Test strip for visual detection of matrix metalloproteinases and method for its production and use

Abstract

The application provides a test paper for visual detection of matrix metalloproteinase, a preparation method and application thereof, and relates to the technical field of test papers. The test paper for visual detection of matrix metalloproteinase comprises a test paper carrier and a modifier grafted to the test paper carrier, wherein the modifier contains a visual marker modified by a peptide segment, and the peptide segment contains a matrix metalloproteinase recognition fragment; the connection between the modifier and the test paper carrier is located on the two sides of a matrix metalloproteinase cleavage site in the matrix metalloproteinase recognition fragment. The test paper solves the problem that there is currently a lack of a simple and convenient MMPs detection product.

Description

Technical Field

[0001] This invention relates to the field of test strip technology, and in particular to a test strip for visually detecting matrix metalloproteinases, its preparation method, and its application. Background Technology

[0002] Matrix metalloproteinases (MMPs) are a highly conserved class of proteases in nature, belonging to the zinc-dependent endopeptidase family. They can degrade almost all components of the extracellular matrix (ECM), as well as myelin, growth factors, cytokines, and cell adhesion molecules. Currently, 26 members of the MMP family have been isolated and identified, numbered MMP 1 to 26. Based on their substrates and fragment homology, MMPs are classified into six classes: collagenases, gelatinases, matrix degraders, matrix lysins, furin-activated MMPs, and other secreted MMPs.

[0003] The extracellular matrix (ECM) provides structural support for vital cellular and tissue units by releasing signaling molecules. The interactions between cells and tissues and the ECM participate in and influence most physiological activities. Under normal physiological conditions, MMP expression levels are extremely low, but significantly upregulated under environmental and pathological conditions such as inflammatory factors, growth factors, high glucose, and oxidative stress. Therefore, MMPs play a crucial role in physiological and pathological processes such as ECM and tissue remodeling, organogenesis and development, angiogenesis, immune inflammation, cell migration, proliferation, and apoptosis. For example, matrix metalloproteinase 9 (MMP 9) plays a vital role in the cleavage of extracellular matrix structural elements during physiological and pathological tissue remodeling and is involved in various diseases related to inflammation and immune cell function, such as cancer, dry eye syndrome, kidney disease, cerebral hemorrhage, and neurological disorders.

[0004] Therefore, assessing MMP expression levels can serve as potential biomarkers for disease diagnosis. Current MMP detection methods primarily rely on ELISA, which requires multiple reagents, is cumbersome, and necessitates specialized equipment and personnel. Therefore, simplifying MMP detection methods and providing a simple and convenient MMP detection product is currently a market need.

[0005] In view of this, the present invention is hereby proposed. Summary of the Invention

[0006] The primary objective of this invention is to provide a test strip for visually detecting matrix metalloproteinases (MMPs) in order to alleviate the current lack of a simple and convenient MMP detection product.

[0007] A second objective of this invention is to provide a method for preparing the above-mentioned test strip.

[0008] A third objective of this invention is to provide a matrix metalloproteinase detection kit.

[0009] The fourth objective of this invention is to provide a method for detecting matrix metalloproteinases (MMPs) for non-diagnostic and non-therapeutic purposes, thereby alleviating the problem of cumbersome operation in existing MMP detection methods.

[0010] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0011] According to one aspect of the present invention, a test strip for visual detection of matrix metalloproteinases is provided, the test strip comprising a test strip carrier and a modifier grafted onto the test strip carrier, the modifier containing a peptide-modified visual marker, the peptide containing a matrix metalloproteinase recognition fragment;

[0012] The connection between the modifier and the test strip carrier is located on either side of the matrix metalloproteinase cleavage site in the matrix metalloproteinase recognition fragment, as is the visual marker.

[0013] Preferably, the test strip carrier and the modifier each contain one of a specific binding pair, and the modifier and the test strip carrier are connected through the specific binding pair;

[0014] Preferably, the specific binding pair is selected from biotin and streptavidin;

[0015] Preferably, the surface of the test strip carrier is modified with streptavidin, and the modification contains biotin.

[0016] Preferably, the visual markers include quantum dots, colloidal gold, colloidal silver, carbon nanoparticles, selenium nanoparticles, fluorescent microspheres, or colored microspheres;

[0017] Preferably, the matrix metalloproteinase recognition fragment is modified with at least one amino acid residue at its C-terminus and / or N-terminus.

[0018] Preferably, the visual marker is colloidal gold, and one end of the peptide contains at least one cysteine ​​residue;

[0019] Preferably, the test paper carrier is selected from nitrocellulose membrane, cellulose acetate membrane, filter paper, polylactic acid membrane or polyacrylonitrile membrane;

[0020] Preferably, the shape of the test strip carrier is selected from square, circular or arc-shaped.

[0021] Preferably, the matrix metalloproteinase is MMP 9, and the amino acid sequence of the matrix metalloproteinase recognition fragment is shown in Seq_1.

[0022] Preferably, the test strip is a test strip for visual detection of MMP 9; the test strip carrier is modified with streptavidin; the modifier contains a peptide-modified colloidal gold, the amino acid sequence of the peptide is shown in Seq_2, and the other end of the peptide is modified with biotin; the modifier is linked to the test strip carrier through the specific binding of biotin and streptavidin.

[0023] According to another aspect of the present invention, the present invention also provides a method for preparing the above-mentioned test strip, comprising attaching the modifier to the test strip carrier.

[0024] Preferably, the test paper carrier is incubated with streptavidin, and then further incubated with BSA solution to obtain a streptavidin-modified test paper carrier; the incubation conditions for the test paper carrier and streptavidin are: 4–37℃ for 1–8 h, and the area-to-volume ratio of the test paper carrier to the streptavidin solution is (5–10):100 mm². 2 / μL; streptavidin concentration is 5–15 μg / mL;

[0025] Preferably, the incubation conditions for further incubation with BSA solution are: incubation at 4–37°C for 0.5–8 h, and the concentration of BSA solution is 1–5% m / v.

[0026] Preferably, the colloidal gold solution is incubated with the biotin-linked peptide, then BSA is added and incubated, the supernatant is collected by centrifugation and resuspended in BSA solution to obtain the modified product;

[0027] The incubation conditions for colloidal gold solution and biotin-linked peptides were: 4–37℃ for 1–8 h, and the mass-to-volume ratio of biotin-linked peptides to colloidal gold solution was 1:(0.5–1.5) μg / mL.

[0028] Preferably, the incubation conditions for adding BSA are: incubation at 4–37°C for 0.5–8 h, BSA solution concentration of 1–5% m / v; and the amount of BSA solution added is 10–30% of the volume of the colloidal gold solution.

[0029] According to another aspect of the present invention, the present invention also provides a matrix metalloproteinase detection kit comprising the above-described test strip.

[0030] According to another aspect of the present invention, the present invention also provides a method for detecting matrix metalloproteinases for non-diagnostic and therapeutic purposes, the method comprising contacting a sample to be tested with the test strip and determining the content of matrix metalloproteinases in the sample to be tested based on observable signal changes of the test strip;

[0031] Preferably, the method includes contacting the sample to be tested with the test strip and determining the content of matrix metalloproteinase in the sample to be tested based on the color change of the test strip.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] The visual detection test strip for matrix metalloproteinases provided by this invention mainly consists of a test strip carrier and modifiers grafted onto the test strip carrier. The modifiers contain peptide-modified visual markers, and these peptides contain matrix metalloproteinase recognition fragments. This invention utilizes the cleavage capability of matrix metalloproteinases to cleave the matrix metalloproteinase recognition fragments, thereby causing the visual markers attached to the test strip carrier to detach from the carrier. The amount of cleaved visual markers changes with the matrix metalloproteinase content in the sample to be tested, and the visual signal on the test strip weakens as the visual markers detach. Therefore, the change in the visual signal on the test strip can reflect the matrix metalloproteinase content in the sample to be tested.

[0034] The main beneficial effects of the visual detection test strip for matrix metalloproteinases provided by this invention include simplified detection procedures and visual detection of MMP content: (a) Simplified detection procedure: When detecting the MMP content in a sample, it is only necessary to fully contact the test strip with the sample and incubate it together. After the detection is completed, the color change of the test strip can be observed with the naked eye or a small detection instrument, without the need for additional detection reagents and procedures. (b) Visual detection of MMP content: This invention uses a visual marker as a signal indicator, and qualitative or semi-quantitative detection of MMP concentration can be achieved through color or fluorescence intensity. Attached Figure Description

[0035] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0036] Figure 1 The test strip provided in this embodiment of the invention is used to detect color changes of different concentrations of MMP 9. Detailed Implementation

[0037] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0038] According to one aspect of the present invention, a test strip for visually detecting matrix metalloproteinases is provided. The test strip mainly comprises a test strip carrier and a modifier grafted onto the test strip carrier. The modifier contains a peptide-modified visual marker, and the peptide contains a matrix metalloproteinase recognition fragment. Furthermore, the junction between the modifier and the test strip carrier is located on either side of the matrix metalloproteinase cleavage site in the matrix metalloproteinase recognition fragment, respectively.

[0039] The detection principle of the visual detection strip for matrix metalloproteinases provided by this invention is as follows: A visual marker is attached to the surface of the test strip carrier. In the absence of a reaction, the surface of the test strip carrier is enriched with the visual marker, resulting in a certain level of observable signal on the test strip surface. When the test strip is used to detect a sample, if matrix metalloproteinases are present in the sample, they recognize and cleave the matrix metalloproteinase recognition fragment on the marker. Since the connection point between the marker and the test strip carrier is located on either side of the matrix metalloproteinase cleavage site in the matrix metalloproteinase recognition fragment, the visual marker is cut off from the test strip carrier after the matrix metalloproteinase cleavage fragment, reducing the observable signal on the test strip. By observing the change in the intensity of the observable signal on the test strip, the presence of matrix metalloproteinases in the sample and the concentration of matrix metalloproteinases in the sample can be detected. The test strip provided by this invention avoids the use of multiple reagents during detection; only sufficient contact between the sample and the test strip is required. The matrix metalloproteinase recognition fragment can be selected according to the specific type of matrix metalloproteinase being detected, and this invention does not impose any limitations on this.

[0040] The visual markers described in this invention refer to markers that provide observable signals. These observable signals may optionally be signals that can be directly observed with the naked eye, such as color-providing visual markers. As the number of color-providing visual markers attached to the test strip carrier decreases, the test strip color becomes lighter. Alternatively, the observable signals may also be signals that can be observed by an instrument, such as fluorescence-providing visual markers. As the number of fluorescence-providing visual markers attached to the test strip carrier decreases, the fluorescence of the test strip weakens. Visual markers include, but are not limited to, quantum dots, colloidal gold, colloidal silver, carbon nanoparticles, selenium nanoparticles, fluorescent microspheres, or colored microspheres, such as colored polystyrene microspheres or colored latex microspheres.

[0041] In some alternative embodiments, the modifier is attached to the test strip carrier via a specific binding pair. A specific binding pair refers to a pair of substances capable of specific binding, such as, but not limited to, an antibody and its specific antigen, streptavidin and biotin, or a ligand and its receptor. One of the specific binding pairs is modified onto both the test strip carrier and the modifier, allowing the modifier to be grafted onto the test strip carrier through the specific binding of the two substances in the specific binding pair.

[0042] In some alternative embodiments, the specific binding pair is selected from biotin and streptavidin, preferably streptavidin is modified on the surface of the test strip carrier, the modifier containing biotin, so that the modifier and the test strip carrier are grafted onto the surface of the test strip carrier through the interaction of streptavidin and biotin.

[0043] In some alternative embodiments, the peptide contained in the modifier, excluding the matrix metalloproteinase recognition fragment, may also be modified by adding arbitrary amino acid residues to the C-terminus and N-terminus of the recognition fragment. For example, amino acid residues that can provide linkage sites with the visualization marker and other N-terminal protein ligands may be added. Preferably, when the visualization marker is colloidal gold, at least one cysteine ​​residue is preferably provided at one end of the peptide to provide a thiol group. The thio group can form a stable Au-S bond with the gold nanoparticle, thereby linking the peptide to the colloidal gold.

[0044] In some alternative embodiments, the test strip carrier is selected from nitrocellulose membranes, cellulose acetate membranes, filter paper, polylactic acid membranes, or polyacrylonitrile membranes. For ease of use, the test strip carrier can be prepared in any size and shape practically usable in the art, such as, but not limited to, square, circular, or arc-shaped.

[0045] In some optional embodiments, the matrix metalloproteinase is MMP 9, and the amino acid sequence of the MMP recognition fragment is PLG↓VRG (Seq_1), with the cleavage site between G and V (arrows indicate positions). The test strip for visual detection of MMP 9 is preferably as follows: the test strip carrier is modified with streptavidin (SA); the modifier contains peptide-modified colloidal gold, preferably with upstream and downstream modifications of PLG↓VRG, resulting in the peptide amino acid sequence GGGPLG↓VRGKGGC (Seq_2), where the C-terminus of GGGPLG↓VRGKGG contains a cysteine ​​residue (C), introducing a thiol group to link the peptide to the colloidal gold; the N-terminus of GGGPLG↓VRGKGGC is linked with biotin (bio), and the modifier structure is as follows: bio-GGGPLG↓VRGKGGC-Au. The modifier is attached to the test strip carrier via biotin and streptavidin specific binding. The structure of the test strip after attachment is as follows: Test strip carrier-SA / bio-GGGPLG↓VRGKGGC-Au.

[0046] According to one aspect of the present invention, the present invention also provides a method for preparing the above-mentioned test strip, comprising attaching the modifier to the test strip carrier.

[0047] In some optional embodiments, the specific binding pair is biotin and streptavidin, the visual marker is colloidal gold, and the test strip is prepared as follows:

[0048] The streptavidin-modified test paper carrier is preferably prepared according to the following method: the test paper carrier is incubated with streptavidin, and then incubated with BSA solution to obtain the streptavidin-modified test paper carrier. The incubation conditions for the test paper carrier and streptavidin are: 4–37℃ for 1–8 h. Specific incubation temperatures and times can be, for example, but are not limited to, 4℃ for 1 h or 37℃ for 8 h. It is understood that as the incubation temperature increases, the incubation time decreases. The area-to-volume ratio of the test paper carrier to the streptavidin solution is (5–10):100 mm². 2 The concentration of streptavidin solution was 5–15 μg / mL. The incubation conditions for BSA solution were: 4–37°C for 0.5–8 h, and the BSA solution concentration was 1–5% m / v. After each step in preparing the streptavidin-modified test paper carrier, the sample was washed with buffer solution.

[0049] The preferred method for introducing colloidal gold into the modified material is as follows: A colloidal gold solution is incubated with a biotin-linked peptide, followed by incubation with BSA. After centrifugation, the supernatant is collected and resuspended in BSA solution to obtain the modified material. The incubation conditions for the colloidal gold solution and the biotin-linked peptide are: 4–37°C for 1–8 h. Specific incubation temperatures and times can be, for example, but are not limited to, 4°C for 1 h or 37°C for 8 h. It is understood that the incubation time decreases as the incubation temperature increases. The mass-to-volume ratio of the biotin-linked peptide to the colloidal gold solution is 1:(0.5–1.5) μg / mL. The colloidal gold solution can be prepared according to general methods in the art, and this invention does not limit this. The incubation conditions for adding BSA are: 4–37°C for 0.5–8 h, with a BSA solution concentration of 1–5% m / v; the amount of BSA solution added is 10–30% of the volume of the colloidal gold solution.

[0050] According to another aspect of the present invention, the present invention also provides a matrix metalloproteinase detection kit comprising the above-described visual detection strip for matrix metalloproteinases. It is understood that the kit may also contain reagents or consumables generally known in the art for kit detection, such as, but not limited to, one or more of negative controls, positive controls, colorimetric cards, diluents, and buffers.

[0051] According to another aspect of the present invention, the present invention also provides a method for detecting matrix metalloproteinases (MMPs) for non-diagnostic and non-therapeutic purposes, comprising contacting a test sample with the test strip to ensure sufficient contact between the MMPs in the test sample and the modified material grafted onto the test strip, and then determining the MMP content in the test sample based on the observable signal change of the test strip, wherein the observable signal change is preferably a color change. Sufficient contact can be achieved, for example, by covering the test strip with the test sample, or by immersing the test strip in the test sample. If the test sample does not meet the conditions for immersing the test strip, the test sample can be pretreated according to generally known methods in the art, such as dilution or extraction, to prepare a sample type suitable for immersion in the test strip. This method can qualitatively or semi-quantitatively determine whether the test sample contains MMPs based on the observable signal change of the test strip, or it can introduce a control, such as a colorimetric card, with a pre-determined correspondence between the observable signal value and the MMP content in the test sample, and determine the MMP content in the test sample based on the control after detection.

[0052] The technical solution and beneficial effects of the present invention will be further described below with reference to preferred embodiments.

[0053] Example

[0054] This embodiment provides a test strip for visually detecting matrix metalloproteinase 9 (MMP 9). The test strip carrier is a circular disc with a diameter of 3 mm. The test strip carrier material is a nitrocellulose membrane, and the surface of the test strip carrier is modified with streptavidin (SA). The surface of the test strip carrier is grafted with a modifier with the following structure: bio-GGGPLG↓VRGKGGC-Au. Among them, PLG↓VRG is the MMP9 recognition fragment. MMP9 can cleave the G and V in PLG↓VRG (Seq_1) between them (i.e., at the position shown by the arrow). The C-terminus and N-terminus of PLG↓VRG are modified to obtain the following peptide: GGGPLG↓VRGKGGC (Seq_2). Biotin (bio) is linked to the N-terminus of GGGPLG↓VRGKGGC. A cysteine ​​(C) residue is linked to its C-terminus, which allows the peptide to be linked with colloidal gold (Au) to obtain the modified bio-GGGPLG↓VRGKGGC-Au.

[0055] The modifier is attached to the streptavidin (SA) modified on the surface of the test strip carrier via a biotin linker at one end, thus grafting the modifier onto the test strip carrier surface. This grafting causes the colloidal gold to bind to the test strip carrier, resulting in a red color on the test strip. When the test strip comes into contact with MMP9, MMP9 recognizes and cleaves the PLG↓VRG peptide, causing the colloidal gold to separate from the test strip carrier, resulting in a lighter color on the test strip. The detection of MMP9 in the sample is achieved through this color change. The preparation method of the test strip in this embodiment is as follows:

[0056] (1) Modification of the test paper carrier: Nitrocellulose membrane (Milipore, NC, 0.45 μm) was cut into circular pieces with a diameter of 3 mm and incubated with 100 μL of streptavidin (Solepro, SA, 10 μg / mL) at 37 °C for 1 h or at 4 °C for 8 h. Subsequently, it was incubated with 3% bovine serum albumin (BSA) solution at 37 °C for 0.5 h. After each step, it was washed three times with 0.01 M PBST solution to obtain streptavidin-modified nitrocellulose membrane (NC-SA).

[0057] (2) Preparation of the modifier:

[0058] (2.1) Preparation of colloidal gold: Add 1 mL of 1% chloroauric acid solution to 100 mL of ultrapure water, heat to boiling, then add 1 mL of 1% sodium citrate solution, continue heating and stirring until the solution turns into a stable red color.

[0059] (2.2) Colloidal gold modification: Take 1 mL of colloidal gold solution and add 100 μL of 10 μg / mL ester peptide (bio-GGGPLG↓VRGKGGC, Qiangyao Biotechnology), incubate at 37℃ for 1 h or at 4℃ for 8 h. Then add 200 μL of 3% m / v BSA solution and incubate at 37℃ for 0.5 h. Then centrifuge at 10,000 rpm, remove the supernatant, resuspend in 200 μL of 1% m / v BSA solution to obtain ester peptide modified colloidal gold (bio-GGGPLG↓VRGKGGC-Au), and store at 4℃.

[0060] (3) The combination of test paper carrier and dye

[0061] The NC-SA membrane was incubated with 100 μL of bio-GGGPLG↓VRGKGGC-Au solution at 37°C for 1 h or at 4°C for 8 h. Since SA specifically binds to bio, Au can be grafted onto the NC membrane, resulting in NC-SA / bio-GGGPLG↓VRGKGGC-Au, and the color of the NC membrane will change from white to red.

[0062] Example of effect

[0063] The concentration was detected using the MMP 9 cleavage characteristics:

[0064] An NC-SA / bio-GGGPLG↓VRGKGGC-Au membrane with a diameter of 3 mm was co-incubated with 100 μL of MMP 9 solutions of different concentrations (0 μg / mL, 1 μg / mL, 2 μg / mL, 4 μg / mL, and 6 μg / mL) at 37 °C. The color changes of the NC membrane at different concentrations were observed, and the results are as follows: Figure 1 As shown. By Figure 1 As can be seen, within the same detection time, the color of the NC membrane fades as the MMP 9 concentration increases. This is because as the MMP 9 concentration increases, more modifiers are cleaved, Au leaves the NC membrane, causing the NC membrane to gradually return to its white color.

[0065] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A test strip for visually detecting matrix metalloproteinases, characterized in that, The test strip includes a test strip carrier and a modification grafted onto the test strip carrier; The test strip is a visual test strip for detecting MMP 9; the test strip carrier is modified with streptavidin; the modifier contains a peptide-modified colloidal gold, the amino acid sequence of which is shown in SEQ ID NO.2, and the other end of which is modified with biotin; the modifier is linked to the test strip carrier by biotin specifically binding to streptavidin.

2. The test strip according to claim 1, characterized in that, The test paper carrier is selected from nitrocellulose membrane, cellulose acetate membrane, filter paper, polylactic acid membrane, or polyacrylonitrile membrane.

3. The test strip according to claim 1, characterized in that, The shape of the test strip carrier is selected from square, circular or arc.

4. The method for preparing the test strip according to any one of claims 1-3, characterized in that, This includes attaching the modification to the test strip carrier.

5. The preparation method according to claim 4, characterized in that, The test strip carrier was incubated with streptavidin, and then further incubated with BSA solution to obtain a streptavidin-modified test strip carrier. The incubation conditions for the test strip carrier and streptavidin were: 4~37℃ for 1~8h, and the area-to-volume ratio of the test strip carrier to the streptavidin solution was (5~10):100 mm². 2 / μL; streptavidin concentration is 5~15 μg / mL.

6. The preparation method according to claim 5, characterized in that, The incubation conditions for further incubation with BSA solution are: 4~37℃ for 0.5~8 h, and BSA solution concentration of 1~5% m / v.

7. The preparation method according to claim 4, characterized in that, The colloidal gold solution was incubated with the biotin-linked peptide, then BSA was added and incubated. After centrifugation, the supernatant was collected and resuspended in BSA solution to obtain the modified product. The incubation conditions for the colloidal gold solution and the biotin-linked peptide were: 4~37℃ for 1~8h, and the mass-to-volume ratio of the biotin-linked peptide to the colloidal gold solution was 1:(0.5~1.5)μg / mL.

8. The preparation method according to claim 7, characterized in that, The incubation conditions for adding BSA are: incubation at 4~37℃ for 0.5~8 h, BSA solution concentration of 1~5% m / v; the amount of BSA solution added is 10~30% of the volume of colloidal gold solution.

9. A matrix metalloproteinase detection kit, characterized in that, The test strip comprises any one of claims 1-3.

10. A method for detecting matrix metalloproteinases for non-diagnostic and non-therapeutic purposes, characterized in that, This includes contacting the sample to be tested with the test strip according to any one of claims 1-3, and determining the content of matrix metalloproteinase in the sample to be tested based on the observable signal change of the test strip.

11. The method according to claim 10, characterized in that, The method includes contacting the sample to be tested with the test strip and determining the content of matrix metalloproteinase in the sample based on the color change of the test strip.

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