Nano-enzyme for specifically recognizing lesion collagen, preparation method and application

Abstract

The invention belongs to the field of biological detection, and particularly relates to a nano-enzyme for specifically recognizing lesion collagen as well as a preparation method and application of the nano-enzyme. The invention firstly provides the polypeptide which is in targeted binding with the lesion collagen, and the polypeptide has excellent binding force to the lesion collagen; secondly, the invention provides the lesion collagen targeting nano-enzyme, and the lesion collagen targeting nano-enzyme is prepared by a one-step method through reaction of the reduction sequence of the lesion collagen targeting polypeptide and Au < 3 + > under mild conditions. The nano-enzyme provided by the invention has the specific recognition capability of the lesion collagen and the catalytic activity of peroxidase, can be applied to qualitative and ultrasensitive quantitative analysis of the lesion collagen, and has a wide application prospect in early screening and noninvasive diagnosis of collagen-related diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection, and in particular relates to a nanozyme that specifically recognizes diseased collagen, a preparation method and an application thereof. Background Art

[0002] Pathological collagen is a key biomarker for diseases such as tumors, fibrosis, and arthritis, and holds great potential for early diagnosis and targeted treatment of these conditions. In tumors, the triple helical structure of collagen is abnormally disrupted by various proteases, resulting in pathological collagen. In fibrotic diseases, elevated collagen expression and cross-linking are accompanied by abnormal increases in pathological collagen. Furthermore, excessive joint friction and enzymatic degradation lead to joint damage, resulting in the production of large amounts of pathological collagen in diseased joint tissue. Therefore, highly sensitive analysis of pathological collagen is crucial for the early screening and non-invasive diagnosis of collagen-related diseases.

[0003] Monoclonal antibodies developed based on immune technology have been used to specifically analyze collagen to achieve disease detection. Patent CN115667295A and patent CN116529602A involve the specific analysis of type XIX collagen and type XXVIII collagen, respectively, for the diagnosis of cancer. Patent CN112505331B involves a type IV collagen detection kit, which provides an important means for the early diagnosis of fibrosis. At the same time, patents CN116355088A and CN114031686A involve monoclonal antibodies targeting the α chain of type IV collagen, which have been used to detect Alport syndrome. However, due to the structural complexity of diseased collagen, these technologies have problems such as insufficient specificity and low binding force.

[0004] (Gly-Pro-Hyp) n Peptide sequences such as F-GOP-10 have been shown to specifically bind to the unhelicaled regions of diseased collagen and have attracted widespread attention as tools for the targeted identification of diseased collagen. However, these probes are prone to self-assembly into triple-helical structures, which in turn lose their ability to bind to diseased collagen. F-GOP-10, the first intrinsically single-stranded peptide probe targeting diseased collagen, has been developed and holds great promise for the specific analysis of diseased collagen. However, the content of diseased collagen in pathological environments is extremely low, and highly sensitive detection of diseased collagen remains a significant challenge. Summary of the Invention

[0005] In response to the above technical problems, the purpose of the present invention is to provide a nanozyme that specifically recognizes diseased collagen, a preparation method and an application.

[0006] Specifically include the following:

[0007] In a first aspect, the present invention provides a polypeptide that targets and binds to diseased collagen, characterized in that the polypeptide comprises a targeting sequence Gly-Hyp-Pro-Gly-Hyp-Hyp-(Gly-Hyp-Pro)6-Gly-Hyp-Hyp-Gly-Hyp-Pro.

[0008] Preferably, the polypeptide further comprises a reducing sequence (Cys) n -(Tyr) m ; wherein n and m are any integer between 1 and 5; the reducing sequence is located at the N-terminus or C-terminus of the targeting sequence.

[0009] Preferably, the reducing sequence is located at the N-terminus of the targeting sequence.

[0010] Preferably, the reducing sequence is Cys-Cys-Tyr.

[0011] Preferably, the reducing sequence and the targeting sequence are connected via Ahx.

[0012] In a second aspect, the present invention provides a diseased collagen-targeted nanozyme, which is composed of a nanozyme and the polypeptide of the first aspect, and the polypeptide is modified on the surface of the nanozyme.

[0013] Preferably, the nanozyme is selected from any one of gold, silver, platinum, iron oxide, manganese oxide, and cerium oxide nanomaterials.

[0014] Preferably, the nanozyme is a gold nanomaterial.

[0015] Preferably, the nanozyme structure is any one of nanospheres, nanoclusters, nanorods, nanoflowers, nanostars and nanocages.

[0016] Preferably, the nanozyme structure is a nanosphere.

[0017] Preferably, the nanozyme has a particle size of 1-100 nm.

[0018] Preferably, the nanozyme particle size is 2-4 nm.

[0019] In a third aspect, the present invention provides a method for preparing a diseased collagen-targeted nanozyme, the preparation method comprising the following steps:

[0020] (1) solid phase synthesis of any of the polypeptides described in the first aspect;

[0021] (2) dissolving the polypeptide synthesized in step (1) in ultrapure water to obtain solution A; dissolving tetrachloroauric acid in ultrapure water to obtain solution B;

[0022] (3) Solution A and solution B are mixed in a molar ratio of 1:10-10:1, the pH of the mixed solution is adjusted to 5-14, and the mixture is stirred at 20-70°C for 1-48 hours to obtain the pathological collagen-targeted nanozyme.

[0023] Preferably, the concentration of solution A is 1 mM.

[0024] Preferably, the molar ratio of solution A to solution B is 1:2.

[0025] Preferably, the reaction temperature is 37°C.

[0026] Preferably, the reaction time is 24 hours.

[0027] Preferably, the pH is 11-14.

[0028] Preferably, the solid phase synthesis method of the diseased collagen targeting polypeptide is:

[0029] (1) Using Rink ammonia resin or chlorine resin as the resin for solid phase synthesis of peptides;

[0030] (2) 4 eq of amino acid, HOBt, and HBTU were dissolved in DMF, activated at low temperature for 10-30 min, and then 4-10 eq of DIEA was added dropwise to the solution to obtain a mixed solution;

[0031] (3) adding the mixed solution prepared in step (2) to the polypeptide resin described in step (1) and reacting in the dark for 2-4 hours;

[0032] (4) treating the resin from step (3) with 20% piperidine DMF solution for 15 min;

[0033] (5) Steps (2), (3) and (4) are repeated until the target lesion collagen-targeted peptide is synthesized;

[0034] (6) The polypeptide resin after the reaction in step (5) is treated with a cutting fluid for 2-4 hours, and then glacial ether is added. The resulting precipitate is the diseased collagen targeting polypeptide, wherein the cutting fluid is composed of trifluoroacetic acid, a free radical scavenger, and water in a volume ratio of 95:2.5:2.5 or trifluoroacetic acid, a free radical scavenger, and dichloromethane in a volume ratio of 90:5:5.

[0035] In a fourth aspect, the present invention provides an application of the diseased collagen targeted nanozyme described in the second aspect in the qualitative or quantitative analysis of diseased collagen.

[0036] In a fifth aspect, the present invention provides a method for quantitatively detecting pathological collagen, the method comprising the following steps:

[0037] (1) Mixing the diseased collagen-targeted nanozyme with the diseased collagen standard and the sample to be tested;

[0038] (2) adding the mixed solution to a binding plate and incubating at 4-25° C. for 1-12 hours; the binding plate is a microplate physically modified with a pathological collagen standard;

[0039] (3) Remove the liquid from the binding plate and wash with PBS;

[0040] (4) color development using peroxidase substrate;

[0041] (5) The microplate reader collects the signal intensity data in the binding plate wells;

[0042] (6) Draw a standard curve based on the test results of the pathological collagen standard and calculate the content of pathological collagen in the sample to be tested.

[0043] Preferably, the peroxidase chromogenic substrate is any one of luminol, 3,3',5,5'-tetramethylbenzidine (TMB), o-phenylenediamine (OPD), 2,2'-azino-bis-3-acetylanilide (ABTS), 4-nitrophenylphosphate disodium (pNPP), 5-bromo-4-chloro-3-indolylphosphate / nitro blue tetrazolium (BCIP / NBT), 3-amino-9-ethylcarbazole (AEC) and tyramine.

[0044] Preferably, the peroxidase chromogenic substrate is luminol.

[0045] Preferably, the concentration of the diseased collagen targeting nanozyme is 800 μg / mL; the volume ratio of the diseased collagen targeting nanozyme to the diseased collagen standard or the sample to be tested is 1:1.

[0046] Preferably, the standard concentration of the diseased collagen in the modified binding plate is 500 μg / mL.

[0047] Preferably, the volume of the mixed solution is 50 μL.

[0048] The beneficial effects of the present invention are:

[0049] ① The present invention first provides a polypeptide that targets and binds to diseased collagen, and the polypeptide has a higher binding affinity to the diseased collagen;

[0050] ② The diseased collagen targeted nanozyme provided by the present invention has both the diseased collagen specific recognition function and the peroxidase catalytic performance;

[0051] ③ The preparation method of the diseased collagen-targeted nanozyme provided by the present invention is simple and rapid;

[0052] The diseased collagen-targeted nanozyme provided by the present invention has strong specificity and high binding affinity in the detection of diseased collagen. It is suitable for qualitative and quantitative analysis of diseased collagen and has broad application prospects in the early diagnosis and non-invasive screening of collagen-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 Gly-Hyp-Pro-Gly-Hyp-Hyp-(Gly-Hyp-Pro)6-Gly-Hyp-Hyp-Gly-Hyp-Pro and (Gly-Hyp-Pro) 10 Binding curves of the sequences to diseased collagen;

[0054] Figure 2 TEM image of 2nm lesion collagen-targeted nanozyme, colorimetric image of catalyzing H2O2 / TMB substrate, and Michaelis-Menten curve; where a is TEM characterization, b is colorimetric image, c is Michaelis-Menten curve of catalyzing H2O2 substrate, and d is Michaelis-Menten curve of catalyzing TMB substrate;

[0055] Figure 3 TEM image of 4nm diseased collagen-targeted nanozyme, colorimetric image of catalyzing H2O2 / TMB substrate, and Michaelis-Menten curve; where a is TEM characterization, b is colorimetric image, c is Michaelis-Menten curve of catalyzing H2O2 substrate, and d is Michaelis-Menten curve of catalyzing TMB substrate;

[0056] Figure 4 TEM image of the 9nm diseased collagen-targeted nanozyme, colorimetric image of the catalytic H2O2 / TMB substrate, and Michaelis-Menten curve; where a is TEM characterization, b is the colorimetric image, c is the Michaelis-Menten curve of the catalytic H2O2 substrate, and d is the Michaelis-Menten curve of the catalytic TMB substrate;

[0057] Figure 5 TEM image of the 12nm diseased collagen-targeted nanozyme, colorimetric image of the catalytic H2O2 / TMB substrate, and Michaelis-Menten curve; where a is TEM characterization, b is the colorimetric image, c is the Michaelis-Menten curve of the catalytic H2O2 substrate, and d is the Michaelis-Menten curve of the catalytic TMB substrate;

[0058] Figure 6TEM image of 15nm diseased collagen-targeted nanozyme, colorimetric image of catalyzing H2O2 / TMB substrate, and Michaelis-Menten curve; where a is TEM characterization, b is colorimetric image, c is Michaelis-Menten curve of catalyzing H2O2 substrate, and d is Michaelis-Menten curve of catalyzing TMB substrate;

[0059] Figure 7 TEM image of the 17nm diseased collagen-targeted nanozyme, colorimetric image of the catalytic H2O2 / TMB substrate, and Michaelis-Menten curve; where a is TEM characterization, b is the colorimetric image, c is the Michaelis-Menten curve of the catalytic H2O2 substrate, and d is the Michaelis-Menten curve of the catalytic TMB substrate;

[0060] Figure 8 Images of 4nm lesion collagen-targeted nanozymes catalyzing TMB, OPD, DAB, and AEC substrates;

[0061] Figure 9 Histograms of PCTE4 binding to lesional collagen, intact type I collagen, intact type II collagen, intact type III collagen, human serum albumin, trypsin, and pepsin;

[0062] Figure 10 TEM images of PCTE4-stained collagen fibers; wherein, a is an image of PCTE4-stained heat-treated collagen fibers, b is an enlarged image of a, c is a TEM image of PCTE4-stained normal collagen fibers, and d is an enlarged image of c;

[0063] Figure 11 Images of PCTE4-stained mouse tail tissue.

[0064] Figure 12 Images of bovine hide tissue stained with PCTE4; a is an image of heat-treated bovine hide stained with PCTE4, b is an image of normal bovine hide stained with PCTE4, c is an image of heat-treated bovine hide stained with gold nanoparticles, and d is an image of bovine hide pre-incubated with Ac-GOP-10 stained with PCTE4;

[0065] Figure 13 Linear curve of quantitative analysis of lesion collagen content using the luminol system.

[0066] Figure 14 Linear curve of TMB system for quantitative analysis of lesion collagen content.

[0067] Figure 15Quantitative analysis of diseased collagen in the presence of intact type I collagen, human serum albumin, vitamin C, vitamin B1, glucose, NaCl, and KCl.

[0068] Figure 16 Chemiluminescent signal curve for quantitative analysis of lesion collagen in a high-protein environment.

[0069] Figure 17 Quantitative analysis of chemiluminescent signal curves of pathological collagen in a high-salt environment.

[0070] Figure 18 Quantitative analysis of the chemiluminescent signal curve of lesion collagen in a high glucose environment.

[0071] Figure 19 Fitting curve of the detected and theoretical concentrations of pathological collagen in serum.

[0072] Figure 20 Fitting curve of the detected and theoretical concentrations of pathological collagen in joint cavity fluid.

[0073] Figure 21 Fitting curve of the detected and theoretical concentrations of pathological collagen in urine.

[0074] Figure 22 Fitting curve of the detected and theoretical concentrations of pathological collagen in saliva.

[0075] Figure 23 Quantitative analysis results of pathological collagen in body fluids of tumor models.

[0076] Figure 24 Quantitative analysis results of pathological collagen in body fluids of fibrosis model.

[0077] Figure 25 Quantitative analysis results of pathological collagen in body fluids of arthritis models. DETAILED DESCRIPTION

[0078] To make the technical means, creative features, objectives and effects of the present invention easier to understand, the present invention is further described below in conjunction with specific embodiments. However, the scope of protection of the present invention is not limited to the following embodiments.

[0079] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the methods belong. Although any methods and compositions similar or equivalent to those described herein can also be used to practice or test the methods and compositions, representative example methods and compositions are now described. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the scope of the present invention, which will be limited only by the appended claims.

[0080] The polypeptides in the following examples were all synthesized by solid phase synthesis.

[0081] Example 1Gly-Hyp-Pro-Gly-Hyp-Hyp-(Gly-Hyp-Pro)6-Gly-Hyp-Hyp-Gly-Hyp-Pro and (Gly-Hyp-Pro) 10 Binding ability to diseased collagen

[0082] 50 μL of 10 μg / mL diseased collagen solution was added to a flat-bottom black microplate and incubated at 4°C for 12 hours. The microwells were then washed three times with 100 μL of PBS. 50 μL of 1 mg / mL BS A solution was added to the microplate and incubated at room temperature for 1 hour. The microwells were washed three times with 100 μL of PBS. 0.1 nM, 1 nM, 3.2 nM, 10 nM, 32 nM, 100 nM, 320 nM, 1 μM, 3.2 μM, 10 μM, 32 μM, and 100 μM of Ac-Gly-Hyp-Pro-Gly-Hyp-Hyp-(Gly-Hyp-Pro)6-Gly-Hyp-Hyp-Gly-Hyp-Pro-NH2 and Ac-(Gly-Hyp-Pro) 10 -NH2 solution in the microwells, incubate at 4°C for 4 hours, and wash the microwells three times with 100 μL PBS. 50 μL FAM-Ahx-Gly-Hyp-Pro-Gly-Hyp-Hyp-(Gly-Hyp-Pro)6-Gly-Hyp-Hyp-Gly-Hyp-Pro-NH2 and FAM-Ahx-(Gly-Hyp-Pro) 10 The -NH2 probe solution was added to the microwells and incubated at 4°C for 4 hours. The microwells were washed three times with 100 μL PBS. The fluorescence intensity was measured using a Swiss Tecan Infinite M200 microplate reader at an excitation wavelength of 493 nm and an emission wavelength of 533 nm. The equilibrium dissociation constant (K) was calculated using the "one site-homogenous" model in GraphPad Prism 9.5.1 software. D ) and draw the binding curve.

[0083] The results are as follows Figure 1 As shown, Gly-Hyp-Pro-Gly-Hyp-Hyp-(Gly-Hyp-Pro)6-Gly-Hyp-Hyp-Gly-Hyp-Pro and (Gly-Hyp-Pro) 10 The binding curves to pathological collagen showed a Z-shaped pattern, indicating that both sequences can bind to pathological collagen. 10 The equilibrium dissociation constant (K D ) values ​​were 12.6 nM and 74.9 nM, respectively, indicating that the Gly-Hyp-Pro-Gly-Hyp-Hyp-(Gly-Hyp-Pro)6-Gly-Hyp-Hyp-Gly-Hyp-Pro sequence has a higher binding affinity to diseased collagen.

[0084] Example 2 Pathological collagen-targeted nanozymes of different particle sizes

[0085] Preparation of pathological collagen-targeted nanozymes of different particle sizes: Weigh 32 mg of pathological collagen-targeted polypeptide Cys-Cys-Tyr-Ahx-Gly-Hyp-Pro-Gly-Hyp-Hyp-(Gly-Hyp-Pro)6-Gly-Hyp-Hyp-Gly-Hyp-Pro-NH2 and dissolve it in 10 mL of ultrapure water, and prepare 10 mL of 2 mM tetrachloroauric acid solution; mix the polypeptide solution with the tetrachloroauric acid solution and divide it into 6 equal parts, and use 0.1 M sodium hydroxide to adjust the pH to 5, 6, 7, 8, 11 and 14, respectively; stir the reaction at 37°C for 24 hours, collect the nanozymes by centrifugation, wash with ultrapure water and air-dry, to obtain six types of pathological collagen-targeted nanozymes.

[0086] Transmission electron microscopy (TEM) characterization: The six types of pathological collagen-targeted nanozymes were dispersed in ultrapure water, and a small amount of the solution was dripped onto the surface of a copper mesh. After incubation for five minutes, the remaining solution was removed and the nanozymes were naturally dried. The morphology was observed using a Talos200s transmission electron microscope.

[0087] Colorimetric characterization of catalytic TMB / H2O2 substrates: 4 μg of each of the six pathological collagen-targeting nanozymes was added to a 200 μL reaction system containing 800 μM TMB and 800 mM H2O2 at pH 4. After 20 minutes of reaction at room temperature, images of the reaction solutions were captured.

[0088] Steady-state kinetic analysis of H2O2 substrate: 10 μg / mL of six nanozyme samples were mixed with 800 μM TMB and different concentrations of H2O2 (0, 200, 500, 1000, 1500, 2000 mM), with a mixed system volume of 200 μL and a pH value of 4. A Swiss Tecan Infinite M200 microplate reader was used to monitor the absorbance changes at a wavelength of 652 nm in real time. According to the Michaelis-Menten kinetic model V = V max [S] / (K m +[S]) to calculate the Michaelis constant K m , where [S] is the substrate concentration, V is the initial reaction rate, and V max is the maximum reaction rate, K m is the Michaelis constant.

[0089] Steady-state kinetic analysis of TMB substrate: Six nanozyme samples at 10 μg / mL were mixed with 800 mM H2O2 and different concentrations of TMB (0, 125, 250, 500, and 1000 μM), with a volume of 200 μL and a pH of 4. Absorbance changes were continuously monitored at a wavelength of 652 nm using an Infinite M200 microplate reader. The reaction data for each group were fitted with the Michaelis-Menten model to obtain K m value.

[0090] The results are as follows Figures 2 to 7 As shown, the prepared nanozymes targeting pathological collagen all exhibited spherical structures. Under reaction conditions of pH 5, 6, 7, 8, 11, and 14, the resulting nanozymes had particle sizes of 17 nm, 15 nm, 12 nm, 9 nm, 4 nm, and 2 nm, respectively. This indicates that the particle size of the pathological collagen-targeted nanozymes can be regulated by pH. Colorimetric results showed that after 20 minutes of reaction with the H2O2 / TMB system, the reaction solution exhibited the deepest blue. As the particle size increased from 4 nm to 17 nm, the color gradually became lighter, indicating that the catalytic activity decreased with increasing particle size.

[0091] Steady-state kinetic analysis showed that the K of 2nm to 17nm lesion collagen-targeted nanozymes for H2O2 substrates m The K values ​​for TMB substrate were 853.8mM, 851.7mM, 1231mM, 1306mM, 1369mM and 1461mM, respectively. m The values ​​were 20.95μM, 21.4μM, 34.83μM, 40.15μM, 44.35μM and 47.41μM, respectively. The results showed that nanozymes with particle sizes of 2nm and 4nm had stronger substrate affinity and higher catalytic efficiency, while the catalytic performance weakened with the increase of particle size.

[0092] In subsequent studies, a 4nm diseased collagen-targeted nanozyme (named PCTE4) was selected for the development of biochips.

[0093] Example 3 Peroxidase Substrate Suitability of PCTE4

[0094] 10 μg PCTE4 was added to 1 mL of TMB / H2O2 system, o-phenylenediamine (OPD) / H2O2 system, 3,3'-diaminobenzidine (DAB) / H2O2 system and 3-amino-9-ethylcarbazole (AEC) / H2O2 system, and images of the solutions before and after the addition of PCTE4 were collected.

[0095] The results are as follows Figure 8 As shown, the original TMB / H2O2 solution is colorless and transparent, but quickly turns dark blue after the addition of PCTE4. Similarly, the OPD / H2O2, DAB / H2O2, and AEC / H2O2 solutions exhibit orange-red, brown, and red, respectively, after the addition of PCTE4. These results demonstrate that PCTE4 exhibits significant catalytic activity in a variety of typical peroxidase substrate systems, effectively promoting substrate oxidation reactions and meeting the substrate applicability requirements for diverse bioanalysis scenarios.

[0096] Example 4 Specificity Study of PCTE4

[0097] Diseased collagen, intact type I collagen (Col I), type II collagen (Col II), type III collagen (Col III), human serum albumin (HSA), trypsin, and pepsin were coated in 96-well plates, and then PCTE4 probe was added for incubation and washing, and color was developed using the TMB / H2O2 substrate system.

[0098] The results are as follows Figure 9 As shown, only the diseased collagen-coated wells produced a distinct blue coloration, while the other wells showed no coloration, demonstrating the excellent targeting specificity of PCTE4. The absorbance intensity of each microwell was measured at 652 nm using a microplate reader. The absorbance of the diseased collagen-coated wells was 1.29 ± 0.021, while the absorbance of the other protein-coated wells was below 0.03, further confirming that PCTE4 specifically binds to diseased collagen, while not binding to intact collagen or other common proteins.

[0099] Example 5 Application of PCTE4 in Collagen Fiber Staining

[0100] (1) Take a small amount of 3 mg / mL type I collagen solution and a type I collagen solution treated at 85°C for 20 min, drop them onto the copper mesh, incubate for 5 minutes, remove the remaining solution and dry naturally.

[0101] (2) A small amount of 200 μg / mL PCTE4 solution was added dropwise onto the copper grids, incubated for 5 minutes, and the remaining solution was removed and allowed to dry naturally.

[0102] (3) Place the copper mesh under a Talos 200s transmission electron microscope to observe its morphology.

[0103] The results are as follows Figure 10 As shown, a large amount of PCTE4 was bound to the surface of collagen fibers after heat treatment, while almost no PCTE4 was found on normal collagen fibers, indicating that PCTE4 can recognize and specifically bind to collagen fibers with destroyed structures.

[0104] Example 6 Application of PCTE4 in Mouse Tail Tissue Staining

[0105] (1) Rat tail tissue was treated with 1% sodium dodecyl sulfate (SDS) solution for 24 hours to denature collagen, and then repeatedly washed with deionized water to remove SDS;

[0106] (2) The SDS-treated rat tail tissue was cut into 5 μm slices using a freezing microtome.

[0107] (3) Treat with 200 μL 3% H2O2 at room temperature for 10 min to eliminate endogenous peroxidase, and then wash with PBS three times (3 min / time).

[0108] (4) Add 0.2 mL of 5% BSA / PBS blocking solution and incubate at room temperature for 30 min.

[0109] (5) 100 μL of 200 μg / mL PCTE4 solution was evenly applied to the SDS-treated rat tail tissue sections, incubated at 4°C for 12 h, and washed three times with PBS (3 min / time).

[0110] (6) The sections were stained with FAM-tyramide, and images were collected using an Olympus IX53 inverted fluorescence microscope.

[0111] The results are as follows Figure 11 As shown, the SDS-treated mouse tail tissue stained with PCTE4 exhibited significant green fluorescence, indicating that PCTE4 could specifically bind to the structurally destroyed collagen in the mouse tail tissue.

[0112] Example 7 Application of PCTE4 in Skin Tissue Staining

[0113] (1) Normal bovine skin tissue and bovine skin tissue treated at 85°C for 20 min were cut into 5 μm slices using a freezing microtome.

[0114] (2) Treat with 200 μL 3% H2O2 at room temperature for 10 min to eliminate endogenous peroxidase, and then wash with PBS three times (3 min / time).

[0115] (3) Add 0.2 mL of 5% BSA / PBS blocking solution and incubate at room temperature for 30 min.

[0116] (4) 100 μL of 200 μg / mL PCTE4 solution was evenly applied to the normal and heat-treated cowhide tissue sections, incubated at 4°C for 12 h, and washed 3 times with PBS (3 min / time); 100 μL of 200 μg / mL gold nanoparticle solution was evenly applied to the heat-treated cowhide tissue sections, incubated at 4°C for 12 h, and washed 3 times with PBS (3 min / time); 100 μL of 50 mM Ac-(Gly-Hyp-Pro) 10 -NH2 solution was evenly covered with normal bovine leather tissue and heat-treated bovine leather tissue sections, incubated at 4°C for 12 h, and washed three times with PBS (3 min / time). Subsequently, 100 μL of 200 μg / mL PCTE4 solution was evenly covered with normal bovine leather tissue and heat-treated bovine leather tissue sections, incubated at 4°C for 12 h, and washed three times with PBS (3 min / time).

[0117] (5) Each group of slices was stained with DAB / H2O2, and images were collected using an Olympus BX63 optical microscope.

[0118] The results are as follows Figure 12 As shown, PCTE4 staining of heat-treated bovine leather tissue showed obvious brown deposits, while normal bovine leather tissue showed no color reaction. At the same time, no color signal was observed in the heated bovine leather tissue stained with gold nanoparticles. This indicates that PCTE4 is highly specific for pathological collagen in bovine leather tissue. 10 After pretreatment with -NH2, PCTE4-stained heated cowhide tissue did not develop color, further confirming the interaction between PCTE4 and Ac-(Gly-Hy p-Pro) 10 -NH2 recognition site identity.

[0119] Example 8 Fitting Linear Curve for Quantitative Analysis of Lesion Collagen

[0120] (1) 50 μL of 800 μg / mL PCTE4 solution was mixed with an equal volume of pathological collagen standard (0.2-20000 ng / mL) and incubated at 4°C for 4 h;

[0121] (3) Take 50 μL of the standard mixture and sample mixture after incubation, add them to the microplate pre-coated with pathological collagen, and continue incubation at 4°C for 4 hours;

[0122] (4) Remove the liquid from the binding plate and wash with PBS three times;

[0123] (5) adding luminol luminescent substrate to develop the signal;

[0124] (6) The microplate reader collects the signal intensity data in the binding plate wells;

[0125] (7) Draw a standard curve based on the test results of the standard sample, and calculate the concentration of pathological collagen in the sample to be tested based on it.

[0126] The results are as follows Figure 13 As shown in Figure 2, the chemiluminescence intensity was negatively correlated with the logarithm of the lesion collagen concentration (R 2 =0.9982), this method can achieve quantitative analysis in the concentration range of 0.1-10000 ng / mL, and the detection limit is as low as 15 pg / mL (50 fM).

[0127] The colorimetric system was constructed by replacing luminol with TMB, and the absorbance of different concentration gradient pathological collagen solutions was analyzed to evaluate the quantitative detection ability of the colorimetric analysis method. Figure 14 As shown in Figure 2, the absorbance and the logarithm of the concentration were linear in the range of 0.32-32000 ng / mL (R 2 =0.9958), with a detection limit of 64 pg / mL.

[0128] Example 9 Stability of quantitative analysis of pathological collagen

[0129] (1) Impurity interference test: 10 μg / mL of pathological collagen (PC), non-PC substances including type I collagen (Col I), human serum albumin (HSA), ascorbic acid, vitamin B1, glucose, urea, sodium chloride (NaCl) and potassium chloride (KCl), and a mixture of non-PC substances were prepared respectively; the pathological collagen quantitative analysis method described in this application was used to detect the concentration of pathological collagen in the above samples.

[0130] The results are as follows Figure 15 As shown, the detected concentration of PC samples was (9782.70 ± 1477.89) ng / mL, while PC was virtually undetectable in non-PC substances and their mixtures. Further analysis of various non-PC interfering substances mixed with PC revealed that the concentrations obtained were nearly identical to those of pure PC samples. This result confirms the excellent selectivity and anti-interference capabilities of this quantitative analysis method.

[0131] (2) Complex environment stability experiment: PC samples with a concentration of 10 μg / mL were tested in environments with HSA concentrations ranging from 0.001% to 10%, NaCl concentrations ranging from 0.1 to 1000 mM, and glucose concentrations ranging from 0.1 to 1000 mM, and the changes in their chemiluminescence signal intensity were collected.

[0132] The results are as follows Figures 16 to 18 As shown in the figure, the chemiluminescence signal of PC sample detection remains stable under high protein, high salt and high sugar environments, indicating that this method has excellent detection stability and is suitable for accurate analysis needs in a variety of actual biological environments.

[0133] Example 10 Precision of quantitative analysis of pathological collagen

[0134] 10, 100, and 1000 ng / mL of pathological collagen standards were added to serum, joint fluid, urine, and saliva, respectively. The pathological collagen concentration in each sample was determined using the pathological collagen quantitative analysis method described in this application. The measured concentration was then curve-fitted with the theoretical concentration, and the fitting degree R was calculated. 2 and the slope of the curve.

[0135] The results are as follows Figure 19 As shown in the figure, in serum matrix, the detection concentrations of the three samples were (11.04±1.76) ng / mL, (101.18±16.39) ng / mL, and (948.79±152.31) ng / mL, respectively, with corresponding spike recovery rates of 110.40%, 101.18%, and 94.79%. The slope of the fitting curve was 0.9697, and R 2 is 0.9985. Figure 20 As shown in the figure, in the joint cavity fluid, the detection concentrations of the three samples were (9.75±1.67) ng / mL, (95.92±16.17) ng / mL, and (999.91±108.42) ng / mL, respectively. The spike recovery rates were 97.50%, 95.92%, and 99.99%, respectively. The slope of the fitting curve was 0.9987, and R 2 is 0.9993. Figure 21 As shown in the figure, in urine matrix, the detection concentrations of the three samples were (10.71±1.71) ng / mL, (96.88±13.08) ng / mL, and (992.21±145.77) ng / mL, with corresponding spike recovery rates of 107.10%, 96.88%, and 99.22%. The slope of the fitting curve was 0.9744, and R 2 is 0.9994. Figure 22As shown in the figure, in saliva samples, the detection concentrations of the three samples were (9.34±2.33) ng / mL, (97.38±12.74) ng / mL, and (1009.50±155.94) ng / mL, respectively. The spike recovery rates were 93.40%, 97.38%, and 100.90%, respectively. The slope of the fitting curve was 0.9928, and R 2 is 0.9995.

[0136] The above results show that this quantitative detection method can accurately detect pathological collagen in complex body fluid matrices such as serum, joint cavity fluid, urine and saliva, and has excellent versatility and accuracy.

[0137] Example 11 Application of body fluid analysis in tumor models

[0138] (1) 50 μL of 400 μg / mL PCTE4 was mixed with 50 μL of standard (0.2, 0.64, 2, 6.4, 20, 64, 200, 640, 2000, 6400, and 20,000 ng / mL of diseased collagen);

[0139] (2) Mix 50 μL of 400 μg / mL PCTE4 with 50 μL of sample (serum dilutions from tumor-bearing and normal mice);

[0140] (3) Add 50 μL of the mixture prepared in steps (1) and (2) to the binding plate and incubate at 4°C for 4 hours;

[0141] (4) Remove the liquid from the binding plate and wash with PBS three times;

[0142] (5) color development using luminol substrate;

[0143] (6) The microplate reader collects the signal intensity data in the binding plate wells;

[0144] (7) Draw a standard curve based on the test results of the standard and calculate the content of pathological collagen in the sample to be tested.

[0145] The standard curve is: y = -8835x + 65450.

[0146] The results are as follows Figure 23 The results showed that the level of pathological collagen in the serum of normal mice was (10.13±5.95) ng / mL, while the level in the serum of tumor model mice was (61.18±21.94) ng / mL. The results showed that there was a significant difference in the levels of pathological collagen in the serum of the two groups of mice. This quantitative analysis method can effectively identify the levels of pathological collagen in tumors and normal conditions, significantly distinguishing tumor model mice from normal mice, and provides a powerful tool for early diagnosis of tumors.

[0147] Example 12 Application of Body Fluid Analysis in Liver Fibrosis Model

[0148] (1) 50 μL of 400 μg / mL PCTE4 was mixed with 50 μL of standard (0.2, 0.64, 2, 6.4, 20, 64, 200, 640, 2000, 6400, and 20,000 ng / mL of diseased collagen);

[0149] (2) Mix 50 μL of 400 μg / mL PCTE4 with 50 μL of the test sample (serum dilutions of normal mice and liver fibrosis model mice);

[0150] (3) Add 50 μL of the mixture prepared in steps (1) and (2) to the binding plate and incubate at 4°C for 4 hours;

[0151] (4) Remove the liquid from the binding plate and wash with PBS three times;

[0152] (5) color development using luminol substrate;

[0153] (6) The microplate reader collects the signal intensity data in the binding plate wells;

[0154] (7) Draw a standard curve based on the test results of the standard and calculate the content of pathological collagen in the sample to be tested.

[0155] The standard curve is: y = -8519x + 62832.

[0156] The results are as follows Figure 24 As shown in the results, the serum levels of pathological collagen in normal mice and liver fibrosis model mice were (7.66±7.04) ng / mL and (70.07±15.90) ​​ng / mL, respectively. This significant difference demonstrates that this quantitative analysis method can accurately distinguish normal mice from liver fibrosis model mice through body fluid sample testing, providing a powerful tool for the early diagnosis of liver fibrosis.

[0157] Example 13 Application of Body Fluid Analysis in Arthritis Model

[0158] (1) 50 μL of 400 μg / mL PCTE4 was mixed with 50 μL of standard (0.2, 0.64, 2, 6.4, 20, 64, 200, 640, 2000, 6400, and 20,000 ng / mL of diseased collagen);

[0159] (2) Mix 50 μL of 400 μg / mL PCTE4 with 50 μL of the test sample (dilution of the joint cavity fluid of normal rabbits and arthritis model rabbits);

[0160] (3) Add 50 μL of the mixture prepared in steps (1) and (2) to the binding plate and incubate at 4°C for 4 hours;

[0161] (4) Remove the liquid from the binding plate and wash with PBS three times;

[0162] (5) color development using luminol substrate;

[0163] (6) The microplate reader collects the signal intensity data in the binding plate wells;

[0164] (7) Draw a standard curve based on the test results of the standard and calculate the content of pathological collagen in the sample to be tested.

[0165] The standard curve is: y = -8670x + 63384.

[0166] The results are as follows Figure 25 As shown, the results of pathological collagen analysis showed that the levels of pathological collagen in the serum of normal rabbits and arthritis model rabbits were (14.79±10.03) ng / mL and (92.85±18.15) ng / mL, respectively. This result indicates that there is a significant difference in the levels of pathological collagen in the joint fluid of arthritis model rabbits and normal rabbits. These results fully demonstrate that this quantitative analysis method can accurately distinguish between normal and arthritis states using body fluid samples, providing a reliable method for the early diagnosis of arthritis.

[0167] The foregoing description is merely a detailed description of a few exemplary embodiments of the present invention. Those skilled in the art will appreciate that various modifications and variations are possible in actual application depending on the specific preparation conditions, and are not intended to limit the present invention. Anything within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A polypeptide that targets and binds to diseased collagen, characterized in that: The polypeptide includes the targeting sequence Gly-Hyp-Pro-Gly-Hyp-Hyp-(Gly-Hyp-Pro)6-Gly-Hyp-Hyp-Gly-Hyp-Pro.

2. The polypeptide according to claim 1, wherein The polypeptide also includes a reducing sequence (Cys) n -(Tyr) m ; wherein n and m are any integer between 1 and 5; the reducing sequence is located at the N-terminus or C-terminus of the targeting sequence.

3. The polypeptide according to claim 1, wherein The reducing sequence and the targeting sequence are connected via Ahx.

4. A diseased collagen-targeted nanozyme, characterized in that: The diseased collagen targeting nanozyme is composed of a nanozyme and a polypeptide according to any one of claims 1 to 3, and the polypeptide is modified on the surface of the nanozyme.

5. The diseased collagen-targeted nanozyme according to claim 4, wherein: The nanozyme is selected from any one of gold, silver, platinum, iron oxide, manganese oxide, and cerium oxide nanomaterials.

6. The diseased collagen-targeted nanozyme according to claim 5, wherein: The nanozyme is a gold nanomaterial.

7. The diseased collagen-targeted nanozyme according to claim 6, wherein: The nanozyme structure is any one of nanospheres, nanoclusters, nanorods, nanoflowers, nanostars and nanocages.

8. The diseased collagen-targeted nanozyme according to claim 7, wherein: The nanozyme particle size is 1-100 nm.

9. A method for preparing a pathological collagen-targeted nanozyme, characterized in that: The preparation method comprises the following steps: (1) solid phase synthesis of the polypeptide according to any one of claims 1 to 3; (2) dissolving the polypeptide synthesized in step (1) in water to obtain solution A; dissolving tetrachloroauric acid in ultrapure water to obtain solution B; (3) Solution A and solution B are mixed in a molar ratio of 1:10-10:1, the pH of the mixed solution is adjusted to 5-14, and the mixture is stirred at 20-70°C for 1-48 hours to obtain the pathological collagen-targeted nanozyme.

10. Use of the diseased collagen targeted nanozyme according to any one of claims 4 to 8 in the qualitative or quantitative analysis of diseased collagen.

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