Biomimetic NANO immunosubstrate material, preparation method therefor and use thereof

Abstract

A biomimetic nano immunosubstrate material, a preparation method therefor and a use thereof. The biomimetic nano immunosubstrate material comprises a PDMS@AgNPs@ZIF-67 biomimetic immunosubstrate and a gold nanorod immunoprobe. The preparation method comprises the following steps: (S10) preparing a ZIF-67 nanomaterial; (S20) preparing a PDMS@AgNPs@ZIF-67 biomimetic polymeric material; (S30) preparing a PDMS@AgNPs@ZIF-67 biomimetic immunosubstrate material; and (S40) synthesizing a gold nanorod immunoprobe. A SERS substrate having both a synergistic enhancement effect and a molecular anchoring capability is constructed on a MOFs-modified biomimetic substrate, thereby improving the efficiency and sensitivity of prostate cancer tests.

Description

Bionic nano-immune base material and preparation method and application thereof TECHNICAL FIELD

[0001] The present application relates to the field of material engineering and nanotechnology, in particular to a bionic nano-immune base material and preparation method and application thereof. BACKGROUND

[0002] Prostate cancer is the most common malignant tumor of the male urogenital system, and its incidence is showing a trend of increasing year by year. As an important screening indicator of prostate cancer, the content of prostate specific antigen (PSA) in serum becomes a key method for measuring the course of prostate cancer. However, it is difficult for traditional detection techniques to achieve efficient and accurate detection of trace PSA in serum. As a new type of spectral detection technology, surface-enhanced Raman scattering (SERS) exhibits unique advantages in detection sensitivity, repeatability of results and convenience of operation. In particular, based on electromagnetic enhancement and chemical enhancement mechanism, the intensity of SERS spectrum can be greatly improved, which is very suitable for the detection of trace target molecules in biological samples. Generally, the detection sensitivity of trace molecules depends on the density and intensity of electromagnetic "hot spots". At present, the mainstream SERS active material is noble metal nanomaterial. Its surface topography and structure are crucial to the formation of electromagnetic "hot spots". Compared with traditional physical and chemical synthesis methods, bionic preparation technology is more effective in creating periodic and uniform surface nanostructures with a large number of SERS active regions. In the past decade, by copying the surface structure of plant leaves and insect wings onto flexible polymer materials, people have manufactured many SERS substrates with various bionic structures. Among existing polymer materials, the advantage of polydimethylsiloxane (PDMS) is that the biological structure imprinted on it can often maintain good integrity without large-scale defects. On the other hand, metal-organic framework (MOFs) materials with high specific surface area can better anchor target molecules and have been used to develop multifunctional SERS substrates. In particular, MOFs can enhance the Raman signal by promoting charge transfer (CT) within organic ligands and target molecules. In addition, MOFs exhibit good chemical stability, which can prevent noble metal oxidation and prolong the service life of SERS substrates. Therefore, it is of great significance to improve the detection efficiency of PSA by studying the construction of SERS substrates with both synergistic enhancement and anchoring molecule capabilities on MOFs modified bionic substrates.

[0003] SUMMARY

[0004] An advantage of the present application is to provide a bionic nano-immune base material and preparation method and application thereof, which can improve the detection sensitivity and detection efficiency and realize early screening of cancer.

[0005] Another advantage of the present application is to provide a kind of bionic nano immunological substrate material and its preparation method and application, the bionic nano immunological substrate material has the complete surface nano structure of period repeatable, it is favorable to output repeatable SERS signal, and PDMS substrate has intrinsic Raman signal, can be as the internal reference signal of to-be-measured signal, effectively improve the accuracy of immunodetection result.

[0006] Another advantage of the present application is to provide a kind of bionic nano immunological substrate material and its preparation method and application, MOFs material is modified on the surface of bionic immunological substrate, by promoting charge transfer (CT) in organic ligand and target molecule to enhance Raman signal, it is favorable to increase the adsorption efficiency of to-be-measured molecule, significantly improve the collection efficiency of PSA in blood, and then improve detection sensitivity, realize the early screening of prostate cancer, it is favorable to measure the course of prostate cancer, to formulate the treatment plan of being conducive to the health of patient early.

[0007] Another advantage of the present application is to provide a kind of bionic nano immunological substrate material and its preparation method and application, MOFs shows good chemical stability, can prevent noble metal oxidation, prolong the service life of SERS substrate, constructs SERS substrate with both synergistic enhancement and anchoring molecule ability on the bionic substrate modified by MOFs, it is important to improve the detection efficiency of PSA.

[0008] Another advantage of the present application is to provide a kind of bionic nano immunological substrate material and its preparation method and application, preparation method is simple, convenient to use, suitable for clinical popularization and application.

[0009] According to an aspect of the present application, the present application provides a kind of preparation method of bionic nano immunological substrate material, comprising the following steps:

[0010] (S10) preparation of ZIF-67 nano material;

[0011] (S20) preparation of PDMS@AgNPs@ZIF-67 bionic polymer material;

[0012] (S30) preparation of PDMS@AgNPs@ZIF-67 bionic immunological substrate material;And

[0013] (S40) synthesis of gold nanorod immunological probe.

[0014] The step (S20) comprises the following steps: (S201) pasting the canna leaf in a culture dish; (S202) mixing the PDMS gel with a curing agent, removing the air bubbles by water bath ultrasonic; (S203) pouring the mixture on the canna leaf in the culture dish, heating to obtain the cured PDMS film, and peeling the PDMS film from the surface of the canna leaf; (S204) depositing silver nanoparticles on the biomimetic PDMS film by magnetron sputtering to obtain the PDMS@AgNPs substrate; (S205) dissolving the ZIF-67 powder obtained in the step (S10) in anhydrous ethanol to obtain a nanomaterial solution of ZIF-67; and (S206) dropping the ZIF-67 ethanol solution on the surface of the PDMS@AgNPs substrate and drying to obtain the PDMS@AgNPs@ZIF-67 biomimetic polymer material.

[0015] In the step (S202), the mass ratio of the PDMS gel to the curing agent is 10:1, in the step (S203), the thickness of the cured PDMS film is 1mm, and in the step (S204), the power of the magnetron sputtering is 40W, and the silver nanoparticles are deposited on the biomimetic PDMS film by magnetron sputtering for 30-60s.

[0016] In the step (S201), the canna leaf is cut into small pieces of 4cm×4cm, and in the step (S203), after the PDMS film is peeled from the surface of the canna leaf, the film is cut into samples of 5mm×5mm for standby.

[0017] The step (S30) comprises the following steps: (S301) coating the PBS solution containing the antibody on the PDMS@AgNPs@ZIF-67 biomimetic polymer material, incubating, and immobilizing the capture antibody; (S302) sequentially washing the substrate with TBS, the PBS solution and deionized water to remove the unreacted antibody, and then adding the PBS buffer solution containing the bovine serum albumin dropwise to react at room temperature; and (S303) sequentially washing the free BSA with TBS, the PBS solution and deionized water to obtain the PDMS@AgNPs@ZIF-67 biomimetic immunological substrate material, which is stored at 4℃ for standby.

[0018] In the step (S301), the PBS solution containing the 0.2mg / mL PSA antibody is coated on the PDMS@AgNPs@ZIF-67 biomimetic material, and the incubation is carried out at 0℃ overnight to immobilize the capture antibody. In the step (S302), the PBS buffer solution containing the bovine serum albumin is added dropwise to react at room temperature for 1h to block the non-specific binding sites.

[0019] The step (S40) comprises the following steps: (S401) adding frozen NaBH4 into the mixed aqueous solution of CTAB and HAuCl4, stirring to prepare Au seeds, and incubating at 25°C; (S402) mixing CTAB solution with AgNO3 to prepare Au NRs growth solution; (S403) adding ascorbic acid, HAuCl4 and incubated seed solution, stirring, and standing overnight to obtain gold nanorod solution; (S404) centrifuging the synthesized Au NRs aqueous solution to remove excess CTAB, and adding MB solution into the Au centrifugate, and centrifuging to remove excess MB; (S405) dissolving the MB modified Au NRs in PBS solution, then adding antibody, incubating at 4°C, centrifuging to remove unbound antibody; (S406) adding PBS buffer solution containing bovine serum albumin, incubating at room temperature, centrifuging to remove excess bovine serum albumin, obtaining Au NRs immunoprobes, dissolving in PBS aqueous solution, and storing at 4°C.

[0020] In the step (S401), 0.4-0.8mL of frozen NaBH4 (0.01M) is quickly added into 20mL of mixed aqueous solution of CTAB (0.05M) and HAuCl4 (0.25M) to prepare Au seeds under vigorous stirring. The obtained seed solution is incubated at 25°C for 2-4h; in the step (S403), 0.1M of 0.35-0.55mL ascorbic acid, 10 -2 mL of HAuCl4 and 160μL of incubated seed solution are sequentially added, gently stirred, and left to stand overnight to obtain gold nanorod solution; in the step (S405), 0.2mg / m of 10-30μL PSA antibody is added into the MB modified Au NRs solution, and incubated at 4°C for 2-4h.

[0021] The step (S10) comprises the following steps: (S101) adding cobalt nitrate hexahydrate and 2-methylimidazole into mixed solutions of methanol and ethanol respectively; (S102) mixing and stirring the above two solutions, and standing for reaction; (S103) washing with ethanol, drying, and obtaining ZIF-67 powder.

[0022] In the step (S101), 43.65-174.6mg of cobalt nitrate hexahydrate and 49.25-197mg of 2-methylimidazole are added into 4-8mL of mixed solution of methanol and ethanol in equal proportions respectively, the above two solutions are mixed and stirred for 20-40min, and left to stand overnight. In the step (S203), washing with ethanol for 2-4times, drying at 80°C for 2-6h, and obtaining ZIF-67 powder.

[0023] According to another aspect of the present application, the present application further provides a kind of bionic nano immune substrate material, including PDMS@AgNPs@ZIF-67 bionic immune substrate and gold nanorod immune probe, wherein the PDMS@AgNPs@ZIF-67 bionic immune substrate is prepared by PDMS@AgNPs@ZIF-67 bionic polymer material.

[0024] According to another aspect of the present application, the present application further provides a kind of bionic nano immune substrate material, including PDMS@AgNPs@ZIF-67 bionic immune substrate and gold nanorod immune probe, wherein the PDMS@AgNPs@ZIF-67 bionic immune substrate is prepared by PDMS@AgNPs@ZIF-67 bionic polymer material.

[0025] Wherein in the application detection process, buffer solution containing different concentrations of to-be-measured cancer marker antigen is added to the PDMS@AgNPs@ZIF-67 bionic immune substrate material, incubated at 37 DEG C for 2-4h, and then washed with TBS, PBS solution and deionized water in sequence to remove excess unreacted to-be-measured antigen, then the gold nanorod immune probe solution is added to the PDMS@AgNPs@ZIF-67 bionic immune substrate adsorbed with to-be-measured antigen, and incubated at 37 DEG C for 2-4h, and then washed to remove excess unreacted gold nanorod immune probe, and then the complex of gold nanorod immune probe and PDMS@AgNPs@ZIF-67 bionic immune substrate obtained after the above immune reaction is measured by Raman spectrometer, and the concentration of to-be-measured antigen is calculated according to the linear relationship between antigen concentration and Raman characteristic peak intensity. BRIEF DESCRIPTION OF DRAWINGS

[0026] Fig. 1 is a scanning electron microscope photograph of the PDMS@AgNPs@ZIF-67 bionic substrate prepared in Example 1 of the present application.

[0027] Fig. 2 is a scanning electron microscope photograph of the gold nanorod prepared in Example 1 of the present application.

[0028] Fig. 3 is a Raman spectrum obtained by Raman detection of the substrate after the PDMS@AgNPs@ZIF-67 bionic immune substrate and gold nanorod immune probe prepared in Example 1 of the present application are subjected to immune reaction with different concentrations of to-be-measured antigen.

[0029] Fig. 4 is a graph showing the change of characteristic peak intensity at frequency shift of 1264cm-1 in the Raman spectrum obtained by immune detection of prostate specific antigen PSA by the PDMS@AgNPs@ZIF-67 bionic immune substrate and gold nanorod immune probe prepared in Example 1 of the present application, and a result graph of internal standard correction. -1

[0030] ​Figure 5 is a scanning electron microscope photo of the PDMS@AgNPs@ZIF-67 biomimetic substrate prepared in Example 2 of the present application.

[0031] Figure 6 is a Raman spectrum obtained by Raman detection of the PDMS@AgNPs@ZIF-67 biomimetic immunosubstrate prepared in Example 2 of the present application and the gold nanorod immunoprobes after immunoreaction with different concentrations of the antigen to be detected.

[0032] Figure 7 is a graph showing the change of the characteristic peak intensity at a frequency shift of 1264 cm-1 in the Raman spectrum of the immunodetection of prostate specific antigen PSA by the PDMS@AgNPs@ZIF-67 biomimetic immunosubstrate prepared in Example 2 of the present application and the gold nanorod immunoprobes with the concentration of the antigen to be detected and a graph showing the results of internal standard correction. -1

[0033] Figure 8 is a scanning electron microscope photo of the PDMS@AgNPs@ZIF-67 biomimetic substrate prepared in Example 3 of the present application.

[0034] Figure 9 is a Raman spectrum obtained by Raman detection of the PDMS@AgNPs@ZIF-67 biomimetic immunosubstrate prepared in Example 3 of the present application and the gold nanorod immunoprobes after immunoreaction with different concentrations of the antigen to be detected.

[0035] Figure 10 is a graph showing the change of the characteristic peak intensity at a frequency shift of 1264 cm-1 in the Raman spectrum of the immunodetection of prostate specific antigen PSA by the PDMS@AgNPs@ZIF-67 biomimetic immunosubstrate prepared in Example 3 of the present application and the gold nanorod immunoprobes with the concentration of the antigen to be detected and a graph showing the results of internal standard correction. -1 DETAILED DESCRIPTION

[0036] The following description is presented to enable any person skilled in the art to practice the present application as claimed. The preferred embodiments disclosed herein are only examples of the present application and alternative embodiments will be apparent to those skilled in the art. The patentable scope of the present application is defined by the appended claims and can include other embodiments that offer optional features separately or in combination with the preferred embodiments disclosed herein.

[0037] To improve the detection efficiency of PSA and better measure the course of prostate cancer, the present application provides a biomimetic nanoimmunosubstrate material and a preparation method thereof for application in the detection of prostate cancer, to improve the detection sensitivity of prostate cancer and realize early screening of prostate cancer.

[0038] The preparation method of the biomimetic nanoimmunosubstrate material comprises the following steps:

[0039] ​​(S10) Preparation of ZIF-67 nanomaterials;

[0040] (S20) Preparation of PDMS@AgNPs@ZIF-67 biomimetic polymer materials;

[0041] (S30) Preparation of PDMS@AgNPs@ZIF-67 biomimetic immunological base materials; and

[0042] (S40) Synthesis of gold nanorod immunoprobes.

[0043] The step (S20) comprises the following steps: (S201) cutting the canna leaf into small pieces and pasting in a culture dish; (S202) stirring the PDMS gel and curing agent mixture in a test tube, removing air bubbles by water bath ultrasonic; (S203) pouring the mixture on the canna leaf in the culture dish, heating to obtain a cured PDMS film, and peeling the PDMS film from the surface of the canna leaf piece; (S204) depositing silver nanoparticles on the biomimetic PDMS film by magnetron sputtering to obtain a PDMS@AgNPs substrate; (S205) dissolving the ZIF-67 powder obtained in step (S10) in anhydrous ethanol to obtain a nanomaterial solution of ZIF-67; (S206) dropping the ZIF-67 ethanol solution onto the surface of the PDMS@AgNPs substrate, drying to obtain a PDMS@AgNPs@ZIF-67 biomimetic polymer material.

[0044] The step (S30) comprises the following steps: (S301) coating the PDMS@AgNPs@ZIF-67 biomimetic polymer material with a PBS solution containing an antibody, incubating, and fixing the immobilized capture antibody; (S302) washing the substrate with TBS, a PBS solution and deionized water in sequence to remove unreacted antibodies, and then adding a PBS buffer solution containing bovine serum albumin and reacting at room temperature.

[0045] The step (S40) comprises the following steps: (S401) adding frozen NaBH4 into a mixed aqueous solution of CTAB and HAuCl4, stirring to prepare Au seeds, and incubating at 25°C; (S402) mixing CTAB solution with AgNO3 to prepare Au NRs growth solution; (S403) adding ascorbic acid, HAuCl4 and incubated seed solution, stirring, and standing overnight to obtain gold nanorod solution; (S404) centrifuging the synthesized Au NRs aqueous solution to remove excess CTAB, and adding MB solution into the Au centrifugate, and centrifuging to remove excess MB; (S405) dissolving the MB-modified Au NRs in PBS solution, then adding antibody, incubating at 4°C, and centrifuging to remove unbound antibody; (S406) adding PBS buffer solution containing bovine serum albumin, incubating at room temperature, centrifuging to remove excess bovine serum albumin, obtaining Au NRs immunoprobes, dissolving in PBS aqueous solution, and storing at 4°C.

[0046] In the application process, a detection step (S50) is further included: dropping buffer solutions containing different concentrations of cancer marker antigens to be detected onto the PDMS@AgNPs@ZIF-67 biomimetic immunosubstrate, incubating at 37°C for 2-4h, and then sequentially washing with TBS, PBS solution and deionized water to remove excess unreacted antigens to be detected, dropping the gold nanorod immunoprobes solution onto the PDMS@AgNPs@ZIF-67 biomimetic immunosubstrate adsorbed with the antigens to be detected, and incubating at 37°C for 2-4h, and then washing to remove excess unreacted gold nanorod immunoprobes, and then using a Raman spectrometer to measure the spectrum of the complex of the gold nanorod immunoprobes and the PDMS@AgNPs@ZIF-67 biomimetic immunosubstrate obtained after the immunoreaction, and then calculating the concentration of the antigen to be detected according to the linear relationship between the antigen concentration and the Raman characteristic peak intensity.

[0047] The raw materials used are all commercially available. The Raman spectrum detector BWS415 used in the examples is purchased from B&W Tek Inc. in the United States. The antigen used in the following examples is prostate specific antigen PSA, but is not limited to prostate specific antigen PSA, and can also be carcinoembryonic antigen CEA, alpha-fetal protein antigen AFP, ferritin antigen and carbohydrate antigen CA199, etc.

[0048] Example 1

[0049] A preparation method of a biomimetic nano immunosubstrate material, comprising the following steps:

[0050] (1) Preparation of ZIF-67 nanomaterial

[0051] First, 43.65 mg of cobalt nitrate hexahydrate and 49.25 mg of 2-methylimidazole were added to a 2:1 mixture of methanol and ethanol, respectively. Then, one solution was slowly added to the other solution under magnetic stirring using a rubber bulb pipette. After the solution was added, the mixture was stirred for 30 min and left to stand overnight. After the reaction time was sufficient, the mixture was centrifuged twice at 8000 r / min using ethanol as the solvent. Then, the solid was dried at 80°C for 2 h to obtain ZIF-67 powder, which was stored at room temperature.

[0052] (2) Preparation of PDMS@AgNPs@ZIF-67 biomimetic polymer material

[0053] The canna leaves collected from the river were cut into small pieces of 4 cm x 4 cm and attached to the culture dish using ultra-thin transparent double-sided tape. Then, the PDMS gel and the curing agent were mixed in a test tube at a mass ratio of 10:1 and stirred manually for 2 min. Then, the mixture was subjected to ultrasonic treatment in a water bath for 3 min to remove the air bubbles. The mixture was poured onto the canna leaves in the culture dish and heated at 80°C for 4 h to obtain a cured PDMS film (about 1 mm thick). Then, the PDMS film was carefully peeled off from the surface of the canna leaf pieces and cut into 5 mm x 5 mm samples for subsequent use. Then, silver nanoparticles were deposited on the biomimetic PDMS film by magnetron sputtering (40 W) for 30 s. Finally, the ZIF-67 powder obtained in step (1) was dissolved in anhydrous ethanol and ultrasonically treated for 2 min to obtain a nanomaterial solution of ZIF-67 with a concentration of 0.075 mg / mL. Then, 10 μL of the synthesized ZIF-67 ethanol solution was dropped onto the surface of the PDMS@AgNPs substrate, and after drying at room temperature, the PDMS@AgNPs@ZIF-67 biomimetic polymer material was obtained.

[0054] (3) Preparation of PDMS@AgNPs@ZIF-67 biomimetic immunosubstrate material

[0055] The PDMS@AgNPs@ZIF-67 biomimetic material was coated with 20 μL of a PBS solution containing 0.2 mg / mL of PSA antibody, and incubated at 0°C overnight to immobilize the capture antibody. Then, the substrate was washed with TBS, PBS solution and deionized water in sequence to remove unreacted antibodies. Then, 10 μL of a PBS buffer solution containing bovine serum albumin was added and reacted at room temperature for 1 h to block the non-specific binding sites. Then, the free BSA was washed with TBS, PBS solution and deionized water in sequence. Finally, the immunosubstrate was stored at 4°C for subsequent detection.

[0056] (4) Synthesis of gold nanorod immunoprobes

[0057] Firstly, 0.4 mL of frozen NaBH4(0.01 M) was quickly added to 20 mL of mixed aqueous solution of CTAB(0.05 M) and HAuCl4(0.25 M) to prepare Au seeds under vigorous stirring. The obtained seed solution was incubated at 25 °C for 2 h. Subsequently, Au NRs growth solution was prepared by mixing CTAB solution(85 mL, 0.1 M) with AgNO3(0.5 L, 10 -2 M). After thorough mixing, ascorbic acid(0.35, 0.1 M), HAuCl4(3 mL, 10 -2 M) and incubated seed solution(160 μL) were added in sequence. The solution was gently stirred and left overnight to obtain gold nanorod solution.

[0058] Subsequently, gold nanorod immunoprobes were prepared. Firstly, 3 mL of synthesized Au NRs aqueous solution was centrifuged to remove excess CTAB. Subsequently, 10 μL of MB solution(10 -5 M) was added to the Au centrifugate. After removing excess MB by centrifugation, the MB-modified Au NRs were dissolved in 1 mL of PBS solution. Then 10 μL of PSA antibody(0.2 mg / mL) was added to the MB-modified Au NRs solution and incubated at 4 °C for 2 h. The unbound PSA antibody was removed by centrifugation, and 10 μL of PBS buffer containing bovine serum albumin was added. After incubation at room temperature for 1 h, the excess bovine serum albumin was removed by centrifugation. The prepared Au NRs immunoprobes were dissolved in 1 mL of PBS aqueous solution and stored at 4 °C.

[0059] Based on the above-mentioned application of PDMS@AgNPs@ZIF-67 biomimetic immunosubstrate material and Au NRs immunoprobes for repeated immunodetection of cancer markers, the following steps are included:

[0060] (1) Immunodetection of cancer markers

[0061] After 20 μL of buffer solution containing different concentrations of cancer marker antigens to be tested was added to the prepared PDMS@AgNPs@ZIF-67 biomimetic immunosubstrate material, it was then incubated at 37 °C for 2 h. After washing with TBS, PBS solution and deionized water to remove excess unreacted antigens to be tested, 20 μL of gold nanorod immunoprobes solution was added to the PDMS@AgNPs@ZIF-67 biomimetic immunosubstrate material adsorbed with the antigens to be tested, and incubated at 37 °C for 2 h. After washing to remove excess unreacted gold nanorod immunoprobes, the complex of gold nanorod immunoprobes and PDMS@AgNPs@ZIF-67 biomimetic immunosubstrate obtained after the above immunoreaction was measured by Raman spectrometer. According to the linear relationship between antigen concentration and Raman characteristic peak intensity, the concentration of the antigen to be tested was calculated.

[0062] Figure 1 shows the scanning electron microscope photos of the PDMS@AgNPs@ZIF-67 biomimetic substrate prepared in this embodiment. As can be seen from Figure 1, the surface of the PDMS film with clear cell biomimetic periodic micro-nano structure is covered with a thin layer of Ag film, on which ZIF-67 nanoparticles are uniformly distributed.

[0063] Figure 2 shows the scanning electron microscope photos of the gold nanorods prepared in this embodiment. As can be seen from Figure 2, the size of the prepared gold nanorods is relatively uniform, and the aspect ratio is 2-3.

[0064] Figure 3 is the Raman spectrum obtained by Raman detection of the substrate after the immunoreaction of the PDMS@AgNPs@ZIF-67 biomimetic immune substrate and gold nanorod immune probes prepared in this embodiment with different concentrations of the antigen to be tested (concentration from 1 microgram per milliliter to 1 picogram per milliliter). As can be seen from Figure 3, as the concentration of the antigen to be tested decreases, the Raman characteristic spectrum intensity of the labeled molecules gradually decreases, and when the concentration of the antigen to be tested decreases to 1 picogram per milliliter, the Raman characteristic peak of the labeled molecules is still very obvious relative to the background signal.

[0065] Figure 4 is a graph showing the change of the characteristic peak intensity at a frequency shift of 1621 cm-1 in the Raman spectrum of the immunodetection of prostate specific antigen PSA by the PDMS@AgNPs@ZIF-67 biomimetic immune substrate and gold nanorod immune probes prepared in this embodiment and the result of internal standard correction. As can be seen by fitting, when the concentration of the antigen to be tested changes from 1 microgram per milliliter to 1 picogram per milliliter, the Raman characteristic peak intensity changes linearly with the concentration. The fitting result shows that this change trend conforms to the linear equation Y = 24041.8 + 2503.5X, and the fitting degree is 0.981. After correction using the intrinsic Raman peak of PDMS at 2905 cm-1 as the internal standard, the change trend conforms to the linear equation Y = 22.7 + 2.3X, and the fitting degree is improved to 0.998, and the detection limit is 210 femtogram per milliliter. -1 -1

[0066] Example 2

[0067] A preparation method of a biomimetic nano-immune substrate material, comprising the following steps:

[0068] (1) Preparation of ZIF-67 nano-material

[0069] ​​First, 87.3 mg of cobalt nitrate hexahydrate and 98.5 mg of 2-methylimidazole were added to an equal proportion of a mixed solution of methanol and ethanol, respectively. Then, the above solution was slowly added to the other solution with a rubber dropper under magnetic stirring. After the solution was added, it was stirred for 30 min, and the mixed solution was allowed to stand overnight. After the reaction time was sufficient, the mixed solution was centrifuged three times at 8000 r / min with ethanol as the solvent. Then, the solid after centrifugation was dried at 80°C for 3 h to obtain a ZIF-67 powder, which was stored at room temperature under dry conditions.

[0070] (2) Preparation of PDMS@AgNPs@ZIF-67 biomimetic polymer material

[0071] The canna leaves collected at the river were cut into small pieces of 4 cm x 4 cm and attached to the culture dish with ultra-thin transparent double-sided tape. Then, the PDMS gel and the curing agent were mixed in a test tube at a mass ratio of 10:1, and then stirred manually for 3 min, and then ultrasonic degassing was performed in a water bath for 5 min. The mixture was poured onto the canna leaves in the culture dish and heated at 80°C for 6 h to obtain a cured PDMS film (about 1 mm thick). Then, the PDMS film was gently peeled off from the surface of the canna leaf pieces and cut into 5 mm x 5 mm samples for subsequent use. Then, silver nanoparticles were deposited on the biomimetic PDMS film by magnetron sputtering (40 W) for 45 s. Finally, the ZIF-67 powder obtained in step (1) was dissolved in anhydrous ethanol and ultrasonically mixed for 3 min to obtain a nanomaterial solution of ZIF-67 with a concentration of 0.075 mg / mL. 20 μL of the synthesized ZIF-67 ethanol solution was dropped onto the surface of the PDMS@AgNPs substrate, and after drying at room temperature, the PDMS@AgNPs@ZIF-67 biomimetic polymer material was obtained.

[0072] (3) Preparation of PDMS@AgNPs@ZIF-67 biomimetic immunosubstrate material

[0073] 30 μL of a PBS solution containing 0.2 mg / mL of PSA antibody was coated on the PDMS@AgNPs@ZIF-67 biomimetic material, and the immobilized capture antibody was incubated at 0°C overnight. The substrate was washed with TBS, PBS solution and deionized water in turn to remove unreacted antibodies, and then 35 μL of a PBS buffer solution containing bovine serum albumin was added and reacted at room temperature for 1 h to block the non-specific binding sites. Then, the free BSA was washed with TBS, PBS solution and deionized water in turn. Finally, the immunosubstrate was stored at 4°C for subsequent detection.

[0074] (4) Synthesis of gold nanorod immunoprobes

[0075] Firstly, 0.6 mL of frozen NaBH4(0.01 M) was quickly added to 20 mL of mixed aqueous solution of CTAB(0.05 M) and HAuCl4(0.25 M) to prepare Au seeds under vigorous stirring. The obtained seed solution was incubated at 25 °C for 2 h. Subsequently, Au NRs growth solution was prepared by mixing CTAB solution(95 mL, 0.1 M) with AgNO3(1.0 mL, 10 -2 M). After thorough mixing, ascorbic acid(0.45 mL, 0.1 M), HAuCl4(3 mL, 10 -2 M) and incubated seed solution(160 μL) were added in sequence. The solution was gently stirred and left overnight to obtain gold nanorod solution.

[0076] Subsequently, gold nanorod immunoprobes were prepared. Firstly, 3 mL of synthesized Au NRs aqueous solution was centrifuged to remove excess CTAB. Subsequently, 10 μL of MB solution(10 -5 M) was added to the Au centrifugate. After removing excess MB by centrifugation, the MB-modified Au NRs were dissolved in 1 mL of PBS solution. Then 10 μL of PSA antibody(0.2 mg / mL) was added to the MB-modified Au NRs solution and incubated at 4 °C for 2 h. The unbound PSA antibody was removed by centrifugation, and 10 μL of PBS buffer containing bovine serum albumin was added. After incubation at room temperature for 1 h, the excess bovine serum albumin was removed by centrifugation. The prepared Au NRs immunoprobes were dissolved in 1 mL of PBS aqueous solution and stored at 4 °C.

[0077] Based on the above-mentioned PDMS@AgNPs@ZIF-67 biomimetic immunosubstrate material and Au NRs immunoprobes, the repeatable immuno-detection application of cancer markers includes the following steps:

[0078] (1) Immuno-detection of cancer markers

[0079] After 20 μL of buffer solution containing different concentrations of cancer marker antigens to be tested was added to the prepared PDMS@AgNPs@ZIF-67 biomimetic immunosubstrate material, it was then incubated at 37 °C for 2 h. After removing the excess unreacted antigens to be tested by washing with TBS, PBS solution and deionized water in sequence, 20 μL of gold nanorod immunoprobes solution was added to the PDMS@AgNPs@ZIF-67 biomimetic immunosubstrate material adsorbed with the antigens to be tested, and incubated at 37 °C for 2 h. After removing the excess unreacted gold nanorod immunoprobes, the complex of gold nanorod immunoprobes and PDMS@AgNPs@ZIF-67 biomimetic immunosubstrate obtained after the above immuno-reaction was measured by Raman spectrometer. According to the linear relationship between the antigen concentration and the Raman characteristic peak intensity, the concentration of the antigen to be tested was calculated.

[0080] Figure 5 shows the scanning electron microscope photos of the PDMS@AgNPs@ZIF-67 biomimetic substrate prepared in this embodiment. As can be seen from Figure 5, the surface of the PDMS film with clear cell biomimetic periodic micro-nano structure is covered with a thin layer of Ag film, on which ZIF-67 nanoparticles are uniformly distributed.

[0081] Figure 6 is the Raman spectrum obtained by Raman detection of the substrate after the immunoreaction of the PDMS@AgNPs@ZIF-67 biomimetic immune substrate prepared in this embodiment and the gold nanorod immune probe with different concentrations of the antigen to be tested (concentration from 100 nanograms per milliliter to 1 picogram per milliliter). As can be seen from Figure 6, as the concentration of the antigen to be tested decreases, the Raman characteristic spectrum intensity of the labeled molecules gradually decreases, and when the concentration of the antigen to be tested decreases to 1 picogram per milliliter, the Raman characteristic peak of the labeled molecules is still very obvious relative to the background signal.

[0082] Figure 7 is a graph showing the change of the characteristic peak intensity at a frequency shift of 1621 cm-1 in the Raman spectrum of the immunodetection of prostate specific antigen PSA by the PDMS@AgNPs@ZIF-67 biomimetic immune substrate and the gold nanorod immune probe prepared in this embodiment and the result of internal standard correction. As can be seen by fitting, when the concentration of the antigen to be tested changes from 100 nanograms per milliliter to 1 picogram per milliliter, the Raman characteristic peak intensity changes linearly with the concentration. The fitting result shows that this change trend conforms to the linear equation Y = 18830.1 + 1865.2X, and the fitting degree is 0.984. After correction using the intrinsic Raman peak of PDMS at 2905 cm-1 as the internal standard, the change trend conforms to the linear equation Y = 18.0 + 1.7X, and the fitting degree is improved to 0.995, and the detection limit is 38 femtograms per milliliter. -1 -1

[0083] Embodiment 3

[0084] A preparation method of a biomimetic nano-immune substrate material, comprising the following steps:

[0085] (1) Preparation of ZIF-67 nano-material

[0086] ​​First, 174.6 mg of cobalt nitrate hexahydrate and 197 mg of 2-methylimidazole were added to an equal proportion of a mixed solution of methanol and ethanol, respectively. Then, the above solution was slowly added to the other solution with a rubber dropper under magnetic stirring. After the solution was added, it was stirred for 40 min, and the mixed solution was allowed to stand overnight. After the reaction time was sufficient, the mixed solution was centrifuged four times at 8000 r / min with ethanol as the solvent. Then, the solid after centrifugation was dried at 80°C for 6 h to obtain a ZIF-67 powder, which was stored at room temperature under dry conditions.

[0087] (2) Preparation of PDMS@AgNPs@ZIF-67 biomimetic polymer material

[0088] The canna leaves collected at the river were cut into small pieces of 4 cm x 4 cm and pasted in a culture dish with ultra-thin transparent double-sided tape. Then, the PDMS gel and the curing agent were mixed in a test tube at a mass ratio of 10:1, and then stirred manually for 5 min, and then ultrasonic degassing was performed in a water bath for 8 min. The mixture was poured onto the canna leaves in the culture dish and heated at 80°C for 8 h to obtain a cured PDMS film (about 1 mm thick). Then, the PDMS film was gently peeled off from the surface of the canna leaf pieces and cut into 5 mm x 5 mm samples for subsequent use. Then, silver nanoparticles were deposited on the biomimetic PDMS film by magnetron sputtering (40 W) for 60 s. Finally, the ZIF-67 powder obtained in step (1) was dissolved in anhydrous ethanol and ultrasonically mixed for 4 min to obtain a nanomaterial solution of ZIF-67 with a concentration of 0.075 mg / mL. 30 μL of the synthesized ZIF-67 ethanol solution was dropped onto the surface of the PDMS@AgNPs substrate, and after drying at room temperature, the PDMS@AgNPs@ZIF-67 biomimetic polymer material was obtained.

[0089] (3) Preparation of PDMS@AgNPs@ZIF-67 biomimetic immunosubstrate material

[0090] 40 μL of a PBS solution containing 0.2 mg / mL of PSA antibody was coated on the PDMS@AgNPs@ZIF-67 biomimetic material, and the immobilized capture antibody was incubated at 0°C overnight. The substrate was washed with TBS, PBS solution and deionized water in turn to remove unreacted antibodies, and then 60 μL of a PBS buffer solution containing bovine serum albumin was added and reacted at room temperature for 1 h to block the non-specific binding sites. Then, the free BSA was washed with TBS, PBS solution and deionized water in turn. Finally, the immunosubstrate was stored at 4°C for subsequent detection.

[0091] (4) Synthesis of gold nanorod immunoprobes

[0092] Firstly, 0.8 mL of frozen NaBH4(0.01 M) was quickly added to 20 mL of mixed aqueous solution of CTAB(0.05 M) and HAuCl4(0.25 M) to prepare Au seeds under vigorous stirring. The obtained seed solution was incubated at 25 °C for 2-4 h. Subsequently, Au NRs growth solution was prepared by mixing CTAB solution(105 mL, 0.1 M) with AgNO3(1.5 mL, 10 -2 M). After thorough mixing, ascorbic acid(0.55 mL, 0.1 M), HAuCl4(7 mL, 10 -2 M) and incubated seed solution(160 μL) were added in sequence. After gentle stirring, the gold nanorod solution was obtained by standing overnight.

[0093] Subsequently, gold nanorod immunoprobes were prepared. Firstly, 7 mL of synthesized Au NRs aqueous solution was centrifuged to remove excess CTAB. Subsequently, 30 μL of MB solution(10 -5 M) was added to the Au centrifugate. After removing excess MB by centrifugation, the MB-modified Au NRs were dissolved in 2 mL of PBS solution. Then 30 μL of PSA antibody(0.2 mg / mL) was added to the MB-modified Au NRs solution and incubated at 4 °C for 2-4 h. The unbound PSA antibody was removed by centrifugation, and 40 μL of PBS buffer solution containing bovine serum albumin was added. After incubation at room temperature for 1 h, the excess bovine serum albumin was removed by centrifugation. The prepared Au NRs immunoprobes were dissolved in 2 mL of PBS aqueous solution and stored at 4 °C.

[0094] Based on the above-mentioned application of PDMS@AgNPs@ZIF-67 biomimetic immunosubstrate material and Au NRs immunoprobes for immunodetection of cancer markers, the following steps are included:

[0095] (1) Immunodetection of cancer markers

[0096] After 20 μL of buffer solution containing different concentrations of cancer marker antigens to be tested was added to the prepared PDMS@AgNPs@ZIF-67 biomimetic immunosubstrate material, it was then incubated at 37 °C for 4 h. After washing with TBS, PBS solution and deionized water to remove excess unreacted antigens to be tested, 20 μL of gold nanorod immunoprobes solution was added to the PDMS@AgNPs@ZIF-67 biomimetic immunosubstrate material adsorbed with the antigens to be tested, and incubated at 37 °C for 4 h. After washing to remove excess unreacted gold nanorod immunoprobes, the complex of gold nanorod immunoprobes and PDMS@AgNPs@ZIF-67 biomimetic immunosubstrate obtained after the above immunoreaction was measured by Raman spectrometer. According to the linear relationship between antigen concentration and Raman characteristic peak intensity, the concentration of the antigen to be tested was calculated.

[0097] Figure 8 shows a scanning electron microscope image of the PDMS@AgNPs@ZIF-67 biomimetic substrate prepared in this embodiment. As can be seen from Figure 8, ZIF-67 and Ag are uniformly coated on the surface of the PDMS film with a periodic micro / nano structure.

[0098] Figure 9 shows the Raman spectra obtained by Raman detection of the substrate after immunization with different concentrations of the PDMS@AgNPs@ZIF-67 biomimetic immunomodulator and gold nanorod immunoprobe prepared in this embodiment (from 100 nanograms per milliliter to 1 picogram per milliliter). As can be seen from Figure 9, the Raman characteristic spectrum intensity of the labeled molecule gradually decreases with decreasing concentration of the antigen until the concentration of the antigen is reduced to 1 picogram per milliliter, at which point the Raman characteristic peak of the labeled molecule remains very obvious relative to the background signal.

[0099] Figure 10 shows the Raman spectrum of the PDMS@AgNPs@ZIF-67 biomimetic immune substrate and gold nanorod immune probe prepared in this embodiment, with a mid-frequency shift of 1621 cm⁻¹. -1 The graphs show the characteristic peak intensity as a function of the analyte concentration and the results of internal standard correction. Fitting analysis reveals that the Raman characteristic peak intensity changes linearly with concentration when the analyte concentration changes from 100 ng / mL to 1 picogram / mL. The fitting results show that this trend conforms to the linear equation Y = 22446.6 + 2329.1X, with a goodness of fit of 0.989. The PDMS peak intensity is located at 2905 cm⁻¹. -1 After correction using the intrinsic Raman peak as an internal standard, the trend of change conforms to the linear equation Y = 22.7 + 2.3X, the goodness of fit is improved to 0.994, and the detection limit is 150 femtograms per milliliter.

[0100] As can be seen from the above embodiments and figures, the biomimetic nano-immune substrate material of the present invention is simple to prepare, has high detection sensitivity and high detection efficiency, and can achieve early screening for prostate cancer. Constructing a SERS substrate with both synergistic enhancement and molecule anchoring capabilities on a MOF-modified biomimetic substrate is of great significance for improving the detection efficiency of PSA.

[0101] Those skilled in the art should understand that the embodiments of the present invention described above and shown in the accompanying drawings are merely examples and do not limit the present invention. The objectives of the present invention have been fully and effectively achieved. The functions and structural principles of the present invention have been demonstrated and explained in the embodiments, and any variations or modifications may be made to the implementation of the present invention without departing from the stated principles.

Claims

1. A method for preparing a biomimetic nano-immunological base material, characterized by, Comprising: (S10) Preparation of ZIF-67 nanomaterials; (S20) Preparation of PDMS@AgNPs@ZIF-67 biomimetic polymer materials; (S30) Preparation of PDMS@AgNPs@ZIF-67 biomimetic immune substrate materials; and (S40) Synthesis of gold nanorod immune probes.

2. The method of claim 1, wherein the step (S20) comprises the steps of: (S201) Paste the canna leaf in a culture dish; (S202) Mix the PDMS gel with the curing agent, and remove the air bubbles by water bath ultrasonic; (S203) Pour the mixture on the canna leaf in the culture dish, heat, obtain the cured PDMS film, and peel the PDMS film from the surface of the canna leaf; (S204) deposit silver nanoparticles on the biomimetic PDMS film by magnetron sputtering to obtain a PDMS@AgNPs substrate; (S205) dissolve the ZIF-67 powder obtained in step (S10) in anhydrous ethanol to obtain a nanomaterial solution of ZIF-67; and (S206) drop the ZIF-67 ethanol solution onto the surface of the PDMS@AgNPs substrate, dry, and obtain a PDMS@AgNPs@ZIF-67 biomimetic polymer material.

3. The preparation method of the biomimetic nano-immune substrate material according to claim 2, wherein in the step (S202), the mass ratio of the PDMS gel to the curing agent is 10:1, in the step (S203), the thickness of the cured PDMS film is 1 mm, and in the step (S204), the power of the magnetron sputtering is 40 W, and the silver nanoparticles are deposited on the biomimetic PDMS film by magnetron sputtering for 30-60 s.

4. The preparation method of the biomimetic nano-immune substrate material according to claim 3, wherein in the step (S201), the canna leaf is cut into small pieces of 4 cm x 4 cm, and after the PDMS film is peeled from the surface of the canna leaf in the step (S203), the film is cut into samples of 5 mm x 5 mm for standby use.

5. The method of claim 2, wherein the step (S30) comprises the steps of: (S301) Coating the PDMS@AgNPs@ZIF-67 biomimetic polymer material with a PBS solution containing antibodies, incubating, and immobilizing the capture antibodies; (S302) sequentially washing the substrate with TBS, a PBS solution, and deionized water to remove unreacted antibodies, and then adding a PBS buffer solution containing bovine serum albumin dropwise for reaction at room temperature; and (S303) sequentially washing the free BSA with TBS, a PBS solution, and deionized water to obtain a PDMS@AgNPs@ZIF-67 biomimetic immune substrate material, which is stored at 4°C for standby use. ​ 6. The preparation method of the biomimetic nano-immune substrate material according to claim 5, wherein in the step (S301), the PBS solution containing 0.2 mg / mL PSA antibodies is coated on the PDMS@AgNPs@ZIF-67 biomimetic material, and the immobilized capture antibodies are incubated at 0°C overnight, and in the step (S302), the PBS buffer solution containing bovine serum albumin is added dropwise for reaction at room temperature for 1 h to block the non-specific binding sites.

7. The method of claim any one of claims 1 to 6, wherein the step (S40) comprises the steps of: (S401) adding the frozen NaBH4 into the mixed aqueous solution of CTAB and HAuCl4, stirring to prepare Au seeds, and incubating at 25℃; (S402) mixing the CTAB solution with AgNO3 to prepare an Au NRs growth solution; (S403) adding ascorbic acid, HAuCl4 and the incubated seed solution, stirring, and standing overnight to obtain a gold nanorod solution; (S404) centrifuging the synthesized Au NRs aqueous solution to remove excess CTAB, and adding an MB solution into the Au centrifugate, and centrifuging to remove excess MB; (S405) dissolving the MB-modified Au NRs in a PBS solution, then adding an antibody, incubating at 4℃, and centrifuging to remove unbound antibody; (S406) adding a PBS buffer solution containing bovine serum albumin, incubating at room temperature, centrifuging to remove excess bovine serum albumin, obtaining an Au NRs immunoprobe, dissolving in a PBS aqueous solution, and storing at 4℃.

8. The method of claim 7, wherein in the step (S401), 0.4-0.8 mL of frozen 0.01 M NaBH4 is added to 20 mL of 0.05 M CTAB and 0.25 M HAuCl4 mixed aqueous solution to prepare Au seeds under vigorous stirring, and the obtained seed solution is incubated at 25 °C for 2-4 h; in the step (S403), 0.1 M 0.35-0.55 mL ascorbic acid, 10 -2 3-7 mL of 0.05 M HAuCl4 and 160 μL of the incubated seed solution are sequentially added, stirred, and left overnight to obtain a gold nanorod solution; and in the step (S405), 0.2 mg / mL 10-30 μL of PSA antibody is added to the MB modified Au NRs solution and incubated at 4 °C for 2-4 h.

9. The method of preparing a biomimetic nanomaterial according to claim 8, wherein the step (S10) comprises the steps of: (S101) adding cobalt nitrate hexahydrate and 2-methylimidazole into a mixed solution of methanol and ethanol, respectively; (S102) mixing and stirring the above two solutions, and standing for reaction; (S103) washing with ethanol, and drying to obtain ZIF-67 powder.

10. The preparation method of the biomimetic nano-immune substrate material according to claim 9, wherein in the step (S101), 43.65-174.6 mg of cobalt nitrate hexahydrate and 49.25-197 mg of 2-methylimidazole are added into a mixed solution of 4-8 mL of methanol and ethanol in equal proportions, the above two solutions are mixed and stirred for 20-40 min, and standing overnight, in the step (S203), washing with ethanol for 2-4 times, drying at 80℃ for 2-6 h, and obtaining ZIF-67 powder.

11. A biomimetic nanoimmunological base material, characterized by, The biomimetic nano-immune substrate material comprises a PDMS@AgNPs@ZIF-67 biomimetic immune substrate and a gold nanorod immunoprobe, wherein the PDMS@AgNPs@ZIF-67 biomimetic immune substrate is prepared from a PDMS@AgNPs@ZIF-67 biomimetic polymer material.

12. The biomimetic nano-immune substrate material according to claim 11, wherein the biomimetic nano-immune substrate material is prepared by the preparation method of claims 1-10.

13. Use of a biomimetic nano-immunological base material, characterized in that, The biomimetic nano-immune substrate material is suitable for application in prostate cancer, colorectal cancer, ovarian cancer or pancreatic cancer specific antigen immune detection.

14. The application of the biomimetic nano-immune substrate material according to claim 13, wherein the biomimetic nano-immune substrate material comprises a PDMS@AgNPs@ZIF-67 biomimetic immune substrate and a gold nanorod immunoprobe, wherein the PDMS@AgNPs@ZIF-67 biomimetic immune substrate is prepared from a PDMS@AgNPs@ZIF-67 biomimetic polymer material.

15. The use of the bionic nano-immune substrate material according to claim 14, wherein in the application detection process, a buffer solution containing different concentrations of the antigen of the cancer marker to be detected is added dropwise to the PDMS@AgNPs@ZIF-67 bionic immune substrate material, incubated at 37°C for 2-4h, and then washed with TBS, PBS solution and deionized water in sequence to remove the excess unreacted antigen to be detected. Then, the gold nanorod immune probe solution is added dropwise to the PDMS@AgNPs@ZIF-67 bionic immune substrate adsorbed with the antigen to be detected, and incubated at 37°C for 2-4h. After washing to remove the excess unreacted gold nanorod immune probe, the complex of the gold nanorod immune probe and the PDMS@AgNPs@ZIF-67 bionic immune substrate obtained after the immune reaction is measured by a Raman spectrometer. According to the linear relationship between the antigen concentration and the Raman characteristic peak intensity, the concentration of the antigen to be detected is calculated.

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