A magnetic probe and its application in preparation and detection of matrix metalloproteinases

Abstract

The present invention belongs to the field of biomedicine technology, and more specifically, to a magnetic probe and its application in the preparation and detection of matrix metalloproteinases. The present invention provides a magnetic probe and its application in the preparation and detection of matrix metalloproteinases, the purpose of which is to utilize the hydrolysis characteristics of matrix metalloproteinases to identify specific oligopeptide sequences and cut them, prepare functionalized signal probes, and combine magnetic separation technology and commercial test strips to solve the problems of high cost, low detection sensitivity and narrow scope of application of the existing technology. The present invention utilizes the high specificity of protease hydrolysis and the high sensitivity of the test strips to the target probe to achieve sensitive detection of MMP-1, with a visual detection limit of 65.5 pg / mL, which has good anti-interference and sensitivity in the application of biological sample matrices.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and more specifically, relates to a magnetic probe and its application in the preparation and detection of matrix metalloproteinases. Background Art

[0002] Matrix metalloproteinases (MMPs) are a class of Zn 2+ A family of neutral proteases, known as matrix metalloproteinases, are generally believed to primarily function in degrading and remodeling the extracellular matrix. During tumor invasion, tumor cells secrete degradative enzymes such as matrix metalloproteinases (MMPs), which degrade the basement membrane and matrix, allowing them to grow around the defect. MMPs, as promoters of tumor cells, regulate numerous genes involved in cellular carcinogenesis and have a significant impact on primary tumor growth. Studies have shown a significant positive correlation between tumor metastasis and the content of MMPs, demonstrating that MMPs can enhance tumor metastasis by degrading the matrix. Furthermore, MMPs can promote angiogenesis, allowing for precise regulation of tumor cell adhesion, detachment, and migration under varying conditions to achieve invasion and metastasis. Therefore, monitoring MMPs can be used to indicate the progression of tumor development and provide an effective basis for early diagnosis.

[0003] Several matrix metalloproteinases have been found to be abnormally expressed in oral squamous cell carcinoma (OSCC) and are involved in its invasion and metastasis. Studies have shown that MMP-1, a member of the matrix metalloproteinase family, can be used as a screening target for oral cancer initiation and has potential for diagnosis. A saliva concentration >2.77 ng / mL can be used as a preliminary diagnostic indicator for OSCC, with a positive rate as high as 78%. Therefore, measuring MMP-1 levels in saliva is crucial for OSCC screening.

[0004] Immunochromatographic assay technology is a mature rapid immunoassay technology that is sensitive, accurate, stable, reliable, safe, and simple. It is also small in size and easy to carry, meeting the needs of rapid, convenient, and popular qualitative testing. The amount of reagents and samples usually used in its use is extremely small, the cost is low, and the results are easy to observe with the naked eye. Operators do not need to undergo special and complex training, and it is environmentally friendly, making it very suitable for individual self-testing. Conventional test strips for tumor marker detection are mostly based on the double antibody sandwich method. However, the use of double antibodies undoubtedly increases costs. At the same time, the overall design and assembly process of the test strips also need to take into account the activity of the antibodies, which correspondingly increases the difficulty of research and development.

[0005] Currently, pregnancy test strips have been successfully commercialized, and their production and sales have been widely covered. They are very easy to obtain for most users, and their costs have been greatly reduced due to their widespread application. Therefore, if the detection target of the pregnancy test strip, human chorionic gonadotropin (hCG), can be converted into a tumor marker through appropriate design, it will greatly reduce the difficulty and cost of research and development. At the same time, it can also achieve universality in detecting different targets, making the detection method simpler. Summary of the Invention

[0006] In response to the above-mentioned defects or improvement needs of the existing technology, the present invention provides a magnetic probe and its application in the preparation and detection of matrix metalloproteinases. Its purpose is to utilize the hydrolysis characteristics of matrix metalloproteinases to identify specific oligopeptide sequences and cut them, thereby preparing a functional signal probe. At the same time, it combines magnetic separation technology with commercial test strips, thereby solving the problems of high cost, low detection sensitivity and narrow scope of application of the existing technology.

[0007] According to a first aspect of the present invention, a method for preparing a magnetic probe is provided, comprising the following steps:

[0008] (1) adding a thiol-modified first single-stranded DNA fragment to an oligopeptide solution, so that the thiol group on the first single-stranded DNA fragment is linked to the oligopeptide to obtain a first probe; the oligopeptide can be cleaved by a matrix metalloproteinase;

[0009] (2) activating the transduction molecule, and then adding a second single-stranded DNA fragment modified with a thiol group, so that the thiol group on the second single-stranded DNA fragment is connected to the transduction molecule to obtain a second probe; the second single-stranded DNA fragment and the first single-stranded DNA fragment are complementary in base pairing;

[0010] (3) Adding magnetic nanoparticles to the first probe to connect the first probe to the magnetic nanoparticles, and then adding the second probe to make the first single-stranded DNA fragment on the first probe and the second single-stranded DNA fragment on the second probe complementary to each other, thereby obtaining the magnetic probe.

[0011] Preferably, in step (1), the amino acid sequence of the oligopeptide is KPLGLARK, PLALWAR, PC(Me)HAK, GPLGYLWAR or PLGC(Me)HA(dR), where (Me) represents C (methylated cysteine) and (dR) represents arginine in the D configuration.

[0012] Preferably, in step (2), the transduction molecule is human chorionic gonadotropin, luteinizing hormone, bilirubin, ascorbic acid or creatinine.

[0013] Preferably, in step (1), biotin and a maleimide group are connected to both ends of the oligopeptide, respectively, and the sulfhydryl group on the first single-stranded DNA fragment is connected to the maleimide group on the oligopeptide;

[0014] In step (2), 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonic acid succinimide ester sodium salt is added to the transduction molecule solution to activate the transduction molecule, and the sulfhydryl group on the second single-stranded DNA fragment is connected to the 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonic acid succinimide ester sodium salt on the transduction molecule.

[0015] Preferably, in step (1), the ratio of the mass of the oligopeptide to the amount of the first single-stranded DNA fragment is (5-12.5 μg):(0.8-4 nmol); in step (2), the ratio of the mass of the transduction molecule to the amount of the second single-stranded DNA fragment is (20-200 μg):(1.5-15 nmol); in step (3), the ratio of the mass of the magnetic nanoparticles, the amount of the first probe and the amount of the second probe is (1-5 mg):(0.2-0.8 nmol):(0.1-0.4 nmol).

[0016] Preferably, in step (3), the size of the modified magnetic nanoparticles is 40 to 80 nm;

[0017] Preferably, the magnetic nanoparticles are carboxyl-modified, amino-modified or streptavidin-modified.

[0018] According to another aspect of the present invention, a magnetic probe prepared by any of the methods described is provided.

[0019] According to another aspect of the present invention, a kit for detecting matrix metalloproteinases is provided, comprising the magnetic probe.

[0020] According to another aspect of the present invention, there is provided a use of the magnetic probe for detecting matrix metalloproteinases. The magnetic probe is added to a sample, and after the reaction, a centrifuge tube containing the sample solution is subjected to magnetic separation. A test strip is immersed in the supernatant, and the test strip can specifically detect the transduction molecules on the magnetic probe. If the detection line on the test strip develops color, the sample contains matrix metalloproteinases; if the detection line on the test strip does not develop color, the sample does not contain matrix metalloproteinases.

[0021] Preferably, a matrix metalloproteinase standard solution with gradient concentration is prepared, a standard curve is drawn between the concentration and the corresponding detection line signal value, and the concentration of the matrix metalloproteinase in the sample is calculated based on the standard curve.

[0022] In general, the above technical solutions conceived by the present invention can achieve the following beneficial effects compared with the prior art:

[0023] (1) The magnetic probe designed in the present invention differs from conventional immunomagnetic probes in that its surface does not recognize and capture targets through immobilized antibodies, but rather is coupled to specific oligopeptide probes to achieve target detection and signal transduction, resulting in extremely high specificity. By magnetically separating the uncleaved signal probes, the present invention can also effectively eliminate the interference of false positive results.

[0024] (2) The present invention avoids the assembly of the test strip and the preparation of the recognition antibody by performing colorimetric determination on the existing test strip, and at the same time effectively avoids matrix interference by utilizing magnetic separation; the present invention utilizes the high specificity of protease hydrolysis and the high sensitivity of the test strip to the target probe to achieve sensitive detection of matrix metalloproteinase MMP-1, with a visual detection limit of 65.5 pg / mL. It has good anti-interference and sensitivity in the application of biological sample matrices and can meet the detection needs in clinical diagnosis.

[0025] (3) The transduction molecules in the magnetic probes provided by the present invention have a wide range of options, and probes labeled with corresponding detection substances can be designed based on different commercial test strips. In addition, the preferred early pregnancy test strips of the present invention cost less than 1 yuan, which is the lowest cost among all types of test strips on the market. Therefore, the preferred hCG-labeled probes of the present invention have great promotional value.

[0026] (4) The present invention has a high selectivity for matrix metalloproteinase MMP-1, which effectively reduces the interference of interfering substances in the sample on the detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Schematic diagram of the synthesis principle of the signal probes SP-DNA1 and hCG-DNA2 of the present invention.

[0028] Figure 2 This is a schematic diagram of the test strip detection principle of MMP-1 of the present invention.

[0029] Figure 3 Flow chart of the quantitative detection of the present invention.

[0030] Figure 4 This is a graph showing the relationship between band intensity and MMP-1 concentration in Example 1 of the present invention and a corresponding physical image of the test strip.

[0031] Figure 5 This is the specific detection of MMP-1 by the test strip of Example 2 of the present invention.

[0032] Figure 6This is a comparison diagram of the test strip in Example 3 of the present invention and the enzyme-linked immunosorbent assay for detecting MMP-1. DETAILED DESCRIPTION

[0033] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely for the purpose of explaining the present invention and are not intended to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

[0034] The present invention provides a method for rapid visual detection of MMP-1, which includes the steps of preparing and modifying magnetic probes. The signal probes are single-stranded DNA1 coupled with oligopeptide (SP-DNA1) and DNA2 coupled with hCG (hCG-DNA2). Magnetic separation is used to eliminate false positive interference generated by free hCG-DNA2 during the analysis process.

[0035] The present invention provides a rapid visual detection method for matrix metalloproteinase (MMP-1), an analytical method based on the specific hydrolysis of oligopeptides by proteases, and a rapid visual detection method for early pregnancy test strips assisted by magnetic separation technology. This method prepares two signal probes: oligopeptide-DNA1 (SP-DNA1) and hCG-DNA2. The other end of the oligopeptide sequence is also labeled with biotin, which stably binds to the prepared and modified Fe3O4-PEI@streptavidin through a biotin-streptavidin interaction. The coupled DNA1 sequence is complementary to the hCG-coupled DNA2 sequence, allowing the hCG-DNA2 to be further bound to the surface of magnetic particles. In the presence of MMP-1, it specifically recognizes and cleaves the oligopeptide sequence, subsequently releasing a hybridization probe containing the hCG-modified DNA. After magnetic separation of the magnetic complex, the hCG probe in the supernatant can be visualized by applying a pregnancy test strip. As the MMP-1 concentration increases, the amount of free hCG probe generated by cleavage increases, allowing the measurement of MMP-1 using the pregnancy test strip. After color development is complete, quantitative detection can be performed using smartphone applications and portable devices developed in our laboratory.

[0036] The present invention comprises a signal probe, a magnetic probe and an early pregnancy test strip for detecting matrix metalloproteinases, wherein the signal probe comprises SP-DNA1 and hCG-DNA2; the magnetic probe is a magnetic complex that sequentially modifies SP-DNA1 and hCG-DNA2; the early pregnancy test strip detects hCG; the early pregnancy test strip comprises a plastic-sealed sample pad, a gold label pad, a nitrocellulose membrane and absorbent paper, and the nitrocellulose membrane is provided with a detection line (T line) and a quality control line (C line).

[0037] The detection method of the present invention specifically includes the following steps.

[0038] 1) Preparation of signal probe:

[0039] Preparation of SP-DNA1 probe: Tris(2-carboxyethyl)phosphine hydrochloride was added to a thiol-modified DNA1 solution and allowed to stand for 30 minutes. The solution was then ultrafiltered and purified eight times using phosphate buffer. The solution was then added to an oligopeptide solution modified with maleimide and biotin at both ends, respectively, and reacted for 6 hours. The solution was then ultrafiltered and purified eight times using phosphate buffer. The final solution was diluted to 200 μL to obtain the prepared SP-DNA1 probe.

[0040] Preparation of hCG-DNA2 probe: Tris(2-carboxyethyl)phosphine hydrochloride was added to a thiol-modified DNA2 solution, allowed to stand for 1 hour, and then ultrafiltration was performed eight times using a phosphate buffer solution. Simultaneously, 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimidyl ester sodium salt was added to the hCG solution, reacted for 1 hour, and then ultrafiltration was performed eight times using a phosphate buffer solution. The two solutions were mixed and reacted for 24 hours, and ultrafiltration was performed eight times using a phosphate buffer solution. The final solution was diluted to 150 μL to obtain the prepared hCG-DNA2 probe.

[0041] The phosphate buffer solution is a mixed solution of 8.1 mM Na2HPO4, 1.9 mM NaH2PO4 and 100 mM NaCl at pH 7.4.

[0042] The concentration of the prepared SP-DNA1 probe was 20 μM, and the concentration of the prepared hCG-DNA2 probe was 10 μM.

[0043] 2) Preparation of magnetic probe:

[0044] Ferrous chloride was dissolved in water, and then ammonia was added and the mixture was exposed to air and stirred continuously for 10 minutes. The resulting suspension was added to a Teflon-lined stainless steel autoclave, and a polyethyleneimine (PEI) aqueous solution was added to the autoclave. After thorough stirring, the mixture was heated at 134°C for 3 hours. After the reaction was completed, the resulting product was cooled to room temperature. The black precipitate was collected by magnetic separation, washed with water and ethanol respectively, and then dried in a vacuum drying oven at 50°C to obtain the prepared magnetic particles (Fe3O4-PEI);

[0045] Fe3O4-PEI was added to an aqueous glutaraldehyde solution and ultrasonically dispersed for 2 hours. The activated magnetic particles were washed with phosphate buffer and resuspended. Streptavidin solution was added to the magnetic suspension and reacted for 2 hours, followed by bovine serum albumin solution to block unmodified sites. An N-terminally biotinylated SP-DNA1 probe was added to the washed suspension and reacted for 2 hours. The resulting magnetic complex was washed and resuspended in Tris-buffered saline, and the hCG-DNA2 probe was added and reacted for 1 hour. The resulting magnetic probe was washed three times and resuspended in TCNB buffer.

[0046] Among them, the size of the Fe3O4-PEI magnetic particles in the magnetic probe is 40 to 80 nm, preferably 60 nm. Generally, the larger the particle size of the magnetic particles, the better the magnetic responsiveness, but it is easy to aggregate, which hinders the reaction at the interface and dispersion in the solution; conversely, the smaller the particle size, the less likely it is to aggregate, but the magnetic responsiveness will be reduced. 60 nm magnetic particles are preferably used to prepare magnetic probes.

[0047] The Tris salt buffer is a mixed solution of 10 mM Tris-HCl, 5 mM MgCl2, 100 mM NaCl, and 5 mM KCl at pH 7.4.

[0048] The TCNB buffer is a mixed solution of 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, and 0.05% (w / v) Brij-35 at pH 7.5.

[0049] Since the concentration of the SP-DNA1 and hCG-DNA2 probes prepared in step (1) is about 2:1, the volume of the SP-DNA1 and hCG-DNA2 probe solutions added is preferably 1:2. By optimizing the ratio of the two signal probes, the optimal binding efficiency is achieved, thereby improving the reaction efficiency.

[0050] 3) Sample Analysis: MMP-1 solutions of varying concentrations were added to the magnetic probe solution and allowed to react for 1 hour. A centrifuge tube containing the sample solution was then inserted into the wells of a magnetic rack and allowed to stand for 15 seconds for magnetic separation. An early pregnancy test strip was immersed in the supernatant and allowed to stand for 10 seconds. After a 10-minute chromatography reaction, red bands were visible on the test and control lines of the test strip. The sample was then photographed with a smartphone, and the MMP-1 concentration in the sample was determined using a smartphone application and portable device developed in our laboratory.

[0051] The amount of magnetic probe used should not be too much or too little, the reaction time between MMP-1 and the magnetic probe should not be too long or too short, and the reaction temperature should not be too high or too low. Using too little or too short a reaction time will result in an incomplete reaction, while using too much or too long a reaction time will waste materials and time. Excessively high or low reaction temperatures will affect the enzymatic activity of MMP-1, resulting in a decrease in the detection signal and affecting the sensitivity of the test strip. Therefore, through repeated experiments, the present invention has optimized the mass concentration of the magnetic probe to be 1 to 5 mg / mL, preferably 4 mg / mL, the reaction time to be 30 to 120 minutes, preferably 75 minutes, and the reaction temperature to be 20 to 55°C, preferably 35°C.

[0052] The magnetic separation time is not less than 15 seconds. The separation of the magnetic probe is very important for the subsequent detection of MMP-1, so the magnetic separation time must be guaranteed to ensure that the magnetic probe can be completely removed to avoid affecting the subsequent detection work.

[0053] Preferably, the mass concentration and molar concentration ratio of the oligopeptide and DNA1 in the signal probe SP-DNA1 is 0.1-2.5 mg / mL:(20-100 μM), preferably 1 mg / mL:100 μM.

[0054] Preferably, the mass concentration and molar concentration ratio of hCG and DNA2 in the signal probe hCG-DNA2 is 0.1-1 mg / mL:10-100 μM, preferably 0.1 mg / mL:10 μM.

[0055] Preferably, the magnetic probe is a carboxylation, an aminoation or a streptavidin-modified magnetic particle, preferably a streptavidin-modified magnetic particle.

[0056] Preferably, the mass concentration and molar concentration ratio of the magnetic particles, SP-DNA1 and hCG-DNA2 in the magnetic probe is 1-5 mg / mL:10-40 μM:1-10 μM, preferably 5 mg / mL:20 μM:10 μM.

[0057] Preferably, the detection method includes the steps of preparing and modifying a magnetic probe, and the signal probes are single-stranded DNA1 coupled with oligopeptide (SP-DNA1) and DNA2 coupled with hCG (hCG-DNA2).

[0058] Preferably, the particle size of Fe3O4-PEI in the magnetic probe in step (2) is 40 to 80 nm.

[0059] Preferably, the magnetic separation time in steps (2) and (3) is not less than 15s.

[0060] Preferably, the brand of the early pregnancy test strips in step (3) is Yuting, David or Blue Cross.

[0061] According to another aspect of the present invention, a method for rapid visual detection of MMP-1 is provided. The detection target MMP-1 can recognize and cleave a specific oligopeptide sequence. hCG in the probe hCG-DNA2 is the detection target of an early pregnancy test strip. The signal probes SP-DNA1 and hCG-DNA2 can be bound to magnetic particles through affinity and hybridization complementarity, respectively. The detection target MMP-1 cleaves the SP-DNA1 probe to produce a free hCG hybridization probe. After magnetic separation, the solution is loaded onto an early pregnancy test strip to achieve color development on the detection line, thereby indirectly completing visual detection of MMP-1.

[0062] The DNA1 sequence is complementary to the DNA2 sequence, and one side of the oligopeptide sequence is labeled with biotin, allowing it to bind to streptavidin magnetic particles. hCG-DNA2 can then be bound to the surface of the magnetic particles. When MMP-1 is present, it specifically recognizes and cleaves the oligopeptide sequence, releasing the hCG-modified DNA hybridization probe. After magnetic separation of the magnetic complex, the hCG probe in the supernatant can be visualized by applying an early pregnancy test strip.

[0063] In some embodiments, the first single-stranded DNA 1 is 5'-GATACGGTCT AGCTTATTGA TATGCTTTTT T-C6-SH-3' (SH represents a thiol-modified 3' end of a DNA sequence), which is complementary to the 5'-GCATATCAAT AAGCTAGACCGTATC-3' sequence of the second single-stranded DNA 2.

[0064] The oligopeptide sequence in the following example is: Biotin-KPLGLARK-maleimide (Biotin is a biotin modification that binds to streptavidin magnetic particles, and maleimide is a maleimide modification that couples to DNA1).

[0065] Figure 1 Schematic diagram of the principle of synthesis of the probe designed for the present invention; Figure 2 This is a schematic diagram of the rapid detection principle of MMP-1 of the present invention.

[0066] The following are specific embodiments:

[0067] Example 1

[0068] A rapid visual detection method for MMP-1 comprises the following steps:

[0069] (1) Preparation of signal probe:

[0070] Preparation of SP-DNA1: Add 10 μL of 10 mM TCEP solution to 40 μL of 100 μM DNA1 aqueous solution and let stand at room temperature for 30 min. Purify the solution using a 3K ultrafiltration tube. Subsequently, add 50 μL of oligopeptide solution (1 mg / mL) to the DNA1 solution and let it react for 6 h. Purify the conjugate using a 10K ultrafiltration tube to remove any remaining unattached oligopeptide. The final solution is diluted to 200 μL and stored in a refrigerator at 4°C until use.

[0071] Preparation of hCG-DNA2: Add 10 μL of TCEP solution (300 μM) and 10 μL of phosphate buffer (10 mM, pH 5.5) to 150 μL of DNA2 aqueous solution (10 μM) and let it stand at room temperature for 1 hour. The resulting solution was purified using a 10K ultrafiltration tube. Simultaneously, 200 μL of hCG (0.1 mg / mL) solution was mixed with 20 μL of sulfo-SMCC aqueous solution (0.1 mg / mL). The resulting solution was incubated at room temperature with gentle shaking for 1 hour and purified using a 3K ultrafiltration tube to remove unreacted coupling agent. The activated hCG and DNA2 solution were then incubated at room temperature for 24 hours. The conjugate was purified using a 10K ultrafiltration tube to remove any remaining unreacted sulfhydryl DNA, and the final volume was adjusted to 150 μL. The resulting hCG-DNA2 conjugate was stored in a refrigerator at 4°C until use.

[0072] (2) Preparation of magnetic probe:

[0073] 1.25 g of FeCl2·4H2O was dissolved in 7.75 mL of water. Under vigorous stirring, 6.25 mL of aqueous ammonia was added, and the resulting suspension was stirred continuously in air for 10 minutes. The suspension was then added to a Teflon-lined stainless steel autoclave (25 mL), and 5 mL of an aqueous solution containing 0.54 g of PEI was added to the autoclave. After thorough stirring, the mixture was heated at 134°C for 3 hours. After the reaction was complete, the resulting product was cooled to room temperature. The black precipitate was collected by magnetic separation, washed with water and ethanol, and dried.

[0074] 5 mg of Fe3O4-PEI was added to 1 mL of 2% glutaraldehyde aqueous solution and ultrasonically dispersed, followed by gentle shaking for 2 hours. 20 μL of streptavidin solution (1 mg / mL) was added to the magnetic suspension, mixed thoroughly, and gently shaken for 2 hours. 1% BSA solution was added and incubated with gentle shaking for 30 minutes to block unmodified sites. 20 μL of N-terminally biotinylated SP-DNA1 probe (~20 μM) was added to the Fe3O4-PEI@streptavidin suspension and gently shaken for 2 hours to obtain Fe3O4-PEI@streptavidin@SP-DNA1. The resulting magnetic complex was washed and resuspended in TBS buffer (10 mM Tris-HCl, 5 mM MgCl2, 100 mM NaCl, 5 mM KCl, pH 7.4). 40 μL of hCG-DNA2 probe (~10 μM) was added and gently shaken for 1 hour. After the reaction, the obtained magnetic probe was washed three times and resuspended in TCNB buffer (50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% (w / v) Brij-35, pH 7.5), and stored at 4°C for future use.

[0075] (3) Drawing of standard curve:

[0076] Prepare 12 standard solutions of MMP-1 concentration gradient, with concentrations of 0 pg / mL, 10.5 pg / mL, 26.2 pg / mL, 65.5 pg / mL, 163 pg / mL, 409 pg / mL, 1.02 ng / mL, 2.56 ng / mL, 6.4 ng / mL, 40 ng / mL, 100 ng / mL, and 500 ng / mL.

[0077] Twelve portions of MMP-1 standard solution of different concentrations were added to the magnetic probe solution, mixed evenly, and reacted for 1 hour. The mixture was then magnetically separated. The early pregnancy test strip was immersed in the supernatant and allowed to stand for 10 seconds. After 10 minutes of reaction, red bands were visible on the test line and quality control line of the test strip. Photos were taken with a smartphone, and quantitative detection was completed using a smartphone application developed by our laboratory ( Figure 3 After the test strip is colored, read the test strip multiple times through the mobile app and select the average of the three readings to reduce the measurement error. After a series of known concentration samples are tested, the signal value results are obtained, and the concentration and the corresponding T line signal value are used to make a standard curve ( Figure 4 ).Depend on Figure 4 It can be seen that the signal value increases with the concentration, and then the signal value begins to decrease with the concentration. When the concentration exceeds 100 ng / mL, it is no longer suitable for quantitative detection. The relationship between the T line signal value (y) and the concentration (x) measured by the present invention conforms to y = 2.377x 0.219When testing samples of unknown concentration, the T-line signal value of the sample to be tested can be measured first, and then substituted into the formula of the standard curve to calculate the sample concentration.

[0078] Figure 4 The results showed that as the MMP-1 concentration increased, the peak value of the test strip detection line increased. Furthermore, when the MMP-1 concentration ranged from 65.5 pg / mL to 100 ng / mL, the MMP-1 concentration index value and the peak value of the test strip detection line showed a good linear relationship; the visual detection limit determined by the test strip was 65.5 pg / mL.

[0079] (4) Testing of samples to be tested

[0080] Fresh saliva samples were collected from healthy volunteers using the spitting method. Rinse the mouth with water for 2 minutes. One minute after rinsing, collect the saliva into a sterile centrifuge tube. The collected saliva sample was centrifuged at 4°C (10,000 rpm for 10 minutes) to remove large particles, diluted to half with water to reduce viscosity, and spiked with a specific amount of MMP-1. This method can detect MMP-1 concentrations as low as 1 ng / mL in the sample.

[0081] Example 2

[0082] Specificity test of the chromatographic test strip of the present invention for MMP-1 and other potential interfering substances in samples:

[0083] First, prepare 1 μmol / L K + 、SCN － , glucose, urea, uric acid, glutamic acid, lysine, immunoglobulin (IgG), amylase, lysozyme and 50 ng / mL MMP-1 standard solution, and then prepare a mixed sample consisting of MMP-1 and other substances so that the MMP-1 content is 50 ng / mL and the concentration of other interfering substances is 1 μmol / L.

[0084] The preparation steps of the magnetic probe were the same as those in step (2) of Example 1. Then, 50 ng / mL of MMP-1 standard solution, 1 μmol / L of K + , 1μmol / L SCN －, 1μmol / L glucose, 1μmol / L urea, 1μmol / L uric acid, 1μmol / L glutamate, 1μmol / L lysine, 1μmol / L immunoglobulin (IgG), 1μmol / L amylase, 1μmol / L lysozyme standard solution and MMP-1 (50ng / mL) and other substances mentioned above (1μmol / L), a total of 12 samples, were mixed evenly and reacted for 1h, and then the 12 mixed solutions were magnetically separated. The early pregnancy test strip was immersed in the supernatant and allowed to stand for 10s. After 10 minutes of reaction, red strips could be seen on the test line and quality control line of the test strip. Photos were taken with a smartphone, and quantitative detection was completed using a smartphone application developed by this laboratory.

[0085] Figure 5 In an experiment to test the specificity of early pregnancy test strips for MMP-1 and other substances, it was found that when the molar concentration of other interfering substances was 1000 times that of MMP-1, the response of MMP-1 to the test strip detection system was much greater than that of other substances; the response of a mixed sample consisting of MMP-1 and all other substances was basically close to that of MMP-1 alone, indicating that this test strip detection system has good anti-interference ability and good specificity.

[0086] Example 3

[0087] Comparison of test strips and ELISA kits:

[0088] Figure 6 The comparison chart of the test strip and the enzyme-linked immunosorbent assay kit for detecting MMP-1 shows that when the samples were measured within the effective concentration range of the enzyme-linked immunosorbent assay kit (0, 0.156, 0.312, 0.625, 1.25, 2.5, 5, 10 ng / mL), the signal value (OD 450 ) and the logarithm of the test strip detection signal can be fitted with a four-parameter curve, showing a good correlation. Theoretically, the signal value of an enzyme-linked immunosorbent assay typically has a certain four-parameter fitting relationship with the analyte concentration. Based on the fitting relationship between the test strip detection signal and the MMP-1 concentration described above, it can be seen that the logarithm of the test strip detection signal should show a good linear relationship with the MMP-1 concentration. Therefore, both methods conform to the four-parameter curve fitting, indicating that the test strip detection method of the present invention has considerable application potential compared to the enzyme-linked immunosorbent assay method.

[0089] It will be easily understood by those skilled in the art that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing a magnetic probe, characterized in that: The following steps are involved: (1) adding a thiol-modified first single-stranded DNA fragment to an oligopeptide solution, so that the thiol group on the first single-stranded DNA fragment is linked to the oligopeptide to obtain a first probe; the oligopeptide can be cleaved by a matrix metalloproteinase; The two ends of the oligopeptide are connected to biotin and maleimide groups respectively, and the sulfhydryl group on the first single-stranded DNA fragment is connected to the maleimide group on the oligopeptide; (2) adding sodium salt of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonyl succinimide ester to the transduction molecule solution to activate the transduction molecule, and then adding a second single-stranded DNA fragment modified with a thiol group, wherein the thiol group on the second single-stranded DNA fragment is linked to the sodium salt of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonyl succinimide ester on the transduction molecule to obtain a second probe; the second single-stranded DNA fragment is complementary to the first single-stranded DNA fragment in base pairing; The transduction molecule is human chorionic gonadotropin, luteinizing hormone or creatinine; (3) Adding magnetic nanoparticles to the first probe, wherein the magnetic nanoparticles are modified with streptavidin, so that the first probe is connected to the magnetic nanoparticles, and then adding the second probe so that the first single-stranded DNA fragment on the first probe is complementary to the second single-stranded DNA fragment on the second probe to obtain the magnetic probe.

2. The method for preparing a magnetic probe according to claim 1, wherein: In step (1), the amino acid sequence of the oligopeptide is KPLGLARK, PLALWAR, PCHAK, GPLGYLWAR or PLGCHA.

3. The method for preparing a magnetic probe according to claim 1, wherein: In step (1), the ratio of the mass of the oligopeptide to the amount of the first single-stranded DNA fragment is (5-12.5 μg):(0.8-4 nmol); in step (2), the ratio of the mass of the transduction molecule to the amount of the second single-stranded DNA fragment is (20-200 μg):(1.5-15 nmol); in step (3), the ratio of the mass of the magnetic nanoparticles, the amount of the first probe and the amount of the second probe is (1-5 mg):(0.2-0.8 nmol):(0.1-0.4 nmol).

4. The method for preparing a magnetic probe according to claim 1, wherein: In step (3), the size of the magnetic nanoparticles is 40 to 80 nm.

5. The magnetic probe prepared by the method according to any one of claims 1 to 4.

6. A kit for detecting matrix metalloproteinases, characterized in that: Comprising the magnetic probe according to claim 5.

7. Use of the magnetic probe according to claim 6 in preparing a reagent for detecting matrix metalloproteinases, characterized in that: The magnetic probe is added to the sample, and after the reaction, the centrifuge tube containing the sample solution is subjected to magnetic separation; a test strip is immersed in the supernatant, and the test strip can specifically detect the transduction molecule on the magnetic probe; If the detection line on the test strip develops color, the sample contains matrix metalloproteinases; if the detection line on the test strip does not develop color, the sample does not contain matrix metalloproteinases.

8. The use according to claim 7, characterized in that Prepare matrix metalloproteinase standard solutions with gradient concentrations, draw a standard curve based on the concentrations and corresponding detection line signal values, and then calculate the concentration of matrix metalloproteinase in the sample based on the standard curve.

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