An electrochemiluminescent peptide sensing method for detecting matrix metalloproteinases

Abstract

This invention belongs to the field of electrochemical analysis technology and relates to an electrochemiluminescence peptide sensing method for detecting matrix metalloproteinases, comprising: 1) mixing a peptide chain solution, an iridium metal complex solution, and a magnetic microparticle MB@SA suspension, adding a protease standard solution, and incubating in one pot; 2) magnetically enriching the magnetic bead complex on the electrode surface and measuring the electrochemiluminescence intensity; 3) changing the volume of the protease standard solution to obtain magnetic bead complexes after reacting with different concentrations of protease and measuring the electrochemiluminescence intensity; obtaining a standard regression equation with protease concentration as the abscissa and the change in electrochemiluminescence intensity as the ordinate; 4) taking the test solution to obtain the corresponding electrochemiluminescence intensity, substituting it into the standard regression equation to obtain the protease concentration. This invention has the advantages of mild probe synthesis conditions, rapid labeling, high sensitivity of the detection method, and ease of operation, realizing rapid, sensitive, and highly selective determination of matrix metalloproteinases.

Description

Technical Field

[0001] This invention belongs to the field of electrochemical analysis technology and relates to an electrochemiluminescent peptide sensing method for detecting matrix metalloproteinases. Background Technology

[0002] Matrix metalloproteinases (MMPs) degrade various protein components in the extracellular matrix, participating in its degradation and transport. MMPs play a crucial role in tumor invasion and metastasis, and cancer development is often closely related to the high expression of multiple MMPs. For example, MMP-2 is highly correlated with tumor development, progression, and metastasis. When the body is stimulated by inflammation or injury, the level of MMP-2 increases significantly. Furthermore, MMP-2 is activated in the brain of a kainic acid rat epilepsy model, and is considered a potential biomarker for human epilepsy. Diseases such as emphysema, chronic obstructive pulmonary disease, and lung injury have also been found to be associated with multiple MMPs (MMP-2, 3, and 9). MMPs can directly degrade collagen and other interstitial tissues in the lungs, leading to lung tissue destruction. Therefore, the measurement of MMPs is of guiding significance for the screening and diagnosis of various diseases. Currently, traditional methods for detecting MMPs mostly employ gelatin polyacrylamide gel electrophoresis and enzyme-linked immunosorbent assay (ELISA), but these methods suffer from cumbersome pretreatment and low detection sensitivity.

[0003] Electrochemiluminescence biosensing is an analytical method that converts biorecognition reactions into electrochemiluminescence signals for quantitative analysis of target analytes. This method boasts advantages such as high sensitivity, low background, and simple instrumentation, and has been widely applied in clinical, environmental, and food fields. While commercially available electrochemiluminescence immunoassay analyzers and their kits are gradually replacing radioimmunoassay in clinical testing, electrochemiluminescence analysis, primarily utilizing antigen / antibody immune reactions, is an affinity-based bioassay and cannot detect cleavage enzymes. Therefore, methods utilizing peptides as molecular recognition substances for cleavage protease analysis have attracted attention in the detection of proteases.

[0004] The synthesis and performance study of electrochemiluminescent signal probes with molecular recognition and signal conversion functions are crucial for electrochemiluminescence biosensing. Currently, in publicly available research on electrochemiluminescence biosensing for protease detection, the electrochemiluminescent probes used are typically prepared based on covalent coupling or by embedding the electrochemiluminescent signal substance into molecular recognition substances such as double-stranded DNA. However, the synthesis of current electrochemiluminescent probes for protease detection has certain limitations. For example, covalent labeling usually requires covalently labeling the electrochemiluminescent signal substance onto the thiol or amino groups at the ends of small peptide chains, leading to reduced recognition ability and electrochemiluminescence efficiency, thus lowering the sensitivity of protease detection. Furthermore, covalent labeling of peptide chains generally requires additional coupling reagents, making the process cumbersome and slow. Embedded labeling typically involves embedding the signal substance into double-stranded DNA, requiring the design of the DNA coupled to the peptide chain. Therefore, developing a novel strategy for synthesizing electrochemiluminescent probes with high luminescence efficiency and high recognition ability, and establishing a rapid, sensitive, and highly selective analytical method for protease detection, is of great significance for disease screening, early diagnosis, and prognostic evaluation. Summary of the Invention

[0005] To address the technical problems of low electrochemiluminescence efficiency, low detection sensitivity, and cumbersome operation in the prior art, this invention provides an electrochemiluminescence peptide sensing method for detecting matrix metalloproteinases.

[0006] To achieve the above objectives, this invention proposes a solvated cyclic iridium metal complex (hereinafter referred to as the iridium metal complex), which, through coordination with a histidine-containing peptide chain, labels the peptide chain to obtain an electrochemiluminescent probe. This probe utilizes a one-pot incubation method to detect matrix metalloproteinases, offering advantages such as convenient probe synthesis, rapid labeling, mild reaction conditions, high sensitivity, and ease of operation. The specific scheme employed in this invention is as follows:

[0007] An electrochemiluminescent peptide sensing method for detecting matrix metalloproteinases includes the following steps:

[0008] Mix peptide chain solution, iridium metal complex solution, and magnetic microparticle MB@SA suspension, add matrix metalloproteinase standard solution, and then dilute to volume with PB buffer solution. Incubate the mixture at a constant temperature to obtain magnetic bead complex.

[0009] 2) Measurement of electrochemiluminescence intensity

[0010] The magnetic bead composite was enriched onto the surface of a magnetic glassy carbon electrode using a magnetic field. The electrode was then placed in an electrolyte, and the electrochemiluminescence intensity of the magnetic bead composite was measured using cyclic voltammetry.

[0011] 3) Obtain the standard regression equation

[0012] 3.1) Referring to step 1), change the volume of the added matrix metalloproteinase standard solution to obtain magnetic bead complexes containing different concentrations of matrix metalloproteinase; referring to step 2), obtain the electrochemiluminescence intensity corresponding to the magnetic bead complexes after the reaction with different concentrations of matrix metalloproteinase.

[0013] 3.2) Using the matrix metalloproteinase concentration as the x-axis and the electrochemiluminescence intensity corresponding to the magnetic bead complex as the y-axis, a standard regression equation was obtained through linear regression:

[0014] (I0-I)=aC MMP +b;

[0015] in:

[0016] I0 represents the electrochemiluminescence intensity of the magnetic bead complex obtained after the mixture reaction when the matrix metalloproteinase content is 0.

[0017] I represents the electrochemiluminescence intensity of the magnetic bead complex obtained after the reaction of the mixture with different matrix metalloproteinase contents;

[0018] C MMP The concentration of matrix metalloproteinases is given in ng / mL.

[0019] a and b are both constants;

[0020] 4) Determine the concentration of matrix metalloproteinases in the sample to be tested.

[0021] Take the sample to be tested and, following the methods in steps 1) to 2), obtain the electrochemiluminescence intensity of the magnetic bead complex obtained after the reaction of the sample mixture. Substitute this intensity into the standard regression equation in step 3) to deduce the concentration of matrix metalloproteinase in the sample to be tested.

[0022] Further specifying that, in step 1), the volume ratio of the peptide chain solution, the iridium metal complex solution, and the magnetic microparticle MB@SA suspension is 3.2:2.5:5; and the total volume after being diluted with PB buffer solution is 100:3 compared with the volume ratio of the iridium metal complex solution.

[0023] Further specifying that in step 1), the concentration of the PB buffer solution is 10 mmol / L; the peptide chain solution is a PB buffer solution containing 1 mg / mL of standard peptide chain; the magnetic microparticle MB@SA suspension is a suspension containing 2.5 mg / mL of streptavidin-functionalized magnetic beads; the iridium metal complex solution is a dimethyl sulfoxide solution containing 1 mmol / L of iridium metal complex; and the protease standard solution is a PB buffer solution containing 100 ng / mL of matrix metalloproteinase.

[0024] Furthermore, the standard peptide chain contains an amino acid sequence that is specifically cleaved by matrix metalloproteinases.

[0025] Further specifying, the structural formula of the iridium metal complex is:

[0026]

[0027] Further specified, the matrix metalloproteinase is MMP-2, MMP-3, MMP-7, or MMP-9.

[0028] Further specifying, in step 1), the conditions for the isothermal incubation reaction are: temperature 37–37.5°C, time 0.5–2 h.

[0029] Further specifying, the PB buffer solution in step 2.1) has a concentration of 10 mmol / L and a volume of 15 μL.

[0030] Further specifying, the electrolyte in step 2.2) is a PBS buffer solution containing 50 mmol / L TPA; the concentration of the PBS buffer solution is 0.1 mol / L.

[0031] Further specifying, in step 2.2), the conditions for cyclic voltammetry are: potential of 0-1.6V and scan rate of 0.1V / s.

[0032] Compared with the prior art, the beneficial effects of the present invention are:

[0033] 1. This invention establishes an electrochemiluminescence peptide sensing method based on coordination labeling, biocleavage, and magnetic enrichment, integrating signal recognition and conversion, and employing a "one-pot" incubation method for matrix metalloproteinase (MMP) detection. During detection, an iridium metal complex, a biotin-labeled standard peptide chain containing a histidine-terminated fragment specifically cleaved by MMPs, streptavidin-functionalized magnetic beads, and an MMP solution are sequentially added to a centrifuge tube, followed by incubation at 37°C for 2 hours. The magnetic bead complex is enriched onto the surface of a magnetic glassy carbon electrode using a magnetic field, and the electrochemiluminescence intensity is detected in a TPA-containing solution. The MMP concentration is determined using a standard curve method. The detection method is simple, easy to perform, and convenient to operate; further, the use of a magnetic enrichment signal amplification strategy significantly improves the detection sensitivity.

[0034] 2. In this invention, an electrochemiluminescent signaling substance (iridium metal complex) is simply mixed with a peptide chain containing histidine. The histidine in the peptide chain competitively coordinates with the iridium in the iridium metal complex, thus obtaining an electrochemiluminescent probe. This labeling method requires no additional coupling reagents, has mild reaction conditions, and enables rapid on-site labeling of electrochemiluminescent probes, avoiding the cumbersome steps of covalent labeling peptide chains with electrochemiluminescent signaling substances in existing technologies. Furthermore, the electrochemiluminescent signal is enhanced after the iridium metal complex coordinates with the peptide chain, avoiding the problem of signal degradation after labeling.

[0035] 3. The matrix metalloproteinase detection method provided by the present invention has the advantages of good linearity, low detection limit and good selectivity. In particular, it has very high sensitivity for the detection of matrix metalloproteinase 2 in the linear range of 1 to 10 ng / mL, and the detection limit is as low as 0.9 ng / mL.

[0036] 4. The electrochemiluminescence peptide sensing method of the present invention uses an iridium metal complex with 3-(2-pyridyl)-benzoic acid as the main ligand as the electrochemiluminescence signal substance. The chemical formula of the iridium metal complex is C1. 26 H 22 ClIrN2O5S is an iridium complex with a carboxyl group introduced at the 3 position of the main ligand phenylpyridine. Therefore, this iridium complex has good water solubility, can be stored for a long time, and has good stability. Attached Figure Description

[0037] Figure 1 This is a schematic diagram of the matrix metalloproteinase detection method provided by the present invention;

[0038] Figure 2 The solubility of the iridium metal complex with 3-(2-pyridyl)-benzoic acid as the main ligand in 10 mmol / L LPB-DMSO (V:V = 99:1);

[0039] Figure 3 Electrochemiluminescence intensity of iridium metal complexes with 3-(2-pyridyl)-benzoic acid as the main ligand after being placed at low temperature for different days;

[0040] Figure 4 Electrochemiluminescence curves of an iridium metal complex with 3-(2-pyridyl)-benzoic acid as the main ligand reacting with a peptide chain containing histidine at the end before and after reaction;

[0041] Figure 5 Electrochemiluminescence curves before and after the reaction of an iridium metal complex with 3-(2-pyridyl)-benzoic acid as the main ligand with a biotin-labeled peptide chain containing histidine at the end, streptavidin-functionalized magnetic beads, and matrix metalloproteinase 2 (MMP-2, 10 ng / mL).

[0042] Figure 6 Electrochemiluminescence curves before and after the reaction of iridium metal complex with 3-(2-pyridyl)-benzoic acid as the main ligand with biotin-labeled peptide chains containing histidine at the end, streptavidin-functionalized magnetic beads, and matrix metalloproteinase 2 at different concentrations.

[0043] Figure 7 The electrochemiluminescence intensity values ​​before and after the reaction are shown for iridium metal complexes with 3-(2-pyridyl)-benzoic acid as the main ligand, biotin-labeled peptide chains with histidine terminals, streptavidin-functionalized magnetic beads, and matrix metalloproteinases 2 (MMP-2, 5 ng / mL), 3 (MMP-3, 20 ng / mL), 7 (MMP-7, 20 ng / mL), 9 (MMP-9, 20 ng / mL), prostate-specific antigen (PSA, 20 ng / mL), and bovine serum albumin (BSA, 20 ng / mL). Detailed Implementation

[0044] The technical solution of the present invention will be further explained and described below with reference to the accompanying drawings and embodiments, but the present invention is not limited to the embodiments described below.

[0045] The one-pot electrochemiluminescence polypeptide sensing method provided by this invention integrates signal recognition and conversion, and is constructed through coordination labeling, bio-cleavage and magnetic enrichment methods.

[0046] Specifically, an electrochemiluminescent probe (peptide chain@iridium metal complex) is formed by coordinating a biotin-labeled molecular recognition substance with histidine residues at the end with an iridium metal complex. This electrochemiluminescent probe is then bound to streptavidin-functionalized magnetic beads (MB@SA) to obtain an iridium metal complex-labeled magnetic probe (MB@SA@peptide chain@iridium metal complex).

[0047] The peptide chain structure used in this invention is labeled with biotin (Bio) at the front end (left end), has histidine H at the end (right end), and contains an amino acid sequence that is specifically cleaved by matrix metalloproteinases in the middle.

[0048] The peptide chains used in this invention and the specific recognition sequences of different matrix metalloproteinases are detailed in Table 1.

[0049] Table 1. Peptide sequences and specific recognition sequences of different matrix metalloproteinases

[0050] matrix metalloproteinases peptide chain Specific recognition sequence MMP-2 Bio-GPLGVRGKHH ~PLGVR~ MMP-3 Bio-GSLTMGGKHH ~SLTMG~ MMP-7 Bio-GLALWRSGKHH ~LALWRS~ MMP-9 Bio-GGRMGLPGKHH ~GRMGLP~

[0051] In this invention, the iridium metal complex used as the electrochemiluminescence signaling substance is a solvated iridium metal complex with 3-(2-pyridyl)-benzoic acid as the main ligand. Specifically, a carboxyl group is introduced at the 3-position of the main ligand phenylpyridine to give it good water solubility. Its chemical structural formula is as follows:

[0052]

[0053] The solubility, stability, and electrochemiluminescence intensity of the electrochemiluminescence signaling substance were experimentally verified.

[0054] 1. Solubility of iridium metal complexes with 3-(2-pyridyl)-benzoic acid as the main ligand.

[0055] See Figure 2 A buffer system was prepared by mixing 10 mmol / L phosphate buffer solution (8.2 mmol / L NaH2PO4 and 1.8 mmol / L Na2HPO4, PB buffer solution) with dimethyl sulfoxide at a volume ratio of 99:1 to form a solution with pH=7.4.

[0056] from Figure 2 It can be seen that strong absorption is observed in the wavelength range below 320 nm; weaker absorption is observed in the 320–500 nm range, which is due to charge transfer from the metal to the ligand. The molar absorptivity of the iridium complex was calculated at this wavelength, in the buffer system, and at different concentrations, using an absorption wavelength of 346 nm. Figure 2 The inner inset shows that the solubility of this iridium complex in the system is 60 μM.

[0057] 2. PB buffer solutions of iridium metal complexes with 3-(2-pyridyl)-benzoic acid as the main ligand can be stored for a long time.

[0058] See Figure 3 An iridium metal complex with 3-(2-pyridyl)-benzoic acid as the main ligand was prepared by mixing 0.1 mol / L PBS buffer solution (82 mmol / L NaH2PO4, 18 mmol / L Na2HPO4 and 0.1 mol / L KCl) with dimethyl sulfoxide at a volume ratio of 99:1 to form a solution with pH=7.4 as a buffer system. After being stored at low temperature (4℃) for 35 days, the electrochemiluminescence intensity of the complex remained relatively stable in the presence of 50 mmol / L TPA, and the relative standard deviation of the electrochemiluminescence intensity value was 3.1%.

[0059] 3. The electrochemiluminescence intensity of iridium metal complexes, which serve as electrochemiluminescence signaling substances, after reacting with histidine-containing peptide chains.

[0060] The specific testing method and steps are as follows:

[0061] (1) Prepare a PB buffer solution with a concentration of 10 mmol / L (8.2 mmol / L NaH2PO4 and 1.8 mmol / L Na2HPO4), a PBS buffer solution with a concentration of 0.1 mol / L (82 mmol / L NaH2PO4, 18 mmol / L Na2HPO4, 0.1 mol / L KCl), a PBS buffer solution with a concentration of 0.1 mol / L containing 50 mmol / L TPA, a dimethyl sulfoxide solution with a concentration of 1 mmol / L iridium complex, and a PB buffer solution with a concentration of 1 mg / mL standard peptide chain (Bio-GPLGVRGKHH).

[0062] (2) In a centrifuge tube, add 2.5 μL of dimethyl sulfoxide solution of iridium metal complex and 2.5 μL of standard peptide chain solution in step (1) and make up to 250 μL with 10 mmol / L PB buffer solution. Incubate at 37°C for 2 hours to obtain the reaction solution.

[0063] (3) Take 100 μL of the reaction solution in step (2) and add it to 900 μL of 0.1 mol / L PBS buffer solution containing 50 mmol / L TPA. Set the potential of the cyclic voltammetry technique to 0-1.6 V and the scan rate to 0.1 V / s, and measure the electrochemiluminescence intensity.

[0064] See Figure 4 The electrochemiluminescence curves of the iridium metal complex reacting with the peptide chain containing histidine on a glassy carbon electrode show that the iridium metal complex produces a weak electrochemiluminescence signal at 1.25V (vs. Ag / AgCl), while after reacting with the peptide chain, it produces a strong electrochemiluminescence signal at 1.1V (vs. Ag / AgCl).

[0065] Meanwhile, compared with the electrochemiluminescence curve of the iridium metal complex alone on the glassy carbon electrode, the electrochemiluminescence signal of the iridium metal complex of the present invention is enhanced by 16.5 times after binding with the peptide chain through ligands.

[0066] Therefore, the electrochemiluminescence signal probe prepared by this invention, which reacts an iridium metal complex with a histidine-containing peptide chain as the main ligand, is simple to operate, does not require additional coupling reagents, and has a high electrochemiluminescence signal.

[0067] Example 1

[0068] This embodiment uses matrix metalloproteinase 2 as an example to illustrate a one-pot electrochemiluminescence peptide sensing method for detecting matrix metalloproteinases.

[0069] The detection principle of matrix metalloproteinase 2 is as follows: After the molecular recognition substance (MB@SA@peptide chain@iridium metal complex) of matrix metalloproteinase 2 synthesized in this invention reacts with the target (matrix metalloproteinase 2), the luminescent short peptide chain fragment of the histidine-coordinated iridium metal complex leaves the magnetic beads and is free in the reaction solution. Through magnetic action, magnetic electrodes are used to enrich the magnetic beads with complete luminescent peptide chains or only residual peptide chains fixed before and after cleavage on the electrode surface, and the electrochemiluminescence signal is detected.

[0070] Before proceeding, prepare the following solution.

[0071] Prepare a 10 mmol / L PB buffer solution. The PB buffer solution contains 8.2 mmol / L NaH2PO4 and 1.8 mmol / L Na2HPO4.

[0072] Prepare a 0.1 mol / L PBS buffer solution. The PBS buffer solution contains 82 mmol / L NaH2PO4, 18 mmol / L Na2HPO4, and 0.1 mol / L KCl.

[0073] Prepare a 0.1 mol / L PBS buffer solution containing 50 mmol / L TPA, denoted as the electrolyte.

[0074] Prepare a 1 mmol / L dimethyl sulfoxide solution of the iridium complex, denoted as the iridium complex solution.

[0075] Prepare a PB buffer solution with a concentration of 1 mg / mL for the standard peptide chain (Bio-GPLGVRGKHH), denoted as the peptide chain solution. The standard peptide chain sequence is Bio-GPLGVRGKHH.

[0076] Prepare a 10 mmol / L PB buffer solution containing 100 ng / mL matrix metalloproteinase 2, and denote it as the matrix metalloproteinase 2 solution.

[0077] A streptavidin-functionalized magnetic bead suspension with a mass concentration of 2.5 mg / mL was prepared and denoted as the magnetic microparticle MB@SA suspension.

[0078] See Figure 1 The electrochemiluminescence peptide sensing method provided in this embodiment includes the following steps.

[0079] 1) "One pot" incubation

[0080] Mix the peptide chain solution, iridium metal complex solution, and MB@SA suspension, add matrix metalloproteinase 2 solution (initial concentration of matrix metalloproteinase MMP-2 is 100 ng / mL), and then bring the volume to a final volume with PB buffer. Incubate the mixture at a constant temperature to obtain the magnetic bead complex.

[0081] Specifically, in a centrifuge tube, 15 μL of MB@SA suspension, 9.6 μL of peptide chain solution, and 7.5 μL of iridium metal complex solution were added sequentially. Then, matrix metalloproteinase 2 solution was added, and the volume was adjusted to 250 μL with PB buffer solution. After incubating the mixture at 37°C for 2 hours, a magnetic bead complex containing matrix metalloproteinase 2 was obtained.

[0082] 2) Measurement of electrochemiluminescence intensity

[0083] The magnetic bead composite was enriched onto the surface of a magnetic glassy carbon electrode using a magnetic field. The electrode was then placed in an electrolyte, and the electrochemiluminescence intensity of the magnetic bead composite was determined using cyclic voltammetry.

[0084] Specifically, after the centrifuge tubes in step 1) have completed the reaction, place them on a magnetic rack for 1 minute. After discarding the supernatant, wash the magnetic bead complex in the centrifuge tubes three times with 10 mmol / L PB buffer solution. Then, use a magnetic separator to separate and discard the washing solution. Finally, disperse the magnetic bead complex in the centrifuge tubes in 15 μL of 10 mmol / L PB buffer solution to obtain a magnetic bead complex suspension.

[0085] The magnetic glassy carbon electrode (Ф=3mm) was placed in the above magnetic bead composite suspension and enriched for 2 min. Then, it was placed in a 0.1 mol / L PBS buffer solution containing 50 mmol / L TPA. The potential of the cyclic voltammetry technique was set to 0-1.6 V and the scan rate was 0.1 V / s. The electrochemiluminescence intensity was measured.

[0086] 3) Obtain the standard regression equation

[0087] 3.1) Change the volume of the matrix metalloproteinase 2 solution added, and obtain a mixture of magnetic bead complexes containing different concentrations of matrix metalloproteinase 2 as in step 1). Then, obtain the electrochemiluminescence intensity of the magnetic bead complexes after the reaction with different concentrations of matrix metalloproteinase 2 according to the method in step 2).

[0088] 3.2) Plotting the final concentration of matrix metalloproteinase 2 on the x-axis and the electrochemiluminescence intensity of the magnetic bead complex on the y-axis, a regression curve was obtained, yielding the standard regression equation:

[0089] (I0-I)=aC MMP-2 +b;

[0090] in:

[0091] I0 represents the electrochemiluminescence intensity of the magnetic bead complex obtained after the mixture reaction when the matrix metalloproteinase 2 content is 0.

[0092] I represents the electrochemiluminescence intensity of the magnetic bead complex obtained after the reaction of the mixture with different matrix metalloproteinase 2 contents;

[0093] C MMP-2 The concentration of matrix metalloproteinase 2 is ng / mL; the concentration here refers to the final concentration after mixing.

[0094] a and b are both constants;

[0095] 4) Determine the concentration of matrix metalloproteinase 2 in the sample to be tested.

[0096] Take the sample to be tested and obtain the mixture after incubation according to step 1); then obtain the electrochemiluminescence intensity corresponding to the magnetic bead complex after the mixture reaction according to step 2); substitute it into the standard regression equation in step 3) to back-calculate the concentration of matrix metalloproteinase 2 in the sample to be tested.

[0097] The reverse calculation process for the concentration of matrix metalloproteinase 2 in the test sample is as follows: first, the concentration of matrix metalloproteinase 2 in the mixture after incubation of the test sample is calculated using the standard regression equation; then, the concentration of matrix metalloproteinase 2 in the test sample is calculated based on the added volume of the test sample.

[0098] In this embodiment, the concentrations of matrix metalloproteinase 2 in the mixture were 0 and 10 ng / mL, respectively (here, concentration refers to the concentration of matrix metalloproteinase 2 in the mixture). Electrochemiluminescence intensity curves were obtained according to steps 1) to 2). See the results below. Figure 5 .

[0099] like Figure 5 As shown, in the absence of matrix metalloproteinase 2 (MMP-2), a strong electrochemiluminescence signal was generated at 1.1 V (vs. Ag / AgCl) on the magnetic electrode. After the addition of MMP-2, the electrochemiluminescence signal at 1.1 V decreased significantly. This indicates that the successful cleavage of the magnetic luminescent peptide by MMP-2 caused the luminescent short peptide chain fragment of the histidine-coordinated iridium metal complex to leave the magnetic beads. Therefore, when plotting the regression curve, the electrochemiluminescence intensity value corresponding to a voltage of 1.1 V was taken from the cyclic voltammetry curve. At this time, the electrochemiluminescence intensity values ​​corresponding to the magnetic bead complexes obtained after the reaction separation of mixtures containing different concentrations of matrix metalloproteinase 2 were all at or close to the highest peak value.

[0100] Further, in step 1), 15 μL of magnetic microparticle MB@SA suspension, 9.6 μL of peptide chain solution, and 7.5 μL of iridium metal complex solution were mixed, and 0, 2.5, 5.0, 10, 12.5, 15, 20, and 25 μL of matrix metalloproteinase 2 solution were added respectively. The mixture was then brought to a final volume of 250 μL with PB buffer solution and incubated at 37°C for 2 hours to obtain multiple mixtures containing different concentrations of matrix metalloproteinase 2.

[0101] Following the method in step 2), the electrochemiluminescence intensity curves corresponding to the magnetic bead complexes obtained after reactions with different concentrations of matrix metalloproteinase 2 were measured. (See [link to relevant documentation]). Figure 6 .exist Figure 6 In the figure, the eight curves from top to bottom are labeled a, b, c, d, e, f, g, and h, representing the added volumes of 0, 2.5, 5.0, 10, 12.5, 15, 20, and 25 μL, respectively; the top curve is the electrochemiluminescence intensity curve corresponding to the added volume of 0 μL.

[0102] See Figure 6 The electrochemiluminescence intensity value corresponding to +1.1V was taken. The concentration of matrix metalloproteinase 2 in the mixture was used as the abscissa, and the change value of electrochemiluminescence intensity (I0-I) corresponding to the magnetic bead complex obtained after the reaction of the mixture containing different concentrations of matrix metalloproteinase 2 was used as the ordinate. The working curve of the change value of electrochemiluminescence intensity and the concentration of matrix metalloproteinase 2 was plotted, and the following standard regression equation was obtained.

[0103] (I0-I)=2848.7C MMP-2 +3789.5

[0104] I0 represents the electrochemiluminescence intensity of the magnetic bead complex obtained after reacting with a mixture containing 0 μL of standard matrix metalloproteinase 2.

[0105] I represents the electrochemiluminescence intensity of the magnetic bead complex obtained after reacting with a mixture containing different amounts of standard matrix metalloproteinase 2.

[0106] C MMP-2 The final concentration of matrix metalloproteinase 2 in the system is given in ng / mL.

[0107] The linear coefficient of the standard regression equation was R = 0.9848. The detection limit was 0.9 ng / mL, which is much lower than the cut-off value for patients with epilepsy (175.4 ng / mL).

[0108] Example 2

[0109] This invention uses matrix metalloproteinase 2 (MMP-2) as an example to verify the selectivity of the provided electrochemiluminescence peptide sensing method for this protease or the interference of other proteases with MMP-2. Examples include MMP-3, MMP-7, MMP-9, PSA, or BSA.

[0110] The specific testing methods are as follows.

[0111] (1) Prepare a 10 mmol / L PB solution (8.2 mmol / L NaH2PO4 and 1.8 mmol / L Na2HPO4), a 0.1 mol / L PBS solution (82 mmol / L NaH2PO4, 18 mmol / L Na2HPO4 and 0.1 mol / L KCl), and a 0.1 mol / L PBS buffer solution containing 50 mmol / L TPA; prepare a 1 mmol / L dimethyl sulfoxide solution of iridium metal complex and a 1 mg / mL peptide chain solution; and prepare a 2.5 mg / mL magnetic microparticle MB@SA suspension.

[0112] Prepare 10 mmol / L PB buffer solutions containing 100 ng / mL matrix metalloproteinase 2 (standard MMP-2 solution), 10 mmol / L PB buffer solutions containing 500 ng / mL matrix metalloproteinase 3 (MMP-3 solution), 10 mmol / L PB buffer solutions containing 500 ng / mL matrix metalloproteinase 7 (MMP-7 solution), 10 mmol / L PB buffer solutions containing 500 ng / mL matrix metalloproteinase 9 (MMP-9 solution), 10 mmol / L PB buffer solutions containing 500 ng / mL prostate-specific antigen (PSA) (standard PSA solution), and 10 mmol / L PB buffer solutions containing 500 ng / mL bovine serum albumin (BSA) (standard BSA solution).

[0113] (2) Take 6 centrifuge tubes and add 15 μL of magnetic microparticle MB@SA suspension, 9.6 μL of peptide chain solution and 7.5 μL of iridium metal complex solution to each centrifuge tube in sequence. Then add 12.5 μL of standard MMP-2 solution, 100 μL of standard MMP-3 solution, 100 μL of standard MMP-7 solution, 100 μL of standard MMP-9 solution, 100 μL of standard PSA solution or 100 μL of standard BSA solution to each centrifuge tube respectively. All centrifuge tubes are diluted to 250 μL with 10 mmol / L PB buffer solution to obtain 6 mixtures, and incubate them at 37℃ for 2 hours.

[0114] Of the six mixtures, the concentration of MMP-2 was 5 ng / mL; the concentrations of MMP-3, MMP-7, MMP-9, PSA, and BSA were all 200 ng / mL.

[0115] (3) Each centrifuge tube that has undergone reaction in step (2) is treated as follows: placed on a magnetic rack for 1 min, the supernatant is discarded, the magnetic bead complex in the centrifuge tube is washed 3 times with 10 mmol / L PB buffer solution, and the washing solution is discarded by separation using a magnetic separator. Finally, the magnetic bead complex in the 6 centrifuge tubes is dispersed in 6 15 μL portions of 10 mmol / L PB buffer solution.

[0116] (4) Place the magnetic glassy carbon electrode (Ф=3mm) in the magnetic bead composite suspension from step (3) for enrichment for 2 min. Then place it in a 0.1mol / L PBS buffer solution containing 50mmol / L TPA. Set the cyclic voltammetry potential to 0-1.6V and the scan rate to 0.1V / s. Measure the electrochemiluminescence curve and obtain the electrochemiluminescence intensity corresponding to a potential of 1.1V. See the results below. Figure 7 .

[0117] Depend on Figure 7 It is known that in the electrochemiluminescence peptide sensing method of the present invention, when 5 ng / mL MMP-2 is added and reacted for 2 hours, the electrochemiluminescence intensity decreases significantly compared with that without MMP-2; while the electrochemiluminescence intensity does not change significantly after reacting with 200 ng / mL of other interfering substances (MMP-3, MMP-7, MMP-9, PSA or BSA) for 2 hours.

[0118] The results show that the electrochemiluminescence peptide sensing method has good selectivity for matrix metalloproteinase 2.

[0119] Example 3

[0120] The electrochemiluminescence peptide sensing method of the present invention was used to detect the MMP-2 content in actual samples (three serum and three plasma samples).

[0121] Referring to the method in Example 1, 25 μL of serum or 25 μL of plasma were incubated with different actual samples using the method in step 1). Then, referring to the method in step 2), the actual electrochemiluminescence intensity of the magnetic bead complex obtained after the reaction of the corresponding mixture was measured. The electrochemiluminescence intensity value corresponding to a potential value of 1.1 V was taken and substituted into the standard regression equation to first obtain the MMP-2 concentration in the mixture corresponding to serum or the MMP-2 concentration in the mixture corresponding to plasma. Then, based on the volume of serum (or plasma) of 25 μL, the MMP-2 content in serum or plasma was calculated by reverse calculation. The results are shown in Table 2.

[0122] Table 2. Results of MMP-2 content detection in serum and plasma

[0123]

[0124] Table 2 shows that the proposed sensing method can detect the content of matrix metalloproteinase 2 in serum and plasma within a linear range. The matrix metalloproteinase 2 content in the three serum samples was 1.4 ng / mL, 1.3 ng / mL, and 0 ng / mL, respectively, with a recovery rate of 99.8% ± 8.0% to 108.0% ± 6.0%. The matrix metalloproteinase 2 content in the three plasma samples was 4.3 ng / mL, 3.0 ng / mL, and 2.5 ng / mL, respectively, with a recovery rate of 93.0% ± 13.0% to 108.6% ± 14.0%. This indicates that the constructed electrochemiluminescence peptide sensing method based on coordination labeling, biocleavage, and magnetic enrichment integrates signal recognition and conversion, and can successfully detect the content of matrix metalloproteinase 2 in actual samples.

[0125] Furthermore, the present invention was compared with existing MMP-2 detection methods, and the results are shown in Table 3.

[0126] Table 3 Comparison of the method of the present invention with other existing methods for detecting MMP-2.

[0127]

[0128]

[0129] The references in the table above are listed below.

[0130] [1]R.Lefkowitz,G. M.Heller,Whole blood assay forelastase,chymotrypsin,matrix metalloproteinase-2,and matrixmetalloproteinase-9activity,Anal.Chem.,2010,82,8251-8258.

[0131] [2]M.Zhao,L.Josephson,Y.Tang,R.Weissleder,Magnetic sensors forprotease assays,Angew.Chem.Int.Ed.,2003,42,1375-1378.

[0132] [3]H.Xu,H.Ye,L.Yu,Y.Chi,X.Liu,G.Chen,Tailor-made peptide sensor fordetection of matrix metalloproteinase 2in blood serum,Anal.Methods,2015,7,5371-5374.

[0133] [4]D.Feng,Y.Zhang,T.Feng,W.Shi,X.Li,H.Ma,Agraphene oxide–peptidefluorescence sensor tailor-made for simple and sensitive detection of matrixmetalloproteinase 2,Chem.Commun.,2011,47,10680-10682.

[0134] [5]X.Wang,Y.Xia,Y.Liu,W.Qi,Q.Sun,Q.Zhao,B.Tang,Dual-luminophore-labeled gold nanoparticles with completely resolved emission for thesimultaneous imaging of MMP-2and MMP-7in living cells under single wavelengthexcitation,Chem.Eur.J.,2012,18,7189-7195.

[0135] [6] E. Song, D. Cheng, Y. Song, M. Jiang, J. Yu, Y. Wang, A graphene oxide-based FRET sensor for rapid and sensitive detection of matrix metalloproteinase 2inhuman serum sample, Biosens. Bioelectron., 2013, 47, 445-450.

[0136] Referring to Table 3, compared with other methods for detecting MMP-2, this electrochemiluminescence peptide sensing method shows advantages in both linear range and detection limit. This method involves a simple mixture of an electrochemiluminescent signal substance (iridium metal complex) and a histidine-containing peptide chain. The histidine in the peptide chain competitively coordinates with the iridium in the iridium metal complex, resulting in a strong electrochemiluminescent signal from the iridium-labeled peptide chain. No additional coupling reagents are required, enabling rapid on-site labeling of the electrochemiluminescent probe, avoiding the cumbersome steps and reduced electrochemiluminescence efficiency issues of covalent labeling of peptide chains with electrochemiluminescent signal substances in existing technologies. During detection, the "one-pot" incubation and magnetic separation methods offer advantages such as simple experimental procedures and batch processing capabilities. The magnetic enrichment signal amplification strategy enhances detection sensitivity, making this method more advantageous than other methods for detecting matrix metalloproteinase 2 listed in Table 3.

[0137] The above describes the detection method for MMP-2 provided by this invention. However, the matrix metalloproteinase detection method of this invention can also be applied to the detection of matrix metalloproteinase 3 (MMP-3), matrix metalloproteinase 7 (MMP-7), and matrix metalloproteinase 9 (MMP-9). During detection, the corresponding peptide chain sequences and specific recognition sequences for different matrix metalloproteinases listed in Table 1 are selected. Specifically, detection can be achieved by replacing the specific recognition sequence in the standard peptide chain sequence from Example 1 with the sequence of the matrix metalloproteinase to be detected.

Claims

1. An electrochemiluminescent peptide sensing method for detecting matrix metalloproteinases, characterized in that, Includes the following steps: 1) "One pot" incubation Mix peptide chain solution, iridium metal complex solution, and magnetic microparticle MB@SA suspension, add matrix metalloproteinase standard solution, and then dilute to volume with PB buffer solution. Incubate the mixture at a constant temperature to obtain magnetic bead complex. The peptide chain is a standard peptide chain with a biotin-labeled front end, a histidine residue at the end, and an amino acid sequence in the middle that is specifically cleaved by matrix metalloproteinases; the magnetic microparticles MB@SA are streptavidin-functionalized magnetic beads. The structural formula of the iridium metal complex is: ； 2) Measurement of electrochemiluminescence intensity The magnetic bead composite was enriched onto the surface of a magnetic glassy carbon electrode using a magnetic field. The electrode was then placed in an electrolyte, and the electrochemiluminescence intensity of the magnetic bead composite was measured using cyclic voltammetry. 3) Obtain the standard regression equation 3.1) Referring to step 1), change the volume of the added matrix metalloproteinase standard solution to obtain magnetic bead complexes after reaction with different concentrations of matrix metalloproteinase; referring to step 2), obtain the electrochemiluminescence intensity corresponding to the magnetic bead complexes after reaction with different concentrations of matrix metalloproteinase. 3.2) Using the matrix metalloproteinase concentration as the x-axis and the electrochemiluminescence intensity corresponding to the magnetic bead complex as the y-axis, a standard regression equation was obtained through linear regression: ； in: The electrochemiluminescence intensity of the magnetic bead complex obtained after the reaction of the mixture is when the matrix metalloproteinase concentration is 0. The electrochemiluminescence intensities of the magnetic bead complexes obtained after the reaction of the mixture at different matrix metalloproteinase concentrations are shown. The concentration of matrix metalloproteinases is given in ng / mL. a、b All are constants; 4) Determine the concentration of matrix metalloproteinases in the sample to be tested. Take the sample to be tested and, following the methods in steps 1) to 2), obtain the electrochemiluminescence intensity of the magnetic bead complex obtained after the reaction of the sample mixture. Substitute this intensity into the standard regression equation in step 3) to back-calculate the concentration of matrix metalloproteinase in the sample to be tested.

2. The electrochemiluminescence peptide sensing method for detecting matrix metalloproteinases according to claim 1, characterized in that, In step 1), the volume ratio of the peptide chain solution, the iridium metal complex solution, and the magnetic microparticle MB@SA suspension is 3.2:2.5:5; the total volume after being diluted with PB buffer solution is 100:3 compared with the volume ratio of the iridium metal complex solution.

3. The electrochemiluminescence peptide sensing method for detecting matrix metalloproteinases according to claim 2, characterized in that, In step 1), the peptide chain solution is a PB buffer solution containing 1 mg / mL of standard peptide chain; the magnetic microparticle MB@SA suspension is a suspension containing 2.5 mg / mL streptavidin-functionalized magnetic beads; the iridium metal complex solution is a dimethyl sulfoxide solution containing 1 mmol / L iridium metal complex; and the matrix metalloproteinase standard solution is a PB buffer solution containing 100 ng / mL matrix metalloproteinase.

4. The electrochemiluminescence peptide sensing method for detecting matrix metalloproteinases according to claim 3, characterized in that, The matrix metalloproteinase is MMP-2, MMP-3, MMP-7, or MMP-9.

5. The electrochemiluminescent peptide sensing method for detecting matrix metalloproteinases according to any one of claims 1-4, characterized in that, In step 1), the conditions for the isothermal incubation reaction are: temperature 37~37.5°C, time 0.5~2h.

6. The electrochemiluminescence peptide sensing method for detecting matrix metalloproteinases according to claim 5, characterized in that, The PB buffer solution in step 1) has a concentration of 10 mmol / L and a volume of 15 μL.

7. The electrochemiluminescence peptide sensing method for detecting matrix metalloproteinases according to claim 6, characterized in that, The electrolyte in step 2) is a PBS buffer solution containing 50 mmol / L tri-n-propylamine (TPA); the concentration of the PBS buffer solution is 0.1 mol / L.

8. The electrochemiluminescent peptide sensing method for detecting matrix metalloproteinases according to claim 7, characterized in that, In step 2), the conditions for cyclic voltammetry are: potential of 0-1.6 V and scan rate of 0.1 V / s.

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