVENTION

Different classes of biological assays have been designed for the detection of a paper- or fiber-based infectious agent, including dipsticks, lateral flow assays (LFA), paper-based analytical devices (pPADs), and plate. microwells. These platforms have several benefits such as speed and costs, as well as being light and requiring a small amount of sample volume. However, they have limitations such as insufficient detection sensitivity, instability of the material (paper or fiber), the need for surface treatment due to the inert nature of the paper or fiber, and a short shelf life after surface activation.

In addition, most paper-based assays use commercially available nitrocellulose (NC). However, there is no great control over the properties of said paper, since the quality of the paper can vary from one manufacturer to another and it is known that nitrocellulose has a WCA of 21 ° to 67 °, this level of hydrophilicity can induce a higher probability of a false positive signal. For that reason, paper analytical platforms generally suffer from insufficient sensitivity. Therefore, it is necessary to develop a paper with an adapted surface morphology, surface properties and adjusted wettability for specific purposes.

For example, W02007 / 106050 describes an ELISA test to detect flavivirus member-specific antibodies from biological samples, and is based on a competition for epitope binding on the envelope protein of captured flavivirus antigen using Anti-flavivirus IgA in the presence of positive flavivirus serum. For its part, document CN205679623U describes a nitrocellulose fiber covered with colloidal gold and a dengue virus antigen detection kit from blood samples.

However, none of the documents suggest the use of a probe that allows a fiber to be suspended vertically without using adhesives to double the contact area in the test. They also do not anticipate the use of a specific reaction arrangement of free radical polymerization, electrospinning and finally a 3D printing or injection molding to obtain high detection sensitivity, stability and useful life of the material.

As a consequence of the foregoing, it has been sought to eliminate the drawbacks presented by the biological tests based on paper or fibers currently used, developing a kit and cylindrical device for the detection of an infectious agent that, in addition to being stable and performing a Diagnostic with a high sensitivity, allows to increase the contact area of the fiber used, without using any adhesive and reducing operating costs. OBJECTS OF THE INVENTION

Taking into account the shortcomings of the prior art, it is an object of the present invention to provide a kit for the detection of an infectious agent without using adhesives with a high sensitivity.

Another object of the present invention is to provide a kit for the detection of an infectious agent that uses a cylindrical device to provide a greater contact area and thus reduce operating costs.

These and other objects are achieved by a kit and cylindrical device for the detection of an infectious agent in accordance with the present invention.

BRIEF DESCRIPTION OF THE INVENTION.

A kit has been invented for the detection of an infectious agent with a cylindrical device, probe type, which allows a detection fiber to be submerged vertically, without using adhesives, to double its contact area and thus obtain a high diagnosis. sensitivity and lower operating cost.

Thus, a first aspect of the present invention is a device for the detection of an infectious agent characterized in that it comprises; i) a female part which in turn comprises at least one slit; and ii) a male piece that in turn comprises at least one extension so that the external faces of each extension fit into each of the grooves of the female piece, and in this way the two pieces can be joined allowing the fastening of a detection fiber vertically at one of its ends, where the device is shaped such that it can be coupled with the wells of a common tray used in tests for the detection of an infectious agent.

Now, a second aspect of the present invention is a kit for the detection of an infectious agent characterized in that it comprises: a) at least one detection fiber which in turn comprises a predetermined antibody for the detection of a predetermined infectious agent; b) at least one device for detecting an infectious agent characterized in that it comprises: i) a female part which in turn comprises at least one slit; and i) a male part that in turn comprises at least one extension so that the external faces of each extension fit into each of the grooves of the female part, and in this way the two parts can be joined allowing the fastening of the detection fiber vertically at one of its ends; and c) a microplate that in turn comprises a plurality of wells and a solution within each well that potentially comprises a biomarker related to a predetermined infectious agent, wherein each device is coupled with one of the wells of the plurality of wells allowing the detection fiber is immersed in the solution. BRIEF DESCRIPTION OF THE DRAWINGS

The novel aspects that are considered characteristic of the present invention will be set forth with particularity in the appended claims. However, some modalities, characteristics and some objects and advantages thereof, will be better understood in the detailed description, when read in connection with the attached drawings, in which:

Figure 1 shows a perspective view of an embodiment of an infectious agent detection device (1100) in accordance with the principles of the present invention.

Figure 2 shows a perspective view of one embodiment of the device for detecting an infectious agent (1100) shown in Figure 1.

Figure 3 shows a side view of an embodiment of a kit for the detection of an infectious agent (1000) in accordance with the principles of the present invention.

Figure 4 shows a perspective photograph of an embodiment of a kit for the detection of an infectious agent (1000) in accordance with the principles of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

A first aspect of the present invention is a device for the detection of an infectious agent characterized in that it comprises: i) a female part which in turn comprises at least one slit; and ii) a male piece that in turn comprises at least one extension so that the external faces of each extension fit into each of the grooves of the female piece, and in this way the two pieces can be joined allowing the fastening of a detection fiber vertically at one of its ends, where the device is shaped such that it can be coupled with the wells of a common tray used in tests for the detection of an infectious agent.

In a preferred embodiment of the present invention, the infectious agent to be detected is related to a tropical disease, more preferably to schistosomiasis, onchocerciasis, lymphatic filariasis, leishmaniasis, leprosy, dengue and severe dengue, yaws, echinococcosis, taeniasis and cysticercosis, rabies, Chagas disease, Buruli ulcer, human African trypanosomiasis, vector-borne diseases, dracunculiasis.

In a preferred embodiment of the present invention, the female part has a half-cylinder shape.

Likewise, said female part preferably comprises two slits.

In a preferred embodiment of the present invention, the male part has a half-cylinder shape.

Likewise, said male part preferably comprises two extensions.

In relation to said female and male parts, preferably both are obtained by 3D printing or injection molding.

When joining both pieces to form a cylindrical shaped device, said cylindrical device preferably has a diameter of 5 mm. In one embodiment of the present invention, the detection fiber is selected from electrospun polymer fibers.

A second aspect of the present invention is a kit for the detection of an infectious agent characterized in that it comprises; a) at least one detection fiber which in turn comprises a predetermined antibody for the detection of a predetermined infectious agent; b) at least one device for detecting an infectious agent characterized in that it comprises: i) a female part which in turn comprises at least one slit; and i) a male part that in turn comprises at least one extension so that the external faces of each extension fit into each of the grooves of the female part, and in this way the two parts can be joined allowing the fastening of the detection fiber vertically at one of its ends; and c) a microplate that in turn comprises a plurality of wells and a solution within each well that potentially comprises a biomarker related to a predetermined infectious agent, wherein each device is coupled with one of the wells of the plurality of wells allowing the detection fiber is immersed in the solution.

The proposed kit represents a tool in the early diagnosis of an infectious agent that has an operation similar to that of a conventional enzyme-linked immunosorbent assay (ELISA), making it convenient for the laboratory technician who performs ELISA on a regular basis.

In a preferred embodiment of the present invention, the infectious agent to be detected is a tropical disease, more preferably it is selected from schistosomiasis, onchocerciasis, lymphatic filariasis, leishmaniasis, leprosy, dengue and severe dengue, yaws, echinococcosis, taeniasis and cysticercosis, rabies, Chagas disease, Buruli ulcer, human African trypanosomiasis, vector-borne diseases, dracunculiasis. Even more preferably the infectious agent is the dengue virus, which is preferably detected through the NS1-type protein, characteristic of the dengue virus and which serves as a biomarker.

In one embodiment of the present invention, the detection fiber is selected from electrospun polymer fibers.

In a preferred embodiment of the present invention, the predetermined antibody is selected from primary and / or secondary antibodies, and more specifically the primary antibody is the NS1 glycoprotein against dengue virus and the secondary antibody is the goat anti-dengue heavy chain. mouse IgG2a.

Likewise, said female part preferably comprises two slits,

Likewise, said male part preferably comprises two extensions. In relation to said female and male parts, preferably both are obtained by 3D printing or injection molding.

In a preferred embodiment of the present invention, the plurality of wells comprise

96 wells.

One of the advantages of the present invention is that the detection fibers can be submerged in a vertical position, which provides an effective interaction with both sides of said fiber, in addition, the present invention makes the application of any type of glue unnecessary, which, in turn, it minimizes the risk of protein deactivation when it comes into contact with adhesive materials.

Referring now to Figure 1, there is shown a perspective view of one embodiment of an infectious agent detection device 1100 in accordance with the principles of the present invention. As can be seen in said figure, the device (1100) comprises a female part (1110) with two slits (lili) and a male part (1120) with 2 extensions (1121), in addition a detection fiber (1200) is shown .

Referring to Figure 2, it shows a perspective view of an assembly modality of the device for the detection of an infectious agent (1100) shown in Figure 1. As can be seen in said figure, the two extensions ( 1121) of the male part (1120) fit with the two slits (lili) of the female part (1110), in such a way that they hold the detection fiber (1200)

Referring to Figure 3, this shows a side view of an embodiment of a kit for the detection of an infectious agent (1000) in accordance with the principles of the present invention. As can be seen in said figure, the kit (1000) comprises a plurality of devices for the detection of an infectious agent (1100); a microplate (1300) with a plurality of wells (1310); and a plurality of detection fibers (1200), where each of the devices (1100) are coupled in each of the wells (1310, in such a way that the detection fiber (1200) is submerged vertically in a solution (1311) potentially containing an infectious agent.

Referring to Figure 4, this shows a perspective photograph of an embodiment of a kit for the detection of an infectious agent (1000) in accordance with the principles of the present invention. As can be seen in said figure, the kit (1000) comprises a device (1100) with a detection fiber (1200) submerged vertically. ¹

The present invention will be better understood from the following examples, which are presented for illustrative purposes only to allow a thorough understanding of the preferred embodiments of the present invention, without implying that there are no other, non-illustrated embodiments that may be put into practice based on the detailed description made above.

Example 1.

A trial was carried out to make cylindrical devices in accordance with the present invention. For this, the female and male parts of the cylindrical device were manufactured using a Fortus® 400 me FDM system in ABS-M30 plastic with a total dimension of 5 mm, so that it can be inserted into one of the plurality of wells of a microplate. The print scheme was designed with SolidWorks® and one hundred pairs of parts were printed for the development of each 96-well microplate.

Once the hundred pairs had been manufactured, a detection fiber, obtained by electrospinning, was placed between a female part and a male part of the cylindrical device, and the above was done with the remaining 95 pairs and thus obtain 96 cylindrical devices, where each one holds a sense fiber.

Finally, each of the 96 cylindrical devices was mounted in each of the 96 wells on the lid of a common microplate.

Example 2.

An assay was performed to prepare dengue virus NS1 protein solutions and antibody solutions according to the present invention, with the following steps: a. Prepare a stock solution of phosphate buffered saline "PBS" (10X) by adding 161.2 g of NaCl, 4.4 g of KCI, 24 g of Na2HPC¡4 and 4 g of KH2PO4 to 2,000 mL of distilled water. b. use dilute phosphate saline buffer (IX) as wash buffer, which is used after coating samples with NS1 protein solution. c. prepare a coating buffer, for the preparation of the NS1 protein solution, with 100 mL of distilled water, 2 mL of 0.2 M sodium carbonate (feCCh) and 23 mL of 0.2 M sodium bicarbonate (NaHCC); d. preparing a blocking buffer to reduce the possibility of non-specific binding, with 3% bovine serum albumin "BSA" in phosphate buffered saline (IX); and. Use 0.05% polysorbate 20 "Tween 20®" in phosphate buffered saline (IX) as wash buffer after coating samples with NS1 glycoprotein primary antibodies against dengue virus (Anti-Dengue virus NS1 glycoprotein primary antibody) and Secondary antibody is goat anti-mouse IgG2a heavy chain "HRP" (Goat anti-mouse IgG2a heavy chain. f. use 1% bovine serum albumin in PBS (IX) as dilution buffer for the preparation of the solutions of antibodies.

Example 3.

An assay was performed to make the diagnosis of dengue through the detection of the biomarker NS1 in accordance with the present invention, with the following steps: a. Wash 3 times, every 5 minutes, each of the plurality of wells of a tray with 200 µl per well of phosphate buffer saline (IX) at room temperature, using a shaker with a stirring speed of 1000 rpm; b. incubate solutions of NS1 at concentrations of: 5, 50, 5xl0 ² , 5xl0 ³ , 5xl0 ⁴ and ^{5x10 s} Pg / mL at 4 ° C for 8 hours, for each concentration 12 positive and 8 negative replicates were made, the negative replicates were prepared in the absence of the NS1 protein; c. loading each of the plurality of wells with 150 µl of the NS1 solutions from the previous step; d. Block with the lid, assembled in Example 1, each of the plurality of wells by adding 200 pL of blocking buffer to each well. Incubation was carried out at 37 ° C for 1 hour; and. prepare a 1: 10,000 stock antibody solution in dilution buffer, each well received 150 pL of stock solution and was blocked and incubated for 2 hours at 37 ° C); F. wash 3 times, every 5 minutes, with 200 µl per well of Tween-20 solution (previous example) at room temperature, using a shaker with a stirring speed of 1000 rpm; g. add 150 pL of secondary antibody solution at 1: 10,000 in dilution buffer and block and incubate for 1 hour at 37 ° C; h. wash 3 times, every 5 minutes, with 200 µl per well of Tween-20 solution (previous example) at room temperature, using a shaker with a stirring speed of 1000 rpm; i. load each of the plurality of wells with 100 µl of Tetramethylbenzidine ELISA substrate "TMB" and block and incubate for 10 minutes; j. Record the absorbance reading at 370 nm according to the manufacturer's instructions.

In accordance with the foregoing, it can be seen that the kit and cylindrical device for the detection of an infectious agent has been designed for the early diagnosis of a tropical disease, and it will be evident to any expert in the field that the kit modalities and cylindrical device for the detection of an infectious agent as described above and illustrated in the accompanying drawings, are only illustrative but not limiting of the present invention, since numerous changes of consideration in its details are possible without departing from the scope of the invention. Therefore, the present invention should not be construed as restricted except as required by the prior art and for the scope of the appended claims.

Claims

NOVELTY OF THE INVENTION CLAIMS

1. A device for detecting an infectious agent characterized in that it comprises: i) a female part which in turn comprises at least one slit; and ii) a male piece that in turn comprises at least one extension so that the external faces of each extension fit into each of the grooves of the female piece, and in this way the two pieces can be joined allowing the fastening of a detection fiber vertically at one of its ends, where the device is shaped such that it can be coupled with the wells of a common tray used in tests for the detection of an infectious agent.

2. The device according to claim 1, further characterized in that the infectious agent is related to a tropical disease.

3. The device according to claim 2, further characterized in that the tropical disease is selected from schistosomiasis, onchocerciasis, lymphatic filariasis, leishmaniasis, leprosy, dengue and severe dengue, yaws, echinococcosis, taeniasis and cysticercosis, rabies, the disease of Chagas disease, Buruli ulcer, human African trypanosomiasis, vector-borne diseases, or guinea worm.

4. The device according to claim 1, further characterized in that the female part has a half-cylinder shape.

5. The device according to claim 1, further characterized in that the female part comprises two slits.

6. The device according to claim 1, further characterized in that the male part has a half-cylinder shape.

7. The device according to claim 1, further characterized in that the male part comprises two extensions.

The device according to claim 1, further characterized in that the female and male parts are obtained by 3D printing or injection molding.

9. The device according to claim 1, further characterized in that at the time of joining both pieces to form a cylindrical device.

The device according to claim 1, further characterized in that the detection fiber is selected from electrospun polymer fibers.

11. A kit for the detection of an infectious agent characterized in that it comprises: a) at least one detection fiber which in turn comprises a predetermined antibody for the detection of an infectious agent; b) at least one device for detecting an infectious agent comprising: i) a female part which in turn comprises at least one slit; and ii) a male part that in turn comprises at least one extension so that the external faces of each extension fit into each of the grooves of the female part, and in this way the two parts can be joined allowing the fastening of the detection fiber vertically at one end; and c) a microplate that in turn comprises a plurality of wells and a solution within each well that potentially comprises a biomarker related to a predetermined infectious agent, where each device is coupled to one of the plurality of wells allowing the detection fiber to be immersed in the solution.

12. The kit according to claim 11, further characterized in that the infectious agent is related to a tropical disease.

The kit according to claim 12, further characterized in that the tropical disease is selected from schistosomiasis, onchocerciasis, lymphatic filariasis, leishmaniasis, leprosy, dengue and severe dengue, yaws, echinococcosis, taeniasis and cysticercosis, rabies, the disease of Chagas disease, Buruli ulcer, human African trypanosomiasis, vector-borne diseases, or guinea worm.

14. The kit according to claim 13, further characterized in that the tropical disease is dengue.

The kit according to claim 11, further characterized in that the detection fiber is selected from electrospun polymer fibers.

16. The kit according to claim 11, further characterized in that the predetermined antibody is selected from primary and / or secondary antibodies.

17. The kit according to claim 16, further characterized in that the primary antibody is the glycoprotein NS1 against dengue virus and the secondary antibody is the goat anti-mouse IgG2a heavy chain.

18. The kit according to claim 11, further characterized in that the female part has a half-cylinder shape.

19. The kit according to claim 11, further characterized in that the female part comprises two slits.

20. The kit according to claim 11, further characterized in that the male part has a half-cylinder shape.

21. The kit according to claim 11, further characterized in that the male part comprises two extensions.

22. The kit according to claim 11, further characterized in that the plurality of wells comprises 96 wells.