CN115651964A

CN115651964A - Magnetic probe and preparation and application for detecting matrix metalloproteinase

Info

Publication number: CN115651964A
Application number: CN202211260257.1A
Authority: CN
Inventors: 赵元弟; 钟子涛
Original assignee: Huazhong University of Science and Technology
Current assignee: Huazhong University of Science and Technology
Priority date: 2022-10-14
Filing date: 2022-10-14
Publication date: 2023-01-31

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a magnetic probe and preparation and application thereof in detecting matrix metalloproteinase. The invention provides a magnetic probe and application of the magnetic probe in preparation and detection of matrix metalloproteinase, and aims to identify and cut a specific oligopeptide sequence by utilizing the hydrolysis characteristic of the matrix metalloproteinase, prepare a functionalized signal probe, and combine a magnetic separation technology and a commercialized test strip, thereby solving the problems of high cost, low detection sensitivity and narrow application range in the prior art. According to the invention, by utilizing the high specificity of proteolytic cleavage and the high sensitivity and reactivity of the test strip to the target probe, the sensitive detection of MMP-1 is realized, the visual detection limit is 65.5pg/mL, and the test strip has good anti-interference performance and sensitivity in the application of a biological sample matrix.

Description

Magnetic probe and preparation and application for detecting matrix metalloproteinase

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a magnetic probe and preparation and application thereof in detecting matrix metalloproteinase.

Background

Matrix Metalloproteinases (MMPs) are a class of Zn ²⁺ The main function of the family of dependent neutral proteases is generally considered to be degradation and remodeling of extracellular matrix, and during tumor invasion, tumor cells secrete degrading enzymes such as matrix metalloproteinase to degrade basement membrane and matrix, and then grow around the defect gap. The matrix metalloproteinase is used as a promoting factor of tumor cells, regulates a plurality of genes related to canceration of cells, and has important influence on the growth of primary tumors. Research shows that the tumor metastasis capacity is in obvious positive correlation with the content of MMPs, and that the MMPs can degrade matrix to enhance the tumor metastasis capacity. In addition, MMPs can promote the formation of new blood vessels, so that the adhesion, detachment and migration of tumor cells can be accurately regulated under different conditions to realize infection and metastasis. Therefore, the monitoring of MMPs can be used for indicating the occurrence and development process of tumors and providing an effective basis for early diagnosis.

Several matrix metalloproteinases have been found to be abnormally expressed in oral squamous cell carcinoma and are involved in the invasion and metastasis processes. Research shows that MMP-1 which is one member of the matrix metalloproteinase family can be used as a screening target for the initiation of oral cancer, is expected to be used for diagnosing oral squamous cell carcinoma, can be used as a primary diagnosis basis for the oral squamous cell carcinoma when the concentration of the MMP-1 in saliva is greater than 2.77ng/mL, and has a positive rate of up to 78%. Therefore, the detection of MMP-1 content in the saliva of a patient is of great importance for the screening of oral squamous cell carcinoma.

The immunochromatographic assay technology is a mature rapid immunoassay technology, has the advantages of sensitivity, accuracy, stability, reliability, safety, simplicity and convenience, small volume and convenient carrying, and can meet the requirements of rapid, convenient and popular qualitative detection. In the using process, the reagent and the sample which are usually used are extremely small in amount, the cost is low, the result can be easily observed by naked eyes, operators do not need to be trained in a special and complicated way, the environment is not polluted, and the kit is very suitable for the independent detection of individuals. The conventional test strip for detecting tumor markers is mostly based on a double-antibody sandwich method, however, the use of double antibodies undoubtedly increases the cost, and meanwhile, the overall design and assembly process of the test strip also needs to consider the activity problem of the antibodies, which correspondingly increases the research and development difficulty.

At present, commercial pregnancy detection test strips are successfully realized, the production and sale of the test strips are covered by a large area, the test strips are easy to obtain for most users, the cost is greatly reduced due to wide application, and therefore if the detection target chorionic gonadotropin (hCG) of the pregnancy test strips can be converted into a tumor marker through proper design, the research and development difficulty and the cost are greatly reduced, meanwhile, the universality of detection aiming at different targets can be realized, and the detection method is simpler.

Disclosure of Invention

Aiming at the defects or improvement requirements of the prior art, the invention provides a magnetic probe and application of preparing and detecting matrix metalloproteinase, and aims to utilize the hydrolysis characteristic of the matrix metalloproteinase, identify and cut a specific oligopeptide sequence, prepare a functional signal probe, and combine a magnetic separation technology and a commercial test strip, thereby solving the problems of high cost, low detection sensitivity and narrow application range of the prior art.

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

(1) Adding a first single-stranded DNA fragment modified by sulfydryl into an oligopeptide solution, and connecting the sulfydryl on the first single-stranded DNA fragment with an oligopeptide to obtain a first probe; the oligopeptides are capable of being cleaved by a matrix metalloproteinase;

(2) Activating the transduction molecule, adding a second single-stranded DNA fragment modified by sulfydryl, and connecting the sulfydryl on the second single-stranded DNA fragment with the transduction molecule to obtain a second probe; the second single-stranded DNA fragment is base complementary paired with the first single-stranded DNA fragment;

(3) And adding magnetic nanoparticles into the first probe to connect the first probe with the magnetic nanoparticles, and adding the second probe to complementarily base pair the first single-stranded DNA fragment on the first probe with the second single-stranded DNA fragment on the second probe to obtain the magnetic probe.

Preferably, in step (1), the amino acid sequence of the oligopeptide is KPLGLARK, plarwar, PC (Me) HAK, gplgylpar or PLGC (Me) HA (dR). (Me) denotes C (cysteine methylation) and (dR) denotes arginine in D configuration.

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

Preferably, in step (1), the two ends of the oligopeptide are respectively connected with biotin and maleimide groups, and the thiol on the first single-stranded DNA fragment is connected with the maleimide group on the oligopeptide;

in the step (2), 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid sulfosuccinimide ester sodium salt is added to the transduction molecule solution to activate the transduction molecule, and the thiol group of the second single-stranded DNA fragment is linked to the 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid sulfosuccinimide ester sodium salt of the transduction molecule.

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

Preferably, in the step (3), the size of the modified magnetic nanoparticles is 40-80 nm;

preferably, the magnetic nanoparticles are carboxylated, aminated or streptavidin modified.

According to another aspect of the present invention, there is provided a magnetic probe prepared by any one of the methods described above.

According to another aspect of the present invention, there is provided a kit for detecting matrix metalloproteinase, comprising the magnetic probe.

According to another aspect of the invention, the application of the magnetic probe for detecting the matrix metalloproteinase is provided, the magnetic probe is added into a sample, and after reaction, a centrifugal tube filled with a sample solution is subjected to magnetic separation; immersing a test strip in the supernatant, wherein the test strip can specifically detect the transduction molecule on the magnetic probe; if the detection line on the test strip is colored, the sample contains matrix metalloproteinase; and if the detection line on the test strip does not develop color, the sample does not contain matrix metalloproteinase.

Preferably, a matrix metalloproteinase standard solution with gradient concentration is prepared, a standard curve is drawn according to the concentration and the corresponding detection line signal value, and then the concentration of the matrix metalloproteinase in the sample is calculated according to the standard curve.

In general, compared with the prior art, the above technical solution contemplated by the present invention can achieve the following beneficial effects:

(1) The magnetic probe designed by the invention is different from the conventional immunomagnetic probe, and the surface of the magnetic probe is not subjected to target recognition and capture through an immobilized antibody, but is coupled with a specific oligopeptide probe to realize target detection and signal transduction, so that the magnetic probe has extremely strong specificity. The invention can effectively eliminate the interference of false positive results by carrying out magnetic separation on the signal probes which are not cut.

(2) The invention not only avoids the assembly of the test strip and the preparation of the recognition antibody, but also well avoids the matrix interference by utilizing magnetic separation through the chromogenic determination on the existing test strip; according to the invention, the high specificity of proteolytic cleavage and the high sensitivity and reactivity of the test strip to the target probe are utilized, the sensitive detection of matrix metalloproteinase MMP-1 is realized, the visual detection limit is 65.5pg/mL, and the test strip has good anti-interference performance and sensitivity in the application of biological sample matrix, and can meet the detection requirement of clinical diagnosis.

(3) The transduction molecules in the magnetic probe provided by the invention have diversified selections, and the probe corresponding to the detection substance marker can be designed according to different commercialized test strips. In addition, the cost of the preferred early pregnancy test strip is less than 1 yuan, and the test strip has the lowest cost in various test strips on the market, so that the preferred hCG labeled probe has good popularization value.

(4) The invention has higher selectivity to matrix metalloproteinase MMP-1, and effectively reduces the interference of interfering substances in a sample on detection.

Drawings

FIG. 1 is a schematic diagram showing the synthesis of the signaling probes SP-DNA1 and hCG-DNA2 of the present invention.

FIG. 2 is a schematic diagram of the test strip for MMP-1 detection of the present invention.

FIG. 3 is a flow chart of the quantitative assay of the present invention.

FIG. 4 is a graph showing the relationship between the band intensity and MMP-1 concentration in example 1 of the present invention and a corresponding test strip object graph.

FIG. 5 shows the specificity of MMP-1 detected by the test strip of example 2 of the present invention.

FIG. 6 is a comparison of the test strip of example 3 of the present invention with enzyme-linked immunosorbent assay for MMP-1 detection.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, 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 illustrative of the invention and do not limit the invention. In addition, the technical features involved in the respective embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

The invention provides a MMP-1 rapid visual detection method, which comprises the steps of preparation and modification of a magnetic probe, wherein the signal probe is a single-stranded DNA1 coupling oligopeptide (SP-DNA 1) and a DNA2 coupling hCG (hCG-DNA 2), and magnetic separation is used for eliminating false positive interference generated in the analysis process of free hCG-DNA2.

The invention provides a rapid visual detection method for matrix metalloproteinase MMP-1, belongs to an analysis method based on protease specificity hydrolysis oligopeptide, and relates to a rapid visual detection method for an early pregnancy test strip assisted by a magnetic separation technology. Two kinds of signal probes, namely oligopeptide-DNA 1 (SP-DNA 1) and hCG-DNA2, are prepared by the method. OligopeptidesThe other end of the sequence is also marked with biotin, and the modified Fe can be prepared through the interaction of biotin-streptavidin ₃ O ₄ -PEI @ streptavidin stably binds, while its coupled DNA1 sequence is complementary to the hCG coupled DNA2 sequence, thus allowing further binding of hCG-DNA2 to the surface of the magnetosphere. When MMP-1 exists, the MMP-1 can specifically recognize oligopeptide sequences and cut the oligopeptide sequences, then a DNA hybridization probe containing hCG modification is released, and after the magnetic separation of the magnetic compound, the hCG probe in the supernatant can be developed through a sample loading early pregnancy test strip. As MMP-1 concentration increases, the amount of free hCG probe cleaved increases, and thus MMP-1 can be assayed by the early pregnancy test strip. After the color development is finished, quantitative detection can be carried out by using the smart phone application and the portable device developed in the laboratory.

The matrix metalloproteinase detection kit comprises a signal probe, a magnetic probe and an early pregnancy test strip, wherein the signal probe comprises SP-DNA1 and hCG-DNA2; the magnetic probe is a magnetic compound for sequentially modifying SP-DNA1 and hCG-DNA2; the detection object of the early pregnancy test strip is hCG; the early pregnancy test strip comprises a sample pad, a gold-labeled pad, a nitrocellulose membrane and absorbent paper which are subjected to plastic package, wherein the nitrocellulose membrane is provided with a detection line (T line) and a quality control line (C line).

The detection method of the invention specifically comprises the following steps.

1) Preparation of a signal probe:

preparation of SP-DNA1 Probe: adding tris (2-carboxyethyl) phosphine hydrochloride into a sulfhydryl-modified DNA1 solution, standing for 30min, then performing ultrafiltration purification for 8 times by using phosphate buffer, then adding the solution into an oligopeptide solution with two ends respectively modified with maleimide and biotin, reacting for 6h, performing ultrafiltration purification for 8 times by using phosphate buffer, and fixing the volume of the final solution to 200 mu L to obtain a prepared SP-DNA1 probe;

preparation of hCG-DNA2 Probe: adding tris (2-carboxyethyl) phosphine hydrochloride into a sulfhydryl modified DNA2 solution, standing for 1h, and then performing ultrafiltration purification for 8 times by using phosphate buffer; meanwhile, adding 4- (N-maleimide methyl) cyclohexane-1-carboxylic acid sulfonic group succinimide ester sodium salt into hCG solution, reacting for 1h, and then performing ultrafiltration purification for 8 times by using phosphate buffer; mixing the two solutions, reacting for 24h, performing ultrafiltration purification for 8 times by using phosphate buffer, and fixing the volume of the final solution to 150 mu L to obtain a prepared hCG-DNA2 probe;

the phosphate buffer solution is 8.1mM Na with pH 7.4 ₂ HPO ₄ 、1.9mM NaH ₂ PO ₄ And 100mM NaCl.

The concentration of the prepared SP-DNA1 probe was 20. Mu.M, and the concentration of the prepared hCG-DNA2 probe was 10. Mu.M.

2) Preparation of magnetic probe:

ferrous chloride was dissolved in water, then ammonia was added and stirring was continued for 10min with exposure to air. Adding the obtained suspension into a stainless steel high-pressure reaction kettle with a teflon lining, and adding a Polyethyleneimine (PEI) aqueous solution into the reaction kettle. After stirring well, it was heated at 134 ℃ for 3h. After completion of the reaction, the resulting product was cooled to room temperature. Collecting black precipitate by magnetic separation, washing with water and ethanol respectively, and drying in vacuum oven at 50 deg.C to obtain prepared magnetic particles (Fe) ₃ O ₄ -PEI)；

Mixing Fe ₃ O ₄ PEI is added into the glutaraldehyde water solution for ultrasonic dispersion and reaction for 2h. The magnetic particles after activation were washed with phosphate buffer and resuspended. Streptavidin solution was added to the magnetic suspension for 2h, followed by bovine serum albumin solution to block unmodified sites. Adding an SP-DNA1 probe of which the N end is marked with biotin into the washed suspension, and reacting for 2 hours. Washing and re-suspending the obtained magnetic compound in a Tris salt buffer solution, adding an hCG-DNA2 probe for reaction for 1h, washing the obtained magnetic probe for three times, and re-suspending the magnetic probe in a TCNB buffer solution;

wherein, fe is contained in the magnetic probe ₃ O ₄ The size of PEI magnetic particles is 40-80 nm, preferably 60nm, generally the larger the particle size of the magnetic particles, the better the magnetic responsiveness, but the aggregation easily occurs to hinder the reaction at the interface from proceeding and the dispersion in the solution; on the contrary, the smaller the particle diameter, the less aggregation is likely to occur, but the magnetic responsiveness is lowered, and it is preferable that 60nm magnetic particles are used for the preparationA magnetic probe.

The Tris salt buffer solution is 10mM Tris-HCl with pH 7.4, and 5mM MgCl ₂ 100mM NaCl,5mM KCl.

The TCNB buffer was 50mM Tris,10mM CaCl at pH 7.5 ₂ 150mM NaCl,0.05% (w/v) Brij-35.

Since the concentration of the SP-DNA1 and hCG-DNA2 probes prepared in step (1) is about 2.

3) Analysis of the sample to be tested: MMP-1 solutions with different concentrations are added into the magnetic probe solution for reaction for 1h. And then, inserting the centrifuge tube filled with the sample solution into a magnetic frame hole, standing for 15s for magnetic separation, immersing the early pregnancy test strip into the supernatant, standing for 10s, after 10min of chromatographic reaction, observing a red strip by a detection line and a quality control line of the test strip, then taking a picture by using a smart phone, and measuring the concentration of MMP-1 in the sample by using the smart phone application and a portable device developed in the laboratory.

The using amount of the magnetic probe is not too much or too little, the reaction time of the MMP-1 and the magnetic probe is not too long or too short, the reaction temperature is not too high or too low, the insufficient reaction can be caused by the too little using amount or the too short reaction time, the waste of materials and time can be caused by the too much using amount or the too long reaction time, and the reduction of detection signals and the detection sensitivity of the test strip can be influenced by the influence of the too high or too low reaction temperature on the enzymatic activity of the MMP-1. Therefore, the invention preferably obtains the mass concentration of the magnetic probe of 1-5 mg/mL, preferably 4mg/mL, the reaction time of 30-120 min, preferably 75min, and the reaction temperature of 20-55 ℃, preferably 35 ℃ through repeated tests.

The magnetic separation time is not less than 15s. The separation of the magnetic probe is very important for the subsequent detection process of MMP-1, so that the magnetic separation time is ensured, the magnetic probe can be thoroughly removed, and the subsequent detection work is prevented from being influenced.

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

Preferably, the mass concentration to molar concentration ratio of hCG to DNA2 in the signal probe hCG-DNA2 is 0.1-1 mg/mL: 10-100. Mu.M, preferably 0.1mg/mL: 10. Mu.M.

Preferably, the magnetic probe is a carboxylated, aminated or streptavidin modified magnetic particle, preferably a streptavidin modified magnetic particle.

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

Preferably, the detection method comprises the steps of preparing and modifying a magnetic probe, wherein the signal probe is a single-stranded DNA1 coupling oligopeptide (SP-DNA 1) and a DNA2 coupling hCG (hCG-DNA 2).

Preferably, fe in the magnetic probe of step (2) ₃ O ₄ -the particle size of PEI is between 40 and 80nm.

Preferably, the magnetic separation time in step (2) (3) is not less than 15s.

Preferably, the test strip of step (3) is Yuting, david or blue cross.

According to another aspect of the invention, a method for rapidly and visually detecting MMP-1 is provided, wherein the detection target MMP-1 can recognize and cut 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 respectively bound to magnetic particles through affinity action and hybridization complementary action, the detection target MMP-1 can cut the SP-DNA1 probe to generate a free hCG hybridization probe, and the solution is loaded onto the early pregnancy test strip after magnetic separation, so that the color development on the detection line can be realized, and the visual detection of MMP-1 is indirectly completed.

The DNA1 sequence is complementary to the DNA2 sequence, the oligopeptide sequence is marked with biotin on one side and can be combined on streptavidin magnetic particles, and then hCG-DNA2 can be combined on the surface of the magnetic particles. When MMP-1 exists, the MMP-1 can specifically recognize oligopeptide sequences and cut the oligopeptide sequences, then a DNA hybridization probe containing hCG modification is released, and after the magnetic separation of the magnetic compound, the hCG probe in the supernatant can be developed through a sample loading early pregnancy test strip.

In some embodiments, the first single-stranded DNA1:5' -GATACGGTCT AGCTTATTGA TATGCTTTTT T-C ₆ -SH-3 '(SH is the modification of thiol group at the 3' end of DNA sequence), which is complementary to the part of 5 'GCATATCAAT AAGCTAGACC GTATC-3' of the second single-stranded DNA2.

Oligopeptide sequence in the following examples: biotin-KPLGLARK-maleimide (Biotin is a Biotin modification in conjunction with streptavidin magnetic particles, maleimide is a maleimide modification of conjugated DNA 1).

FIG. 1 is a schematic diagram of the synthesis of a probe designed according to the present invention; FIG. 2 is a schematic diagram of the rapid detection of MMP-1 of the present invention.

The following are specific examples:

example 1

A rapid visual detection method of MMP-1 comprises the following steps:

(1) Preparation of a signal probe:

preparation of SP-DNA 1: mu.L of TCEP solution (10 mM) was added to 40. Mu.L of an aqueous DNA1 solution (100. Mu.M), and the mixture was allowed to stand at room temperature for 30min. The solution was purified using a 3K ultrafiltration tube. Subsequently, 50. Mu.L of an oligopeptide solution (1 mg/mL) was added to the DNA1 solution for standing reaction for 6 hours, the conjugate was purified using a 10K ultrafiltration tube to remove the remaining unlinked oligopeptide, and the final solution was made up to 200. Mu.L, and the resulting SP-DNA1 conjugate was stored in a refrigerator at 4 ℃ until use.

Preparation of hCG-DNA 2: mu.L of TCEP solution (300. Mu.M) and 10. Mu.L of phosphate buffer (10 mM, pH 5.5) were added to 150. Mu.L of aqueous DNA2 solution (10. Mu.M), and allowed to stand at room temperature for 1 hour. The resulting solution was purified using a 10K ultrafiltration tube. Meanwhile, 200. Mu.L of a solution of hCG (0.1 mg/mL) and 20. Mu.L of an aqueous solution of sulfo-SMCC (0.1 mg/mL) were mixed, and the resulting solution was incubated at room temperature for 1 hour with gentle shaking, purified with a 3K ultrafiltration tube to remove unreacted coupling agent, and then the activated hCG was incubated with the DNA2 solution at room temperature for 24 hours at rest. The conjugate was purified with 10K to remove the remaining unreacted thiol DNA, and the final solution was made up to 150. Mu.L, and the resulting hCG-DNA2 conjugate was stored in a refrigerator at 4 ℃ until use.

(2) Preparation of magnetic probe:

1.25g of FeCl ₂ ·4H ₂ O was dissolved in 7.75mL of water. Under vigorous stirring, 6.25mL of aqueous ammonia was added and the resulting suspension was left to stir for 10min under exposure to air. The suspension was then charged to a Teflon lined stainless steel autoclave (25 mL) and 5mL of an aqueous PEI solution containing 0.54g was added to the autoclave. After stirring well, it was heated at 134 ℃ for 3h. After completion of the reaction, the resulting product was cooled to room temperature. The black precipitate was collected by magnetic separation and washed with water and ethanol, respectively, and dried.

Mixing 5mg of Fe ₃ O ₄ PEI was added to 1mL of a 2% aqueous glutaraldehyde solution for ultrasonic dispersion, followed by reaction with gentle shaking for 2 hours. mu.L of streptavidin solution (1 mg/mL) was added to the magnetic suspension, mixed well and reacted for 2h with gentle shaking. Add 1% BSA solution and gently shake incubation for 30min to block unmodified sites. To Fe ₃ O ₄ Adding 20 muL of SP-DNA1 probe (20 muM) of N-end labeled biotin into the-PEI @ streptavidin suspension, and slightly oscillating for 2h to obtain Fe ₃ O ₄ -PEI @ streptavidin @ SP-DNA1. The resulting magnetic complexes were washed and resuspended in TBS buffer (10 mM Tris-HCl,5mM MgCl) ₂ 100mM NaCl,5mM KCl, pH 7.4) and 40. Mu.L of hCG-DNA2 probe (. About.10. Mu.M) was added, and the reaction was gently shaken for 1 hour. After completion of the reaction, the resulting magnetic probe was washed three times and resuspended in TCNB buffer (50mM Tris,10mM CaCl) ₂ 150mM NaCl,0.05% (w/v) Brij-35, pH 7.5), and stored at 4 ℃ until use.

(3) Drawing a standard curve:

12 standard MMP-1 concentration gradient standard solutions were prepared, with concentrations of 0pg/mL,10.5pg/mL,26.2pg/mL,65.5pg/mL,163pg/mL,409pg/mL,1.02ng/mL,2.56ng/mL,6.4ng/mL,40ng/mL,100ng/mL,500ng/mL in that order.

Adding 12 parts of MMP-1 standard solutions with different concentrations into the magnetic probe solution, uniformly mixing, reacting for 1h, performing magnetic separation on the mixed solution, immersing the early pregnancy test strip into the supernatant, and standing for 10sAfter reaction for 10min, red strips can be seen in the detection line and the quality control line of the test strip, a picture is taken by using a smart phone, and quantitative detection is completed by using the smart phone developed in the laboratory (figure 3). After the test strip is developed, reading is carried out for multiple times through the mobile phone app, and the average value of the three readings is selected to reduce the determination error. After a series of samples with known concentration are detected, signal value results are obtained, and a standard curve is drawn by the concentration and the corresponding signal value of the T line (figure 4). As can be seen from FIG. 4, the signal value increased with increasing concentration, and then the signal value began to decrease with increasing concentration, and exceeding this concentration (100 ng/mL) is no longer suitable for quantitative determination. The relation between the measured T-line signal value (y) and the concentration (x) of the invention accords with y =2.377x ^0.219 The fitting formula of (1). When a sample with unknown concentration is detected, the T-line signal value of the sample to be detected can be firstly measured, and then the T-line signal value is substituted into the formula of the standard curve to calculate the concentration of the sample.

FIG. 4 shows that the peak of the test strip detection line increases with increasing MMP-1 concentration. When the concentration of MMP-1 is 65.5 pg/mL-100 ng/mL, the index value of the concentration of MMP-1 and the peak value of the test strip detection line are in good linear relation; the visual detection limit measured by the test strip is 65.5pg/mL.

(4) Detection of a sample to be tested

Fresh saliva samples of healthy volunteers were collected by expectoration. Rinsing with clear water for 2min, and collecting saliva generated in oral cavity into a sterile centrifuge tube after 1 min. The collected saliva sample was centrifuged at 4 ℃ for 10min (10000rpm) to remove large particulate matter, diluted one-fold with water to reduce viscosity, and a labeled amount of MMP-1 was added to the saliva sample. MMP-1 with the concentration as low as 1ng/mL can be measured by the sample to be measured according to the method.

Example 2

The chromatographic test strip of the invention is used for specificity experiments of potential interference substances in MMP-1 and other samples:

firstly, 1 mu mol/L of K is prepared respectively ⁺ 、SCN ^－ Glucose, urea, uric acid, glutamic acid, lysine, immunoglobulin (IgG), amylase, lysozyme and 50ng/mL MMP-1 standard solution, and then preparing MMP-1 and other substancesThe samples were mixed so that MMP-1 was 50ng/mL and the concentrations of other interfering substances were all 1. Mu. Mol/L.

The procedure for preparing the magnetic probe was the same as in (2) of example 1, after which 50ng/mL of MMP-1 standard solution and 1. Mu. Mol/L of K were added to the magnetic probe solution ⁺ 、1μmol/L SCN ^－ 1 mu mol/L glucose, 1 mu mol/L urea, 1 mu mol/L uric acid, 1 mu mol/L glutamic acid, 1 mu mol/L lysine, 1 mu mol/L immunoglobulin (IgG), 1 mu mol/L amylase, a standard solution of 1 mu mol/L lysozyme, and a mixed sample consisting of MMP-1 (50 ng/mL) and other substances (1 mu mol/L), wherein 12 samples are mixed uniformly and then reacted for 1h, then the 12 mixed solutions are subjected to magnetic separation, the early pregnancy test strip is immersed in the supernatant and is kept still for 10s, red strips can be seen from the test line and the quality control line of the test strip after 10min reaction, a picture is taken by using a smart phone, and quantitative detection is completed by using the smart phone developed in the laboratory.

FIG. 5 is a specificity test of the test strip for early pregnancy to MMP-1 and other substances, and it can be found that under the condition that the molar concentration of other interfering substances is 1000 times that of MMP-1, the response of MMP-1 to the test strip detection system is far greater than that of other substances; the response of the mixed sample composed of MMP-1 and other substances is basically close to the situation of single MMP-1, which shows that the test strip detection system has better anti-interference capability and good specificity.

Example 3

And (3) comparing the test strip detection with the enzyme-linked immunosorbent assay kit:

FIG. 6 is a comparison graph of MMP-1 detection by the test strip and the ELISA kit, and the results show that the signal values (OD) of the samples in the effective concentration range (0, 0.156,0.312,0.625,1.25,2.5,5, 10 ng/mL) of the ELISA kit are determined ₄₅₀ ) The log value of the test signal of the test strip can be fitted through a four-parameter curve, and the correlation is good. Theoretically, the signal value of the ELISA usually has a certain four-parameter fitting relationship with the analyte concentration, and the logarithmic value of the test strip detection signal can be known from the fitting relationship between the detection signal of the test strip and the MMP-1 concentrationThe test strip and the MMP-1 concentration should present a good linear relation, so the two methods accord with four-parameter curve fitting, and the test strip detection method has considerable application potential compared with an enzyme-linked immunosorbent assay method.

It will be understood by those skilled in the art that the foregoing is only an exemplary embodiment of the present invention, and is not intended to limit the invention to the particular forms disclosed, since various modifications, substitutions and improvements within the spirit and scope of the invention are possible and within the scope of the appended claims.

Claims

1. The preparation method of the magnetic probe is characterized by comprising the following steps of:

(2) Activating the transduction molecule, adding a second single-stranded DNA fragment modified by sulfydryl, and connecting the sulfydryl on the second single-stranded DNA fragment with the transduction molecule to obtain a second probe; the second single-stranded DNA fragment is base complementary to the first single-stranded DNA fragment;

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

3. The method of preparing a magnetic probe according to claim 1 or 2, wherein in step (2), the transduction molecule is human chorionic gonadotropin, luteinizing hormone, bilirubin, ascorbic acid, or creatinine.

4. The method for preparing a magnetic probe according to claim 1, wherein in step (1), the two ends of the oligopeptide are respectively connected with biotin and maleimide groups, and the thiol group on the first single-stranded DNA fragment is connected with the maleimide group on the oligopeptide;

in the step (2), the 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid sulfosuccinimidyl ester sodium salt is added into the transduction molecule solution to activate the transduction molecule, and the thiol group on the second single-stranded DNA fragment is connected with the 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid sulfosuccinimidyl ester sodium salt on the transduction molecule.

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

6. The method for preparing a magnetic probe according to claim 1, wherein in the step (3), the modified magnetic nanoparticle has a size of 40 to 80nm;

7. A magnetic probe prepared according to any one of claims 1 to 6.

8. A kit for detecting matrix metalloproteinase, comprising the magnetic probe of claim 7.

9. The use of a magnetic probe according to claim 7 for the detection of matrix metalloproteinases, wherein the magnetic probe is added to a sample and after reaction the centrifuge tube containing the sample solution is subjected to magnetic separation; immersing a test strip in the supernatant, wherein the test strip can specifically detect the transduction molecules on the magnetic probe; if the detection line on the test strip is colored, the sample contains matrix metalloproteinase; and if the detection line on the test strip does not develop color, the sample does not contain matrix metalloproteinase.

10. The use of claim 9, wherein a gradient concentration of matrix metalloproteinase standard solution is prepared, a standard curve is drawn by the concentration and the corresponding detection line signal value, and the concentration of matrix metalloproteinase in the sample is calculated according to the standard curve.