A kit for targeted detection of collagen in a solution and its application in diagnosis of liver fibrosis

Abstract

The application belongs to the technical field of biological detection, and particularly relates to a kit for targeted detection of collagen in a solution and application thereof in diagnosis of liver fibrosis, comprising a microneedle chip, capture probes fixed on the microneedle chip, and free detection probes; the microneedle chip is obtained by mixing an activated PEGDA solution with collagen capture probes and performing light curing 3D printing; the capture probe sequence is D-(Gly-Pro-Hyp)n-Ahx-Cys-X or D-(Gly-Pro-Pro)n-Ahx-Cys-X, n is an integer between 8 and 20, and X is a nanoparticle; the detection probe sequence is Y-CTP-X-R, X is a nanoparticle, R is a Raman signal molecule, and Y-CTP is a different type of collagen targeting probe; the kit is simple and convenient to operate, can simultaneously detect multiple collagen markers in a solution, has high specificity, a low detection limit, can be used for non-invasive early diagnosis of liver fibrosis, and has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological detection, and particularly relates to a kit for targeted detection of collagen in a solution and application thereof in diagnosis of liver fibrosis. BACKGROUND

[0002] Liver fibrosis is a pathological condition caused by various pathogenic factors such as hepatitis B virus, hepatitis C virus, alcoholism, metabolic syndrome, etc., leading to abnormal proliferation of connective tissue in the liver. Any liver damage will have liver fibrosis during the process of repair and healing. If the process of fibrosis continues for a long time, it will eventually lead to cirrhosis. Therefore, liver fibrosis is an important factor for predicting chronic liver damage and disease progression, and it is crucial to diagnose liver fibrosis in a timely manner. At present, the main methods for diagnosing liver fibrosis include liver biopsy and serum molecular marker detection. As a non-invasive detection method, serum molecular marker detection is low in cost, simple and convenient, and therefore attracts much attention in the field of liver fibrosis diagnosis.

[0003] Collagen is an important biomarker for serum molecular detection of liver fibrosis. Patent CN105911296A discloses a chemiluminescent immunoassay kit for type IV collagen and a preparation method thereof; patent WO2021193763A1 discloses a method for determining type IV collagen 7S domain fragment and a kit for use thereof. These patents provide a fluorescence detection method for single component type IV collagen, but liver fibrosis is the result of the joint action of multiple types of collagen. Therefore, it is still a great challenge to develop a more efficient method for simultaneously detecting different types of collagen in blood.

[0004] Photocuring 3D printing is a new type of free-form fabrication technology that uses a digital-controlled layer-by-layer deposition scheme to produce complex components and architectural materials. This technology starts with a 3D model designed by computer-aided design (CAD), which is sliced by a special program and provided to a 3D printer in a multi-layer slicing manner. Photocuring technology has the advantages of customizable model, high selection precision, fast polymerization speed, and fast printing speed. 3D printing can be applied to biological detection to achieve uniform and firm distribution of various functional components in the chip.

[0005] The inventors accidentally found a kit for targeted detection of collagen in a solution and its application in the diagnosis of liver fibrosis during the research, which comprises a 3D printed microneedle chip, a capture probe fixed on the microneedle chip and a free detection probe; the microneedle chip is obtained by mixing an activated PEGDA solution with a collagen capture probe and light curing 3D printing; the sequence of the capture probe is D-(Gly-Pro-Hyp)n-Ahx-Cys-X or D-(Gly-Pro-Pro)n-Ahx-Cys-X, n is an integer between 8 and 20, wherein X is a nanoparticle; the sequence of the detection probe is Y-CTP-X-R, X is a nanoparticle, R is a Raman signal molecule, and Y-CTP is a different type of collagen targeting probe; the kit is simple and convenient to operate, can simultaneously detect multiple collagen markers in a solution, has high specificity, low detection limit, can be used for non-invasive early diagnosis of liver fibrosis, and has a wide application prospect. SUMMARY

[0006] In view of the above technical problems, the present application provides a kit for targeted detection of collagen in a solution, which comprises a 3D printed microneedle chip, a capture probe fixed on the microneedle chip and a free detection probe; the microneedle chip is obtained by mixing an activated PEGDA solution with a collagen capture probe and light curing 3D printing; the sequence of the capture probe is D-(Gly-Pro-Hyp)n-Ahx-Cys-X or D-(Gly-Pro-Pro)n-Ahx-Cys-X, n is an integer between 8 and 20, wherein X is a nanoparticle; the sequence of the detection probe is Y-CTP-X-R, X is a nanoparticle, R is a Raman signal molecule, and Y-CTP is a different type of collagen targeting probe.

[0007] Preferably, Y is type I, type II, type III or type IV collagen.

[0008] Preferably, X is a transition metal nanoparticle and / or a semiconductor nanoparticle.

[0009] Preferably, X is an Au, Ag or Cu nanoparticle.

[0010] Preferably, the activation method of the PEGDA solution is: taking PEGDA, adding 0.1%wt photoinitiator phenyl bis(2,4,6-trimethylbenzoyl) phosphine oxide, and stirring to obtain a uniform solution.

[0011] Preferably, the light curing printing parameters are: layer height 25μm, base layer number 5, base layer exposure 2.1s, slice layer number 90, slice layer exposure 2.1s, light intensity 2mW / cm 2 .

[0012] Preferably, the Raman signal molecule is selected from any one of 4-MBA, 4-MBN, 4-EBT, S-(4-ethynylphenyl) ethanethioate, S-(4-((trimethylsilyl)ethynyl)phenyl) ethanethioate and S-(4-cyanophenyl) ethanethioate.

[0013] Preferably, the capture probe sequence is prepared by solid phase synthesis.

[0014] Preferably, the solid phase synthesis comprises the following steps:

[0015] a. 80-120 mg of resin is added to a reactor with a sieve plate, and the resin is swelled with 2-8 mL of dichloromethane;

[0016] b. The N-terminal Fmoc protecting group is removed from a 15-25% piperidine / N,N- dimethylformamide (DMF) solution, and the removal of the protecting group is detected by a color reaction;

[0017] c. 4 eq of an N-terminal Fmoc-protected amino acid, 4 eq of HOBt and 4 eq of HBTU are dissolved in DMF, and after being activated at low temperature for 10-30 min, 6 eq of DIEA is added dropwise to the solution, which is then added to the reactor and reacted for 1-6 hrs;

[0018] d. After the reaction is completed, the reaction solution is removed from the reactor, and the resin is washed with 2-8 mL of DMF and DCM for 2-4 times, respectively, and the complete condensation of the amino acid is detected by a color reaction. The resin is treated with a 15-25% piperidine / DMF solution for 3 times, with 5 min, 5 min and 15 min, respectively, and the resin is washed with 5 mL of DMF and DCM for 3 times, respectively, and the complete removal of the protecting group is detected by a color reaction;

[0019] e. Then steps c) and d) are repeated until the collagen polypeptide of the target sequence is synthesized. Then 20-30% acetic anhydride is added to the reactor, and after the complete reaction is detected by a color reaction, the resin is washed with 3-8 mL of DMF and DCM for 2-4 times, respectively;

[0020] f. The resin is washed with DCM and methanol alternately for 2-4 times, respectively, and then the resin is dried, a cleavage solution is added, the components of the cleavage solution are TFA:TIS:water with a mass ratio of 90:5:5, and the reaction is carried out for 1-6 hrs;

[0021] g. The reaction solution is added with ice ether, the polypeptide is precipitated, and then the precipitate is collected by centrifugation. The precipitate is dissolved in TFA, and an excess of ice ether is added to precipitate and collect the precipitate by centrifugation. The precipitate is washed with ice ether for 2-4 times and then dried to obtain a crude peptide. The crude peptide is purified by reverse phase liquid chromatography to obtain a pure peptide, and then the solution is freeze-dried to obtain the probe.

[0022] The second object of the present application is to provide the kit for detecting the collagen in blood / early stage of liver fibrosis.

[0023] (1) The capture probe of collagen is prepared by a solid-phase synthesis method, and the microneedle chip is prepared by light curing printing;

[0024] (2) The sample is added to the microneedle chip, and the excess sample is washed after the combination is completed;

[0025] (3) The prepared detection probe is added to the microneedle chip obtained in step (2), combined, and washed after the combination is completed;

[0026] (4) Spectral acquisition is performed on a confocal Raman spectrometer.

[0027] The third object of the present application is to provide the application of the kit in detecting the collagen in blood.

[0028] The fourth object of the present application is to provide the application of the kit in detecting the collagen in blood at the early stage of liver fibrosis.

[0029] The present application provides a kit for targeted SERS detection of collagen in solution, which comprises a 3D-printed microneedle chip, a capture probe fixed on the microneedle chip and a free detection probe; the collagen polypeptide capture probe is mixed with PEGDA for printing to obtain a regularly shaped microneedle chip; a charged amino acid is introduced into the sequence of the targeting polypeptide probe to adjust the overall charge characteristics of the polypeptide to obtain a stable single-molecule capture probe; the collagen polypeptide probe is prepared by a solid-phase synthesis method to synthesize a collagen polypeptide with a specific sequence, and the Cys is combined with a nanoparticle; SERS is used for simultaneous detection of collagen; the polypeptide detection probe is connected with SERS nanoparticles to assemble into a SERS polypeptide probe; solution collagen is captured; in-vitro serum detection is performed; liver fibrosis staging is performed; the kit preparation method is simple, the shape is variable, multiple markers can be detected simultaneously, the specificity is high, the SERS signal continuously increases with the increase of the collagen concentration, the system is a good enrichment and detection method for collagen in solution, the linear detection range is 10-400 ng / mL, and the detection limit is as low as 3.09 ng / mL; the kit can be used for diagnosing or assisting in diagnosing early liver fibrosis, has high diagnostic accuracy, and has good clinical application prospects. BRIEF DESCRIPTION OF DRAWINGS

[0030] The present application will be further described below in combination with the drawings and examples.

[0031] Figure 1 Characterization of the 3D-printed microneedle chip;

[0032] Figure 23D-printed microneedle chip to capture and detect type I / IV collagen in solution;

[0033] Figure 3 Specificity of 3D-printed microneedle chip to detect type I / IV collagen in solution;

[0034] Figure 4 Linearity of 3D-printed microneedle chip to detect type I / IV collagen in solution;

[0035] Figure 5 Linearity of 3D-printed microneedle chip to detect type I / IV collagen in blood;

[0036] Figure 6 Detection of type I and IV collagen in mouse liver fibrosis tissue and blood; DETAILED DESCRIPTION

[0037] The scope of protection of the present application is illustrated in detail below with examples, and it should be understood that the scope of protection of the present application is not limited by the following examples.

[0038] Example 1 3D-printed microneedle chip (MC) and characterization

[0039] 1. Design of collagen capture probe

[0040] The sequence of the collagen capture probe designed this time is as follows: D-(Gly-Pro-Hyp)8-Ahx-Cys-X, wherein X is Ag nanoparticle;

[0041] 2. Preparation of collagen capture probe

[0042] a. 100 mg of Rink amino resin was added to a reactor with a sieve plate, and the resin was swelled with 5 mL of dichloromethane;

[0043] b. The N-terminal Fmoc protecting group was removed from a 20% piperidine / N,N- dimethylformamide (DMF) solution, and a color reaction was used to detect complete removal of the protecting group;

[0044] c. The N-terminal Fmoc-protected amino acid (4 eq) was dissolved in DMF with HOBt (4 eq) and HBTU (4 eq), and after 20 min of low-temperature activation, DIEA (6 eq) was added dropwise to the solution, which was then added to the reactor and reacted for 3 hrs.

[0045] d. After the reaction was completed, the reaction solution was extracted from the reactor, and the resin was washed with 5 mL of DMF and DCM for 3 times, respectively. The complete condensation of amino acids was detected by color reaction, and the resin was treated with 20% piperidine / DMF solution for 3 times, 5 min, 5 min and 15 min, respectively. The resin was washed with 5 mL of DMF and DCM for 3 times, respectively, and the complete removal of the protective group was detected by color reaction;

[0046] e. Repeat steps c, d until the target sequence of collagen polypeptide D-(Gly-Pro-Hyp)8-Ahx-Cys-X is synthesized. Add 25% acetic anhydride to the reactor, and detect the complete reaction by color reaction. Wash the resin with 5 mL of DMF and DCM for 3 times, respectively.

[0047] f. The resin is washed with DCM and methanol alternately for 3 times. The resin is extracted dry, and the cleavage solution (TFA:TIS:water = 90:5:5) is added. The reaction is carried out for 3 hrs.

[0048] g. The reaction solution is added to ice ethyl ether to precipitate the polypeptide. The precipitate is collected by centrifugation, dissolved with a small amount of TFA, and precipitated again by adding excess ice ethyl ether and collecting the precipitate by centrifugation. The precipitate is washed with ice ethyl ether for 2 times and air-dried to obtain the crude peptide. The crude peptide is purified by reverse phase liquid chromatography to obtain the pure peptide. The solution is freeze-dried to obtain the probe D-(Gly-Pro-Hyp)8-Ahx-Cys(D-PCTP).

[0049] h. Take 1 mL of Ag nanoparticles, and add 200 μL of D-PCTP (0.5 mM) probe preheated at 85°C for 20 min and quenched with ice water. Stir for 1-4 hrs, and remove the unbound polypeptide by centrifugation at 5000 rpm for 5 min to obtain the capture probe Ag@PCTP-D.

[0050] 3. PEGDA activation: take a certain mass of PEGDA, add 0.1% wt of the photoinitiator phenyl bis(2,4,6-trimethylbenzoyl)phosphine oxide to it, and magnetically stir for 48 h until a uniform solution is obtained. Stir magnetically at room temperature for standby.

[0051] 4. 3D printed microneedle chip (MC): mix the activated PEGDA solution with the Ag@PCTP-D capture probe (v / v = 4:1), mix thoroughly, and avoid light for standby. Take 1 ml of the mixed and uniformly mixed printing material and place it on the 3D printing sample table (EFL). Set the corresponding parameters and start the light curing printing. The specific parameters are: layer height 25 μm, base layer number 5, base layer exposure 2.1 s, slice layer number 90, slice layer exposure 2.1 s, light intensity 2 mW / cm2.

[0052] 5. MC characterization: collect the microneedle chip image by upright fluorescence microscope. SEM element analysis and Mapping verify the presence of PEGDA and polypeptide in the chip.

[0053] As shown in Figure 1 a is the dispersion state of the capture probe Ag@PCTP-D characterized by TEM, the TEM Mapping element distribution of the capture probe, S represents that the polypeptide probe is uniformly distributed around the Ag nanoparticle, and exists in the form of a single particle. b is the DLS characterization of the capture probe Ag@PCTP-D, indicating that the particle size of the capture probe is about 70 nm, further indicating the uniformity of the capture probe in the solution. c is the regular array of the microneedle chip recorded by fluorescence microscope, and the morphology of the single microneedle is characterized by SEM, further characterized by Mapping and element analysis, C represents the components of the whole chip and the signal of PCTP-D, N represents the position of the Ag@PCTP-D capture nanoprobe, indicating that the capture probe is uniformly distributed in the microneedle chip.

[0054] Example 2 Characterization of microneedle chip capture detection of type I / IV collagen

[0055] 1. The detection probe is DDD-KLWVLPK-Ahx-Cys-X-R1 (S-IV), DD-LRELHLNNN-Ahx-Cys-X-R2 (S-I), wherein X is an Ag nanoparticle, R1 is S-(4-cyanophenyl) ethyl thioacid ester, and R2 is S-(4-ethynylphenyl) ethyl thioacid ester;

[0056] 2. Collagen detection

[0057] Both type I and type IV collagen used for MC capture collagen need to be denatured by preheating at 70°C for 10 min. First, type I collagen (Col-I) or type IV collagen (Col-IV) is added to the MC, and after the binding is completed, it is washed, and then the detection probe S-I or S-IV is added, and after the detection is completed, it is washed to characterize the SERS signals of MC+Col-I+S-I and MC+Col-IV+S-IV. At the same time, the SERS signal of MC+H2O+S-I is detected as a control, and the SERS signal of MC and the SERS signal of MC combined with collagen (MC+Col-I or MC+Col-IV).

[0058] The specific process is as follows: the collagen type I and collagen type IV solution is preheated at 70°C for 10 min to denature. The MC is incubated with (1) water, (2) 400 ng / mL collagen type I, and (3) 400 ng / mL collagen type IV in an untreated 24-well plate for 4 h, and the unbound collagen is washed away with PB (10 mM, pH 7.4) buffer solution, and the washing is performed for 3 times, each time for 3 min. 600 μL of probe S-I is added to the well plate (1) and (2), and S-IV is added to the well plate (1) and (3), and the incubation is performed at 4°C for 4 h. Then, 600 μL of PB solution is used for washing for 3 times, each time for 3 min, to remove the unbound detection probe. The 800-2600 cm -1 Spectrum collection is performed on a confocal Raman spectrometer, the excitation light is 633 nm, and the laser intensity is 0.258 mW.

[0059] As shown in Figure 2 a, from bottom to top, the SERS spectra of MC, MC+Col-I, MC+H2O+S-I, and MC+Col-I+S-I are shown, and the characteristic peak is 2102 cm -1 . Only when the to-be-detected substance is Col-I and the detection probe is S-I, the characteristic peak signal appears, and under other conditions, no characteristic peak signal appears. Figure 2 c, from top to bottom, the SERS signals of MC, MC+Col-IV, MC+H2O+S-IV, and MC+Col-IV+S-IV are shown, and the characteristic peak is 2227 cm -1 . Only when the to-be-detected substance is Col-IV and the detection probe is S-IV, the characteristic peak signal appears, and under other conditions, no characteristic peak signal appears, and the detection signal is the signal of MC, and the detection signals of the two kinds of collagen are significantly different from the signals under other comparison conditions. The results prove that the MC can capture the denatured collagen in the solution, and the two kinds of SERS detection probes can detect the collagen type IV and collagen type I, respectively. In addition, the signal intensity of the collagen type I at 2102 cm -1 is obviously greater than that of other samples -1 . Figure 2 b, d).

[0060] The specificity of this method was verified by comparing the capture and detection results of BSA, lysozyme, trypsin, pepsin, type I collagen, and type IV collagen on MC. Specifically, MC was incubated with BSA, lysozyme, trypsin, pepsin, type I collagen, and type IV collagen in untreated 24-well plates for 4 h. Unbound collagen was washed away with PB (10 mM pH 7.4) buffer solution three times, 3 min each time. 600 μL of probe SI was added to each well, and the plates were incubated at 4 °C for 4 h. Afterwards, the plates were washed three times with 600 μL PB solution for 3 min each time to remove unbound detection probes. Similarly, MC was incubated with BSA, lysozyme, trypsin, pepsin, type I collagen, and type IV collagen in untreated 24-well plates for 4 h. Unbound collagen was washed away with PB (10 mM pH 7.4) buffer solution three times, 3 min each time. 600 μL of probe S-IV was added to each well of the plate and incubated at 4 °C for 4 h. The plates were then washed three times with 600 μL of PB solution for 3 min each time to remove unbound detection probes. Confocal Raman spectroscopy was used to acquire samples from 800 to 2600 cm⁻¹. -1 Spectrum, through the characteristic peak 2102 cm⁻¹ -1 and 2227cm -1 Compare the protein capture detection results.

[0061] like Figure 3 As shown in Figure a, the proteins to be detected from top to bottom are type I collagen, BSA, pepsin, trypsin, and lysozyme. The detection probe is SI. Type I collagen showed a good detection characteristic signal at 2102 cm⁻¹. -1 The characteristic peak signal appears, while other proteins do not show characteristic peak signals. b is the characteristic signal at 2102 cm⁻¹. -1 The signal peak intensity values ​​were compared, and it can be seen that the signal of type I collagen is significantly stronger than that of other proteins, indicating that SI has a specific binding to type I collagen. Figure c shows, from top to bottom, the proteins to be detected are type IV collagen, BSA, pepsin, trypsin, and lysozyme, with SI as the detection probe. Type I collagen shows a good detection characteristic signal at 2102 cm⁻¹. -1 The characteristic peak signal appears, while other proteins do not show characteristic peak signals. d represents the characteristic signal at 2227 cm⁻¹. -1 The signal peak intensity values ​​were compared, and it can be seen that the signal of type IV collagen is significantly stronger than that of other proteins, indicating that S-IV has a specific binding to type IV collagen.

[0062] Example 3: Linearity of Detection of Type I and Type IV Collagen in Solution

[0063] Collagen type I was prepared in a gradient concentration solution of 10, 20, 50, 100, 200, 400 ng / mL. MC was incubated with heat denatured collagen type I in an untreated 24-well plate at 4°C for 4 h. 600 μL of PB solution was used to wash away the unbound protein, and the washing was repeated 3 times, each for 3 min. 600 μL of probe S-I was added to the well plate, and incubated at 4°C for 4 h. Then, 600 μL of PB solution was used to wash away the unbound SERS polypeptide probe, and the washing was repeated 3 times, each for 3 min. Collagen type IV was prepared in a gradient concentration solution of 10, 20, 50, 100, 200, 400 ng / mL. 3D-printed MC was incubated with heat denatured collagen type IV in an untreated 24-well plate at 4°C for 4 h. 600 μL of PB solution was used to wash away the unbound collagen, and the washing was repeated 3 times, each for 3 min. 600 μL of SERS polypeptide probe S-IV was added to the well plate, and incubated at 4°C for 4 h. Then, 400 μL of PB solution was used to wash away the unbound SERS polypeptide probe, and the washing was repeated 3 times, each for 3 min.

[0064] Collagen type I was prepared in a gradient concentration solution of 10, 20, 50, 100, 200, 400 ng / mL. MC was incubated with heat denatured collagen type I in an untreated 24-well plate at 4°C for 4 h. 600 μL of PB solution was used to wash away the unbound protein, and the washing was repeated 3 times, each for 3 min. 600 μL of probe S-I was added to the well plate, and incubated at 4°C for 4 h. Then, 600 μL of PB solution was used to wash away the unbound SERS polypeptide probe, and the washing was repeated 3 times, each for 3 min. Collagen type IV was prepared in a gradient concentration solution of 10, 20, 50, 100, 200, 400 ng / mL. 3D-printed MC was incubated with heat denatured collagen type IV in an untreated 24-well plate at 4°C for 4 h. 600 μL of PB solution was used to wash away the unbound collagen, and the washing was repeated 3 times, each for 3 min. 600 μL of SERS polypeptide probe S-IV was added to the well plate, and incubated at 4°C for 4 h. Then, 400 μL of PB solution was used to wash away the unbound SERS polypeptide probe, and the washing was repeated 3 times, each for 3 min.

[0065] As shown in Figure 4 a, the SERS signal of the sample system of different concentrations of collagen type I (10, 20, 50, 100, 200, 400 ng / mL) was measured, and the characteristic peak was 2102 cm -1 . The SERS signal was continuously enhanced with the increase of the concentration of collagen type I. b is the linear regression curve (R -1 = 0.97) of the signal intensity at 2102 cm 2 measured by different concentrations of collagen type I, showing that the linear range of this method is 10-400 ng / mL. c is the SERS signal of the sample system of different concentrations of collagen type IV (10, 20, 50, 100, 200, 400 ng / mL), and the characteristic peak is 2227 cm -1 . The SERS signal was continuously enhanced with the increase of the concentration of collagen type IV. d is the linear regression curve (R -1 = 0.97) of the signal intensity at 2227 cm 2 measured by different concentrations of collagen type IV, and the detection range of collagen type IV is 10-400 ng / mL. These results show that this system is a good enrichment and detection method for collagen type I and IV in solution.

[0066] As shown in Figure 5The normal mouse blood samples were added with different concentrations of collagen type I and collagen type IV to obtain the linear relationship curve for detecting collagen type I and collagen type IV in serum samples. a is the SERS spectrum of normal mouse serum added with different concentrations of collagen type I (10, 20, 50, 100, 200, 400 ng / mL). b is the linear regression curve (R = 0.96) of the signal intensity at 2102 cm-1 measured by different concentrations of collagen type I, which shows that the linear range of the method is 10-400 ng / mL, and the detection limit is as low as 4.36 ng / mL. c is the SERS spectrum of normal mouse serum added with different concentrations of collagen type IV (10, 20, 50, 100, 200, 400 ng / mL). d is the linear regression curve (R = 0.97) of the signal intensity at 2227 cm-1 measured by different concentrations of collagen type IV, which shows that the linear range of the method is 10-400 ng / mL, and the detection limit is as low as 3.09 ng / mL. -1 2 -1 2

[0067] Example 4 Capture and detection of collagen in liver tissue extract and blood of liver fibrosis mice

[0068] 1. Construction of liver fibrosis model

[0069] Take 4 mL of carbon tetrachloride and 2 mL of olive oil for injection in a round-bottom flask and magnetically stir for 10 minutes to fully mix the mixture. Draw 1 mL with a syringe for standby. Inject the CCl4 mixture through the abdominal cavity to construct a liver fibrosis mouse model, twice a week, and continuously inject for 2, 4, 6, and 8 weeks to obtain S1-S4 liver fibrosis mice.

[0070] Randomly divide 80 KM mice into a model group of 70 mice and a control group of 10 mice. The model group is injected with the mixture at 70 μL / 100 g of body weight for 8 weeks; the experimental group of 10 mice is injected with olive oil at 70 μL / 100 g of body weight for 8 weeks. After each week of injection, randomly select two mice and sacrifice them on the same day, remove the liver tissue, paraffin-embed, and make pathological sections for HE staining. After determining the stages of the remaining mice, randomly take 10 mice for eye blood sampling, sacrifice them, and take liver tissue for standby until the sampling of the S0-S4 model mice is completed.

[0071] 2. Extraction of collagen from liver tissue

[0072] ​​​​Liver tissue was minced and soaked overnight in a 20% NaCl aqueous solution. Collagen was extracted from the liver tissue using a pepsin-acetic acid solution over 3 days. NaCl granules were added with stirring to induce salting out, and the precipitate was allowed to set overnight. The precipitate was obtained by centrifugation at 3000 rpm for 10 min and then dialyzed to remove salt. The collagen was reconstituted with acetic acid solution. The resulting liver fibrosis tissue extracts (stages 1-4) were characterized by SDS-PAGE and the hydroxyproline concentration was determined. The diluted collagen solution was stored at 4°C for later use.

[0073] 3. Detection of collagen in tissue extracts

[0074] The collagen capture chip and diluted tissue extract were incubated in untreated 24-well plates at 4°C for 4 h. Unbound proteins were washed three times with 600 μL LPB (10 mM pH 7.4) buffer for 3 min each time. A mixed solution of SI and S-IV probes was added to the wells, and the plates were incubated at 4°C for 4 h for specific detection of the captured proteins. The plates were then washed three times with 400 μL PB buffer for 3 min each time to remove unbound detection probes. The samples were then subjected to confocal Raman spectroscopy at 800–2600 cm⁻¹. -1 Spectral acquisition, with characteristic peaks at 2227 and 2102 cm⁻¹ -1 The intensity is determined by the concentration of type IV and type I collagen in the tissue extract.

[0075] 4. Serum collagen detection

[0076] Whole blood was collected from the eyeballs of mice with liver fibrosis stages S0-S4, and heparin was added as an anticoagulant. The serum was obtained by centrifugation at 3000 rpm for 10 min. Serum solutions from stages S1, S2, S3, and S4 were incubated with a microneedle chip for 4 h, and excess serum was washed away with PB. Serum from different stages was then incubated with the microneedle chip in an untreated 24-well plate at 4°C for 4 h. Unbound proteins were washed away with 600 μL PB solution three times, 3 min each time. 600 μL of SI and S-IV probes were mixed and added to the wells, and incubated at 4°C for 4 h. The plates were then washed three times with 600 μL PB buffer for 3 min each time to remove unbound detection probes. Data were collected at 800-2600 cm⁻¹ using a confocal Raman spectrometer. -1 The spectral signal consists of characteristic peaks at 2102 and 2227 cm⁻¹. -1 The intensity was used to obtain the concentrations of type I and type IV collagen in the serum.

[0077] like Figure 6 Figure a shows the capture and detection of collagen in mouse liver tissue extracts. From bottom to top, the results represent the detection results for liver fibrosis stages S1, S2, S3, and S4. As the degree of liver fibrosis increases, [the collagen content increases by 2102 cm]. -1 Representative type I collagen and 2227cm -1The concentrations of type I and type IV collagens in the liver tissue extracts of mice at different stages S1-S4 were calculated. The concentrations of type I collagen in the liver tissue extracts of mice at different stages S1-S4 were 18.0 ng / mL, 46.4 ng / mL, 254.5 ng / mL and 406.7 ng / mL, respectively, after dilution by 1000 times. The concentrations of type IV collagen in the liver tissue extracts of mice at different stages S1-S4 were 17.2 ng / mL, 34.5 ng / mL, 95.3 ng / mL and 249.7 ng / mL, respectively, after dilution by 1000 times. The proportions of type I collagen in the liver tissue were 0.323 mg / g, 0.856 mg / g, 5.15 mg / g and 7.929 mg / g, respectively. The proportions of type IV collagen in the liver tissue were 0.309 mg / g, 0.636 mg / g, 1.929 mg / g and 4.87 mg / g, respectively. The capture and detection of type I and type IV collagens in the serum of mice at different stages S1-S4 showed that the SERS signal of type I collagen was very low, indicating that there was almost no type I collagen in the serum. The SERS signal intensity of type IV collagen corresponded to a concentration of 5.44 ng / mL for S1, 10.97 ng / mL for S2, 29.24 ng / mL for S3 and 36.65 ng / mL for S4. The concentrations of type I and type IV collagens in the serum of mice at different stages of liver fibrosis are shown in the line graph.

[0078] The application provides a kit for targeted SERS detection of collagen in a solution, which comprises a 3D-printed microneedle chip, a capture probe fixed on the microneedle chip and a free detection probe. The collagen polypeptide capture probe is mixed with PEGDA for printing to obtain a regularly shaped microneedle chip. Charged amino acids are introduced into the sequence of the targeted polypeptide probe to adjust the overall charge characteristics of the polypeptide to obtain a stable monomolecular capture probe. The collagen polypeptide probe is prepared by synthesizing a collagen polypeptide with a specific sequence by a solid-phase synthesis method and combining Cys with a nanoparticle. The SERS is used for simultaneous detection of collagen. The polypeptide detection probe is connected with SERS nanoparticles to assemble into a SERS polypeptide probe. Solution collagen is captured. Serum is detected in vitro. Liver fibrosis is staged. The kit has the advantages of simple preparation method, variable shape, simultaneous detection of multiple markers, high specificity, continuously enhanced SERS signal with the increase of collagen concentration, and the like. The linear detection range is 10-400 ng / mL, and the detection limit is as low as 3.09 ng / mL. The kit can be used for diagnosis or auxiliary diagnosis of early liver fibrosis and has high diagnostic accuracy and good clinical application prospect.

[0079] Finally, it is to be explained that the above embodiments are only used to illustrate the technical solutions of the present application but not to limit the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or equivalently replaced without departing from the purpose and scope of the present application, and all of them should be covered in the scope of the claims of the present application.

Claims

1. A kit for the targeted detection of collagen in a solution, characterized in that, The kit comprises a 3D-printed microneedle chip, a capture probe fixed on the microneedle chip and a free detection probe; the microneedle chip is obtained by mixing an activated PEGDA solution with the capture probe and performing light curing printing; the capture probe sequence is D-(Gly-Pro-Hyp)n-Ahx-Cys-X or D-(Gly-Pro-Pro)n-Ahx-Cys-X, n is an integer between 8 and 20, wherein X is a nanoparticle; the detection probe sequence is Y-CTP-X-R, X is a nanoparticle, R is a Raman signal molecule, Y-CTP is a targeting probe of different types of collagen, and D is aspartic acid.

2. The kit of claim 1, wherein X is a transition metal nanoparticle and / or a semiconductor nanoparticle.

3. The kit of claim 2, wherein X is an Au, Ag or Cu nanoparticle.

4. The kit of claim 1, wherein The activation method of the PEGDA solution is as follows: taking PEGDA, adding 0.1%wt of a photoinitiator phenyl bis(2,4,6-trimethylbenzoyl)phosphine oxide, and stirring to obtain a uniform solution.

5. The kit of claim 1, wherein The light curing printing parameters are: layer height 25 μm, base layer number 5, base layer exposure 2.1 s, slice layer number 90, slice layer exposure 2.1 s, light intensity 2 mW / cm 2 .

6. The kit of claim 1, wherein The Raman signal molecule is selected from any one of 4-MBA, 4-MBN, 4-EBT, S-(4-ethynylphenyl) ethanethioate, S-(4-((trimethylsilyl)ethynyl)phenyl) ethanethioate and S-(4-cyanophenyl) ethanethioate.

7. The kit of claim 1, wherein Y is collagen type I, II, III or IV.

8. The kit of claim 1, wherein The capture probe sequence is prepared by a solid-phase synthesis method.

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