Bionic nano immune substrate material as well as preparation method and application thereof

Abstract

The invention discloses a bionic nano immune substrate material as well as a preparation method and application thereof. The bionic nano immune substrate material comprises a PDMS (at) AgNPs (at) ZIF-67 bionic immune substrate and a gold nanorod immune probe. The preparation method comprises the following steps: (S10) preparing a ZIF-67 nano material; (S20) preparing a PDMS (Polydimethylsiloxane) (at) AgNPs (at) ZIF-67 bionic polymeric material; (S30) preparation of a PDMS (Polydimethylsiloxane) (at) AgNPs (at) ZIF-67 bionic immune substrate material; and (S40) synthesizing the gold nanorod immune probe. The SERS substrate which has a synergistic enhancement effect and a molecule anchoring capability is constructed on the MOFs modified bionic substrate, so that the efficiency and the sensitivity of prostatic cancer examination are improved.

Description

Technical Field

[0001] The present invention relates to the fields of material engineering and nanotechnology, and in particular to a bionic nanoimmune base material and a preparation method and application thereof. Background Art

[0002] Prostate cancer is the most common malignant tumor of the male genitourinary system, with an increasing incidence rate. As an important screening marker for prostate cancer, the level of prostate-specific antigen (PSA) in serum has become a key method for measuring the progression of prostate cancer. However, traditional detection techniques struggle to efficiently and accurately detect trace levels of PSA in serum. As a novel spectroscopic detection technique, surface-enhanced Raman scattering (SERS) offers unique advantages in sensitivity, reproducibility, and ease of use. In particular, SERS spectral intensity can be significantly enhanced through electromagnetic and chemical enhancement mechanisms, making it ideal for detecting trace amounts of target molecules in biological samples. Typically, the sensitivity of trace molecule detection depends on the density and intensity of electromagnetic "hotspots." Currently, the mainstream SERS-active materials are noble metal nanomaterials. Their surface morphology and structure are crucial for the formation of electromagnetic "hotspots." Compared with traditional physical and chemical synthesis methods, biomimetic fabrication techniques are more effective in creating periodic and uniform surface nanostructures with numerous SERS-active areas. Over the past decade, many SERS substrates with various biomimetic structures have been fabricated by replicating the surface structures of plant leaves and insect wings onto flexible polymer materials. Among existing polymer materials, the advantage of polydimethylsiloxane (PDMS) is that the biological structures imprinted on it tend to maintain good integrity without large-scale defects. On the other hand, metal-organic frameworks (MOFs) materials with higher specific surface areas can better anchor target molecules and have been used to develop multifunctional SERS substrates. In particular, MOFs can enhance Raman signals by promoting charge transfer (CT) between organic ligands and target molecules. In addition, MOFs exhibit good chemical stability, which can prevent the oxidation of precious metals and extend 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 molecular anchoring capabilities on MOF-modified biomimetic substrates. Summary of the Invention

[0003] One advantage of the present invention is that it provides a biomimetic nanoimmune substrate material and its preparation method and application, which can improve the sensitivity and efficiency of detection and achieve early screening of cancer.

[0004] Another advantage of the present invention is that it provides a biomimetic nanoimmune substrate material, a preparation method and application thereof. The biomimetic nanoimmune substrate material has a complete surface nanostructure that can be repeated periodically, which is conducive to outputting repeatable SERS signals. The PDMS substrate has an intrinsic Raman signal, which can be used as an internal reference signal for the signal to be tested, effectively improving the accuracy of the immune detection results.

[0005] Another advantage of the present invention is that it provides a biomimetic nano-immune substrate material and its preparation method and application. The modification of MOFs material on the surface of the biomimetic immune substrate enhances the Raman signal by promoting charge transfer (CT) between organic ligands and target molecules, which is beneficial to increase the adsorption efficiency of the molecules to be tested, significantly improve the collection efficiency of PSA in the blood, and thus improve the detection sensitivity, realize early screening of prostate cancer, and help measure the course of prostate cancer so as to formulate a treatment plan that is beneficial to the patient's health as soon as possible.

[0006] Another advantage of the present invention is that it provides a biomimetic nanoimmune substrate material, its preparation method and application. MOFs exhibit good chemical stability, can prevent the oxidation of precious metals, and extend the service life of the SERS substrate. Constructing a SERS substrate with both synergistic enhancement and molecular anchoring capabilities on the MOFs-modified biomimetic substrate is of great significance for improving the detection efficiency of PSA.

[0007] Another advantage of the present invention is that it provides a bionic nano-immune substrate material and a preparation method and application thereof. The preparation method is simple, easy to use, and suitable for clinical promotion and application.

[0008] According to one aspect of the present invention, the present invention provides a method for preparing a biomimetic nanoimmune substrate material, comprising the following steps:

[0009] (S10) Preparation of ZIF-67 nanomaterials;

[0010] (S20) Preparation of PDMS@AgNPs@ZIF-67 biomimetic polymer materials;

[0011] (S30) Preparation of PDMS@AgNPs@ZIF-67 biomimetic immune substrate materials; and

[0012] (S40) Synthesis of gold nanorod immunoprobes.

[0013] The step (S20) includes the following steps: (S201) placing a canna leaf in a culture dish; (S202) mixing PDMS gel with a curing agent, and removing bubbles by water bath ultrasound; (S203) pouring the mixture on the canna leaf in the culture dish, heating it to obtain a 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 a PDMS@AgNPs substrate; (S205) dissolving the ZIF-67 powder obtained in step (S10) in anhydrous ethanol to obtain a ZIF-67 nanomaterial solution; and (S206) dripping the ZIF-67 ethanol solution onto the surface of the PDMS@AgNPs substrate, drying it, and obtaining a PDMS@AgNPs@ZIF-67 biomimetic polymer material.

[0014] In the step (S202), the mass ratio of PDMS gel to 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 silver nanoparticles are deposited on the biomimetic PDMS film by magnetron sputtering for 30-60 seconds.

[0015] In the step (S201), the canna leaves are cut into small pieces of 4 cm×4 cm. In the step (S203), the PDMS film is peeled off from the surface of the canna leaves and cut into 5 mm×5 mm samples for later use.

[0016] The step (S30) includes the following steps: (S301) coating a PBS solution containing an antibody on the PDMS@AgNPs@ZIF-67 biomimetic polymer material, incubating, and immobilizing the capture antibody; (S302) rinsing the substrate with TBS, PBS solution, and deionized water in sequence to remove unreacted antibodies, and then adding a PBS buffer solution containing bovine serum albumin to react at room temperature; and (S303) rinsing free BSA with TBS, PBS solution, and deionized water in sequence to obtain a PDMS@AgNPs@ZIF-67 biomimetic immune substrate material, which is stored at 4°C for future use.

[0017] In step (S301), a PBS solution containing 0.2 mg / mL PSA antibody was applied to the PDMS@AgNPs@ZIF-67 biomimetic material and incubated overnight at 0°C to immobilize the capture antibody. In step (S302), a PBS buffer solution containing bovine serum albumin was added dropwise and reacted at room temperature for 1 hour to block nonspecific binding sites.

[0018] The step (S40) includes the following steps: (S401) adding frozen NaBH4 to a mixed aqueous solution of CTAB and HAuCl4, stirring to prepare Au seeds, and incubating at 25°C; (S402) mixing the CTAB solution with AgNO3 to prepare an AuNRs growth solution; (S403) adding ascorbic acid, HAuCl4 and incubation seed solution, stirring, and standing overnight to obtain a gold nanorod solution; (S404) centrifuging the synthesized Au NRs aqueous solution to remove excess CTAB, adding MB solution to the Au centrifuge solution, and centrifuging to remove excess MB; (S405) dissolving the MB-modified Au NRs in PBS solution, then adding antibodies, incubating at 4°C, and centrifuging to remove unbound antibodies; (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 immune probe, dissolving it in a PBS aqueous solution, and storing it at 4°C.

[0019] In the step (S401), 0.4-0.8 mL of frozen NaBH4 (0.01 M) was quickly added to a 20 mL aqueous solution of CTAB (0.05 M) and HAuCl4 (0.25 M) and stirred vigorously to prepare Au seeds. The resulting seed solution was incubated at 25°C for 2-4 hours. In the step (S403), 0.1 M ascorbic acid, 0.35-0.55 mL of HAuCl4, and 10 mL of CTAB (0.05 M) were added in sequence. -2 3-7 mL of HAuCl4 and 160 μL of incubation seed solution were gently stirred and allowed to stand overnight to obtain a gold nanorod solution; in the step (S405), 10-30 μL of PSA antibody at 0.2 mg / m was added to the MB-modified Au NRs solution and incubated at 4°C for 2-4 h.

[0020] The step (S10) includes the following steps: (S101) adding cobalt nitrate hexahydrate and 2-methylimidazole to a mixed solution of methanol and ethanol respectively; (S102) mixing and stirring the above two solutions, and standing them for reaction; (S103) washing with ethanol and drying to obtain ZIF-67 powder.

[0021] In step (S101), 43.65-174.6 mg of cobalt nitrate hexahydrate and 49.25-197 mg of 2-methylimidazole are respectively added to 4-8 mL of a mixture of methanol and ethanol in equal proportions. The two solutions are mixed and stirred for 20-40 minutes and allowed to stand overnight. In step (S203), the mixture is washed with ethanol 2-4 times and dried at 80°C for 2-6 hours to obtain ZIF-67 powder.

[0022] According to another aspect of the present invention, the present invention also provides a biomimetic nanoimmune substrate material, including a PDMS@AgNPs@ZIF-67 biomimetic immune substrate and a gold nanorod immune probe, wherein the PDMS@AgNPs@ZIF-67 biomimetic immune substrate is prepared from a PDMS@AgNPs@ZIF-67 biomimetic polymer material.

[0023] According to another aspect of the present invention, the present invention also provides an application of a biomimetic nano-immune base material, wherein the biomimetic nano-immune base material is suitable for application in prostate cancer, colorectal cancer, ovarian cancer or pancreatic cancer specific antigen immunodetection.

[0024] During the application detection process, a buffer solution containing different concentrations of the cancer marker antigen to be tested is added dropwise to the PDMS@AgNPs@ZIF-67 biomimetic immune substrate material, incubated at 37°C for 2-4 hours, and rinsed with TBS, PBS solution and deionized water in sequence to remove excess unreacted antigen to be tested. Then, the gold nanorod immune probe solution is added dropwise to the PDMS@AgNPs@ZIF-67 biomimetic immune substrate adsorbed with the antigen to be tested, and incubated at 37°C for 2-4 hours to wash away excess unreacted gold nanorod immune probe. The complex of the gold nanorod immune probe and the PDMS@AgNPs@ZIF-67 biomimetic immune substrate obtained after the above immune reaction is spectrally measured using a Raman spectrometer, and the concentration of the antigen to be tested is calculated based on the linear relationship between the antigen concentration and the Raman characteristic peak intensity. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a scanning electron microscope photograph of the PDMS@AgNPs@ZIF-67 biomimetic substrate prepared in Example 1 of the present invention.

[0026] Figure 2 This is a scanning electron microscope photograph of the gold nanorods prepared in Example 1 of the present invention.

[0027] Figure 3 This is a Raman spectrum obtained by performing Raman detection on the substrate after the PDMS@AgNPs@ZIF-67 biomimetic immune substrate and gold nanorod immune probe prepared in Example 1 of the present invention undergo immune reaction with different concentrations of the antigen to be tested.

[0028] Figure 4 The frequency shift 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 Example 1 of the present invention is 1264 cm -1 The graph of the characteristic peak intensity changing with the concentration of the antigen to be tested and the result of internal standard correction are shown.

[0029] Figure 5 This is a scanning electron microscope photograph of the PDMS@AgNPs@ZIF-67 biomimetic substrate prepared in Example 2 of the present invention.

[0030] Figure 6 This is a Raman spectrum obtained by performing Raman detection on the substrate after the PDMS@AgNPs@ZIF-67 biomimetic immune substrate and gold nanorod immune probe prepared in Example 2 of the present invention undergo immune reactions with antigens to be tested at different concentrations.

[0031] Figure 7 The frequency shift 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 Example 2 of the present invention is 1264 cm -1 The graph of the characteristic peak intensity changing with the concentration of the antigen to be tested and the result of internal standard correction are shown.

[0032] Figure 8 This is a scanning electron microscope photograph of the PDMS@AgNPs@ZIF-67 biomimetic substrate prepared in Example 3 of the present invention.

[0033] Figure 9 This is a Raman spectrum obtained by performing Raman detection on the substrate after the PDMS@AgNPs@ZIF-67 biomimetic immune substrate and gold nanorod immune probe prepared in Example 3 of the present invention undergo immune reaction with different concentrations of the antigen to be tested.

[0034] Figure 10 The frequency shift 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 Example 3 of the present invention is 1264 cm -1 The graph of the characteristic peak intensity changing with the concentration of the antigen to be tested and the result of internal standard correction are shown. DETAILED DESCRIPTION

[0035] The following description is intended to disclose the present invention so that those skilled in the art can implement the present invention. The preferred embodiments described below are for illustrative purposes only, and those skilled in the art will readily appreciate other obvious variations. The basic principles of the present invention defined in the following description may be applied to other embodiments, variations, improvements, equivalents, and other technical solutions that do not depart from the spirit and scope of the present invention.

[0036] In order to improve the detection efficiency of PSA and better measure the course of prostate cancer, the present invention provides a bionic nano-immune substrate material and its preparation method, which can be applied to the detection of prostate cancer, improve the detection sensitivity of prostate cancer, and realize early screening of prostate cancer.

[0037] The preparation method of the bionic nano-immune substrate material comprises the following steps:

[0038] (S10) Preparation of ZIF-67 nanomaterials;

[0039] (S20) Preparation of PDMS@AgNPs@ZIF-67 biomimetic polymer materials;

[0040] (S30) Preparation of PDMS@AgNPs@ZIF-67 biomimetic immune substrate materials; and

[0041] (S40) Synthesis of gold nanorod immunoprobes.

[0042] The step (S20) includes the following steps: (S201) cutting the canna leaves into small pieces and sticking them in a culture dish; (S202) stirring the PDMS gel and curing agent mixture in a test tube and removing bubbles by water bath ultrasound; (S203) pouring the mixture on the canna leaves in the culture dish, heating it to obtain a solidified PDMS film, and peeling the PDMS film from the surface of the canna leaves; (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 ZIF-67 nanomaterial solution; (S206) dripping the ZIF-67 ethanol solution onto the surface of the PDMS@AgNPs substrate, drying it, and obtaining a PDMS@AgNPs@ZIF-67 biomimetic polymer material.

[0043] The step (S30) includes the following steps: (S301) coating a PBS solution containing an antibody on the PDMS@AgNPs@ZIF-67 biomimetic polymer material, incubating, and immobilizing the capture antibody; (S302) washing the substrate with TBS, PBS solution, and deionized water in sequence to remove unreacted antibodies, and then adding a PBS buffer solution containing bovine serum albumin to react at room temperature.

[0044] The step (S40) includes the following steps: (S401) adding frozen NaBH4 to a mixed aqueous solution of CTAB and HAuCl4, stirring to prepare Au seeds, and incubating at 25°C; (S402) mixing the CTAB solution with AgNO3 to prepare an AuNRs growth solution; (S403) adding ascorbic acid, HAuCl4 and incubation seed solution, stirring, and standing overnight to obtain a gold nanorod solution; (S404) centrifuging the synthesized Au NRs aqueous solution to remove excess CTAB, adding MB solution to the Au centrifuge solution, and centrifuging to remove excess MB; (S405) dissolving the MB-modified Au NRs in PBS solution, then adding antibodies, incubating at 4°C, and centrifuging to remove unbound antibodies; (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 immune probe, dissolving it in a PBS aqueous solution, and storing it at 4°C.

[0045] During the application process, it also includes a detection step (S50): a buffer solution containing different concentrations of the cancer marker antigen to be tested is added dropwise to the PDMS@AgNPs@ZIF-67 biomimetic immune substrate material, incubated at 37°C for 2-4 hours, and rinsed with TBS, PBS solution and deionized water in sequence to remove excess unreacted antigen to be tested. Then, the gold nanorod immune probe solution is added dropwise to the PDMS@AgNPs@ZIF-67 biomimetic immune substrate adsorbed with the antigen to be tested, and incubated at 37°C for 2-4 hours to wash away excess unreacted gold nanorod immune probes. The complex of the gold nanorod immune probe obtained after the above immune reaction and the PDMS@AgNPs@ZIF-67 biomimetic immune substrate is spectrally measured using a Raman spectrometer, and the concentration of the antigen to be tested is calculated based on the linear relationship between the antigen concentration and the Raman characteristic peak intensity.

[0046] All raw materials used are commercially available. The Raman spectrometer BWS415 used in the examples was purchased from B&W Tek Inc., USA. The antigen used in the following examples is prostate-specific antigen (PSA), but is not limited to PSA. Other antigens include carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), ferritin, and carbohydrate antigens such as CA199.

[0047] Example 1

[0048] A method for preparing a biomimetic nanoimmune substrate material comprises the following steps:

[0049] (1) Preparation of ZIF-67 nanomaterials

[0050] First, 43.65 mg of cobalt nitrate hexahydrate and 49.25 mg of 2-methylimidazole were added to 2 mL of a mixture of equal proportions of methanol and ethanol. One solution was then slowly added to the other using a rubber-tipped pipette under magnetic stirring. After the addition of each solution, the mixture was stirred for 30 minutes and allowed to stand overnight. After sufficient reaction time, the mixture was centrifuged twice at 8000 rpm using ethanol as the solvent. The solid was then dried at 80°C for 2 hours to obtain ZIF-67 powder, which was then stored in a dry place at room temperature.

[0051] (2) Preparation of PDMS@AgNPs@ZIF-67 biomimetic polymer materials

[0052] Canna leaves collected from the river were cut into 4 cm × 4 cm pieces and attached to a Petri dish with ultrathin transparent double-sided tape. Subsequently, a mixture of PDMS gel and curing agent at a mass ratio of 10:1 was manually stirred in a test tube for 2 minutes, followed by 3 minutes of ultrasonication in a water bath to remove bubbles. The mixture was poured onto the canna leaves in the Petri dish and heated at 80°C for 4 hours to obtain a cured PDMS film (approximately 1 mm thick). The PDMS film was then delicately peeled from the canna leaf surface and cut into 5 mm × 5 mm samples for subsequent use. Subsequently, silver nanoparticles were deposited on the biomimetic PDMS film for 30 seconds by magnetron sputtering (40 W). Finally, the ZIF-67 powder obtained in step (1) was dissolved in anhydrous ethanol and ultrasonicated for 2 min to allow uniform mixing to obtain a ZIF-67 nanomaterial solution with a concentration of 0.075 mg / mL. 10 μL of the synthesized ZIF-67 ethanol solution was dropped onto the surface of the PDMS@AgNPs substrate and dried at room temperature to obtain the PDMS@AgNPs@ZIF-67 biomimetic polymer material.

[0053] (3) Preparation of PDMS@AgNPs@ZIF-67 biomimetic immune substrate materials

[0054] A 20 μL solution of 0.2 mg / mL PSA antibody in PBS was applied to the PDMS@AgNPs@ZIF-67 biomimetic material and incubated overnight at 0°C to immobilize the capture antibody. Unreacted antibody was removed by rinsing the substrate with TBS, PBS, and deionized water. Nonspecific binding sites were then blocked by adding 10 μL of bovine serum albumin in PBS buffer at room temperature for 1 hour. Free BSA was then rinsed with TBS, PBS, and deionized water. The immunosubstrate was stored at 4°C for subsequent detection.

[0055] (4) Synthesis of gold nanorod immunoprobes

[0056] First, 0.4 mL of frozen NaBH4 (0.01 M) was quickly added to a 20 mL aqueous solution of CTAB (0.05 M) and HAuCl4 (0.25 M) under vigorous stirring to prepare Au seeds. The resulting seed solution was incubated at 25°C for 2 h. Subsequently, CTAB solution (85 mL, 0.1 M) was mixed with AgNO3 (0.5 L, 10 -2 M) to prepare the Au NRs growth solution. After thorough mixing, ascorbic acid (0.35, 0.1 M), HAuCl4 (3 mL, 10 -2 M) and incubate seed solution (160 μL), gently stir, and let stand overnight to obtain a gold nanorod solution.

[0057] Then, gold nanorod immunoprobes were prepared. First, 3 mL of the synthesized Au NRs aqueous solution was centrifuged to remove excess CTAB. Then, 10 μL of MB solution (10 -5 After removing excess MB by centrifugation, the MB-modified Au NRs were dissolved in 1 mL of PBS. 10 μL of PSA antibody (0.2 mg / mL) was then added to the MB-modified Au NRs solution and incubated at 4°C for 2 h. Unbound PSA antibody was then removed by centrifugation, and 10 μL of PBS buffer containing bovine serum albumin was added. After incubation at room temperature for 1 h, excess bovine serum albumin was removed by centrifugation. The prepared Au NRs immunoprobe was dissolved in 1 mL of PBS aqueous solution and stored at 4°C.

[0058] The repeatable immunodetection application of cancer markers based on the PDMS@AgNPs@ZIF-67 biomimetic immunoassay substrate and the Au NRs immunoprobe includes the following steps:

[0059] (1) Immunodetection of cancer markers

[0060] 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 immune substrate material, and then incubated at 37°C for 2 hours. After rinsing with TBS, PBS solution and deionized water in sequence to remove excess unreacted antigens to be tested, 20 μL of gold nanorod immune probe solution was added to the PDMS@AgNPs@ZIF-67 biomimetic immune substrate adsorbed with the antigens to be tested, and incubated at 37°C for 2 hours to remove excess unreacted gold nanorod immune probes. The complex of the gold nanorod immune probe obtained after the above immune reaction and the PDMS@AgNPs@ZIF-67 biomimetic immune substrate was spectrally measured using a Raman spectrometer. The concentration of the antigen to be tested was calculated based on the linear relationship between the antigen concentration and the Raman characteristic peak intensity.

[0061] Figure 1 The scanning electron microscope image of the PDMS@AgNPs@ZIF-67 biomimetic substrate prepared in this example is shown. Figure 1 It can be seen that ZIF-67 and Ag are evenly coated on the surface of the PDMS film with a clear cell biomimetic periodic micro-nanostructure, which is covered by a thin layer of Ag film on which ZIF-67 nanoparticles are evenly distributed.

[0062] Figure 2 The scanning electron microscope photo of the gold nanorods prepared in this example is shown. Figure 2 It can be seen that the prepared gold nanorods have relatively uniform sizes, with an aspect ratio of 2-3.

[0063] Figure 3 The Raman spectra are obtained by using the PDMS@AgNPs@ZIF-67 biomimetic immune substrate and gold nanorod immune probe prepared in this example to conduct an immune reaction with different concentrations of the antigen to be tested (concentrations range from 1 μg / ml to 1 pg / ml) and then performing Raman detection on the substrate. Figure 3 It can be seen that as the concentration of the antigen to be tested decreases, the intensity of the Raman characteristic spectrum of the labeled molecule gradually decreases. When the concentration of the antigen to be tested drops to 1 picogram per milliliter, the Raman characteristic peak of the labeled molecule is still very obvious relative to the background signal.

[0064] Figure 4 The frequency shift of the Raman spectrum of the immunodetection of prostate-specific antigen PSA by the PDMS@AgNPs@ZIF-67 biomimetic immune substrate and gold nanorod immune probe prepared in this example is 1621 cm -1 The graph of the characteristic peak intensity changes with the concentration of the antigen to be tested and the result of internal standard correction. Through fitting, it can be seen that when the concentration of the antigen to be tested changes from 1 microgram per milliliter to 1 picogram per milliliter, the intensity of the Raman characteristic peak changes linearly with the concentration. The fitting results show that this change trend conforms to the linear equation Y = 24041.8 + 2503.5X, with a fitting degree of 0.981. PDMS is used at 2905cm -1 After the intrinsic Raman peak of was used as the internal standard for correction, the change trend was consistent with the linear equation Y=22.7+2.3X, the fitting degree was improved to 0.998, and the detection limit was 210 femtograms per milliliter.

[0065] Example 2

[0066] A method for preparing a biomimetic nanoimmune substrate material comprises the following steps:

[0067] (1) Preparation of ZIF-67 nanomaterials

[0068] First, 87.3 mg of cobalt nitrate hexahydrate and 98.5 mg of 2-methylimidazole were added to 6 mL of a mixture of equal proportions of methanol and ethanol. One solution was then slowly added to the other using a rubber-tipped pipette under magnetic stirring. After the addition of each solution, the mixture was stirred for 30 minutes and allowed to stand overnight. After sufficient reaction time, the mixture was centrifuged three times at 8000 rpm using ethanol as the solvent. The solid was then dried at 80°C for 3 hours to obtain ZIF-67 powder, which was then stored in a dry place at room temperature.

[0069] (2) Preparation of PDMS@AgNPs@ZIF-67 biomimetic polymer materials

[0070] Canna leaves collected from the river were cut into 4 cm × 4 cm pieces and attached to a Petri dish with ultrathin transparent double-sided tape. Subsequently, a mixture of PDMS gel and curing agent at a mass ratio of 10:1 was manually stirred in a test tube for 3 minutes, followed by 5 minutes of ultrasonication in a water bath to remove bubbles. The mixture was poured onto the canna leaves in the Petri dish and heated at 80°C for 6 hours to obtain a cured PDMS film (approximately 1 mm thick). The PDMS film was then delicately peeled from the canna leaf surface and cut into 5 mm × 5 mm samples for subsequent use. Subsequently, silver nanoparticles were deposited on the biomimetic PDMS film for 45 seconds by magnetron sputtering (40 W). Finally, the ZIF-67 powder obtained in step (1) was dissolved in anhydrous ethanol and ultrasonicated for 3 min to allow uniform mixing to obtain a ZIF-67 nanomaterial solution 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 dried at room temperature to obtain the PDMS@AgNPs@ZIF-67 biomimetic polymer material.

[0071] (3) Preparation of PDMS@AgNPs@ZIF-67 biomimetic immune substrate materials

[0072] A 30 μL PBS solution containing 0.2 mg / mL PSA antibody was applied to the PDMS@AgNPs@ZIF-67 biomimetic material and incubated overnight at 0°C to immobilize the capture antibody. Unreacted antibody was removed by rinsing the substrate with TBS, PBS, and deionized water. Nonspecific binding sites were then blocked by adding 35 μL of PBS buffer containing bovine serum albumin (BSA) at room temperature for 1 hour. Free BSA was then rinsed with TBS, PBS, and deionized water. The immunosubstrate was stored at 4°C for subsequent testing.

[0073] (4) Synthesis of gold nanorod immunoprobes

[0074] First, 0.6 mL of frozen NaBH4 (0.01 M) was quickly added to a 20 mL aqueous solution of CTAB (0.05 M) and HAuCl4 (0.25 M) under vigorous stirring to prepare Au seeds. The resulting seed solution was incubated at 25°C for 2 h. Subsequently, CTAB solution (95 mL, 0.1 M) was mixed with AgNO3 (1.0 mL, 10 -2 M) to prepare the Au NRs growth solution. After thorough mixing, ascorbic acid (0.45 mL, 0.1 M), HAuCl4 (3 mL, 10 -2 M) and incubate seed solution (160 μL), gently stir, and let stand overnight to obtain a gold nanorod solution.

[0075] Then, gold nanorod immunoprobes were prepared. First, 3 mL of the synthesized Au NRs aqueous solution was centrifuged to remove excess CTAB. Then, 10 μL of MB solution (10 -5 After removing excess MB by centrifugation, the MB-modified Au NRs were dissolved in 1 mL of PBS. 10 μL of PSA antibody (0.2 mg / mL) was then added to the MB-modified Au NRs solution and incubated at 4°C for 2 h. Unbound PSA antibody was then removed by centrifugation, and 10 μL of PBS buffer containing bovine serum albumin was added. After incubation at room temperature for 1 h, excess bovine serum albumin was removed by centrifugation. The prepared Au NRs immunoprobe was dissolved in 1 mL of PBS aqueous solution and stored at 4°C.

[0076] The repeatable immunodetection application of cancer markers based on the PDMS@AgNPs@ZIF-67 biomimetic immunoassay substrate and the Au NRs immunoprobe includes the following steps:

[0077] (1) Immunodetection of cancer markers

[0078] 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 immune substrate material, and then incubated at 37°C for 2 hours. After rinsing with TBS, PBS solution and deionized water in sequence to remove excess unreacted antigens to be tested, 20 μL of gold nanorod immune probe solution was added to the PDMS@AgNPs@ZIF-67 biomimetic immune substrate adsorbed with the antigens to be tested, and incubated at 37°C for 2 hours to remove excess unreacted gold nanorod immune probes. The complex of the gold nanorod immune probe obtained after the above immune reaction and the PDMS@AgNPs@ZIF-67 biomimetic immune substrate was spectrally measured using a Raman spectrometer. The concentration of the antigen to be tested was calculated based on the linear relationship between the antigen concentration and the Raman characteristic peak intensity.

[0079] Figure 5 The scanning electron microscope image of the PDMS@AgNPs@ZIF-67 biomimetic substrate prepared in this example is shown. Figure 5 It can be seen that ZIF-67 and Ag are evenly coated on the surface of the PDMS film with a clear cell biomimetic periodic micro-nanostructure, which is covered by a thin layer of Ag film on which ZIF-67 nanoparticles are evenly distributed.

[0080] Figure 6 The Raman spectra are obtained by using the PDMS@AgNPs@ZIF-67 biomimetic immune substrate and gold nanorod immune probe prepared in this example to conduct an immune reaction with different concentrations of the antigen to be tested (concentrations range from 100 nanograms per milliliter to 1 picogram per milliliter) and then performing Raman detection on the substrate. Figure 6 It can be seen that as the concentration of the antigen to be tested decreases, the intensity of the Raman characteristic spectrum of the labeled molecule gradually decreases. When the concentration of the antigen to be tested drops to 1 picogram per milliliter, the Raman characteristic peak of the labeled molecule is still very obvious relative to the background signal.

[0081] Figure 7 The frequency shift of the Raman spectrum of the immunodetection of prostate-specific antigen PSA by the PDMS@AgNPs@ZIF-67 biomimetic immune substrate and gold nanorod immune probe prepared in this example is 1621 cm -1 The graph of the characteristic peak intensity changing with the concentration of the antigen to be tested and the result of internal standard correction are shown. Through fitting, it can be seen that when the concentration of the antigen to be tested changes from 100 nanograms per milliliter to 1 picogram per milliliter, the intensity of the Raman characteristic peak changes linearly with the concentration. The fitting results show that this change trend conforms to the linear equation Y = 18830.1 + 1865.2X, with a fitting degree of 0.984. PDMS is used at 2905 cm -1 After the intrinsic Raman peak of was used as the internal standard for correction, the change trend was consistent with the linear equation Y=18.0+1.7X, the fitting degree was improved to 0.995, and the detection limit was 38 femtograms per milliliter.

[0082] Example 3

[0083] A method for preparing a biomimetic nanoimmune substrate material comprises the following steps:

[0084] (1) Preparation of ZIF-67 nanomaterials

[0085] First, 174.6 mg of cobalt nitrate hexahydrate and 197 mg of 2-methylimidazole were added to 8 mL of a mixture of equal proportions of methanol and ethanol. One solution was then slowly added to the other using a rubber-tipped pipette under magnetic stirring. After the addition of each solution, the mixture was stirred for 40 minutes, and the mixture was allowed to stand overnight. After sufficient reaction time, the mixture was centrifuged four times at 8000 rpm using ethanol as the solvent. The solid was then dried at 80°C for 6 hours to obtain ZIF-67 powder, which was then stored in a dry place at room temperature.

[0086] (2) Preparation of PDMS@AgNPs@ZIF-67 biomimetic polymer materials

[0087] Canna leaves collected from the river were cut into 4 cm × 4 cm pieces and attached to a Petri dish with ultrathin transparent double-sided tape. Subsequently, a mixture of PDMS gel and curing agent at a mass ratio of 10:1 was manually stirred in a test tube for 5 minutes, followed by 8 minutes of ultrasonication in a water bath to remove bubbles. The mixture was poured onto the canna leaves in the Petri dish and heated at 80°C for 8 hours to obtain a cured PDMS film (approximately 1 mm thick). The PDMS film was then delicately peeled from the canna leaf surface and cut into 5 mm × 5 mm samples for subsequent use. Subsequently, silver nanoparticles were deposited on the biomimetic PDMS film by magnetron sputtering (40 W) for 60 seconds. Finally, the ZIF-67 powder obtained in step (1) was dissolved in anhydrous ethanol and ultrasonicated for 4 min to allow uniform mixing to obtain a ZIF-67 nanomaterial solution 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 dried at room temperature to obtain the PDMS@AgNPs@ZIF-67 biomimetic polymer material.

[0088] (3) Preparation of PDMS@AgNPs@ZIF-67 biomimetic immune substrate materials

[0089] 40 μL of a PBS solution containing 0.2 mg / mL PSA antibody was applied to the PDMS@AgNPs@ZIF-67 biomimetic material and incubated overnight at 0°C to immobilize the capture antibody. The substrate was rinsed sequentially with TBS, PBS, and deionized water to remove unreacted antibody. Then, 60 μL of a PBS buffer solution containing bovine serum albumin was added dropwise and reacted at room temperature for 1 hour to block nonspecific binding sites. Free BSA was then rinsed sequentially with TBS, PBS, and deionized water. The immunosubstrate was stored at 4°C for subsequent detection.

[0090] (4) Synthesis of gold nanorod immunoprobes

[0091] First, 0.8 mL of chilled NaBH4 (0.01 M) was quickly added to a 20 mL aqueous solution of CTAB (0.05 M) and HAuCl4 (0.25 M) under vigorous stirring to prepare Au seeds. The resulting seed solution was incubated at 25°C for 2-4 h. Subsequently, CTAB solution (105 mL, 0.1 M) was mixed with AgNO3 (1.5 mL, 10 -2 M) to prepare the Au NRs growth solution. After thorough mixing, ascorbic acid (0.55 mL, 0.1 M), HAuCl4 (7 mL, 10 -2 M) and incubate seed solution (160 μL), gently stir, and let stand overnight to obtain a gold nanorod solution.

[0092] Then, gold nanorod immunoprobes were prepared. First, 7 mL of the synthesized Au NRs aqueous solution was centrifuged to remove excess CTAB. Then, 30 μL of MB solution (10 -5 After removing excess MB by centrifugation, the MB-modified Au NRs were dissolved in 2 mL of PBS. 30 μL of PSA antibody (0.2 mg / mL) was then added to the MB-modified Au NRs solution and incubated at 4°C for 2-4 hours. Unbound PSA antibody was then removed by centrifugation, and 40 μL of PBS buffer containing bovine serum albumin was added. After incubation at room temperature for 1 hour, excess bovine serum albumin was removed by centrifugation. The prepared Au NRs immunoprobe was dissolved in 2 mL of PBS aqueous solution and stored at 4°C.

[0093] The cancer marker immunoassay application based on the PDMS@AgNPs@ZIF-67 biomimetic immune substrate material and the Au NRs immune probe includes the following steps:

[0094] (1) Immunodetection of cancer markers

[0095] 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 immune substrate material, and then incubated at 37°C for 4 hours. After rinsing with TBS, PBS solution and deionized water in sequence to remove excess unreacted antigens to be tested, 20 μL of gold nanorod immune probe solution was added to the PDMS@AgNPs@ZIF-67 biomimetic immune substrate adsorbed with the antigens to be tested, and incubated at 37°C for 4 hours to wash away excess unreacted gold nanorod immune probes. The complex of the gold nanorod immune probe obtained after the above immune reaction and the PDMS@AgNPs@ZIF-67 biomimetic immune substrate was spectrally measured using a Raman spectrometer. The concentration of the antigen to be tested was calculated based on the linear relationship between the antigen concentration and the Raman characteristic peak intensity.

[0096] Figure 8 The scanning electron microscope image of the PDMS@AgNPs@ZIF-67 biomimetic substrate prepared in this example is shown. Figure 8 It can be seen that ZIF-67 and Ag are uniformly coated on the surface of the PDMS film with periodic micro-nanostructure.

[0097] Figure 9 The Raman spectra are obtained by using the PDMS@AgNPs@ZIF-67 biomimetic immune substrate and gold nanorod immune probe prepared in this example to conduct an immune reaction with different concentrations of the antigen to be tested (concentrations range from 100 nanograms per milliliter to 1 picogram per milliliter) and then performing Raman detection on the substrate. Figure 9 It can be seen that as the concentration of the antigen to be tested decreases, the intensity of the Raman characteristic spectrum of the labeled molecule gradually decreases. When the concentration of the antigen to be tested drops to 1 picogram per milliliter, the Raman characteristic peak of the labeled molecule is still very obvious relative to the background signal.

[0098] Figure 10 The frequency shift of the Raman spectrum of the immunodetection of prostate-specific antigen PSA by the PDMS@AgNPs@ZIF-67 biomimetic immune substrate and gold nanorod immune probe prepared in this example is 1621 cm -1 The graph of the characteristic peak intensity changing with the concentration of the antigen to be tested and the result of internal standard correction are shown. Through fitting, it can be seen that when the concentration of the antigen to be tested changes from 100 nanograms per milliliter to 1 picogram per milliliter, the intensity of the Raman characteristic peak changes linearly with the concentration. The fitting results show that this change trend conforms to the linear equation Y = 22446.6 + 2329.1X, with a fitting degree of 0.989. PDMS is used at 2905 cm -1 After the intrinsic Raman peak of was used as the internal standard for correction, the change trend was consistent with the linear equation Y=22.7+2.3X, the fitting degree was improved to 0.994, and the detection limit was 150 femtograms per milliliter.

[0099] As demonstrated by the above examples and accompanying figures, the biomimetic nanoimmune substrate material of the present invention is simple to prepare, exhibits high sensitivity and efficiency, and is capable of early screening for prostate cancer. The construction of a SERS substrate on a MOF-modified biomimetic substrate, which exhibits both synergistic enhancement and molecular anchoring capabilities, is of great significance for improving the detection efficiency of PSA.

[0100] Those skilled in the art will appreciate that the embodiments of the present invention described above and shown in the accompanying drawings are intended to be illustrative only and are not intended to limit the present invention. The objectives of the present invention have been fully and effectively achieved. The functional and structural principles of the present invention have been demonstrated and illustrated in the embodiments. Any variations or modifications may be made to the embodiments of the present invention without departing from the principles described.

Claims

1. A method for preparing a biomimetic nanoimmune substrate material, characterized in that: include: (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 immunoprobes.

2. The method for preparing the biomimetic nanoimmune substrate material according to claim 1, wherein the step (S20) comprises the following steps: (S201) placing a canna leaf in a petri dish; (S202) mixing the PDMS gel with the curing agent and removing bubbles by water bath sonication; (S203) pouring the mixture on a canna leaf in a culture dish and heating it to obtain a solidified PDMS film, and then 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 a PDMS@AgNPs substrate; (S205) dissolving the ZIF-67 powder obtained in the above step (S10) in anhydrous ethanol to obtain a ZIF-67 nanomaterial solution; and (S206) taking the ZIF-67 ethanol solution and dropping it onto the surface of the PDMS@AgNPs substrate and drying it to obtain a PDMS@AgNPs@ZIF-67 biomimetic polymer material.

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

4. The method for preparing the biomimetic nanoimmune substrate material according to claim 3, wherein in the step (S201), the canna leaves are cut into small pieces of 4 cm×4 cm, and in the step (S203), the PDMS film is peeled off from the surface of the canna leaves and cut into 5 mm×5 mm samples for later use.

5. The method for preparing the biomimetic nano-immune substrate material according to claim 2, wherein the step (S30) comprises the following steps: (S301) coating a PBS solution containing an antibody on the PDMS@AgNPs@ZIF-67 biomimetic polymer material, incubating, and immobilizing the capture antibody; (S302) sequentially rinsing the substrate with TBS, PBS solution, and deionized water to remove unreacted antibodies, and then adding a PBS buffer solution containing bovine serum albumin to react at room temperature; and (S303) sequentially rinsing free BSA with TBS, PBS solution, and deionized water to obtain a PDMS@AgNPs@ZIF-67 biomimetic immune substrate material, which was stored at 4°C for future use.

6. The method for preparing a biomimetic nanoimmune substrate material according to claim 5, wherein in step (S301), a PBS solution containing 0.2 mg / mL PSA antibody is applied to the PDMS@AgNPs@ZIF-67 biomimetic material and incubated overnight at 0°C to immobilize the capture antibody, and in step (S302), a PBS buffer solution containing bovine serum albumin is added dropwise and reacted at room temperature for 1 hour to block nonspecific binding sites.

7. The method for preparing the biomimetic nano-immune substrate material according to any one of claims 1 to 6, wherein the step (S40) comprises the following steps: (S401) adding frozen NaBH4 to a mixed aqueous solution of CTAB and HAuCl4, stirring to prepare Au seeds, and incubating at 25°C; (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 AuNRs aqueous solution to remove excess CTAB, adding MB solution to the Au centrifuge solution, and centrifuging to remove excess MB; (S405) dissolving the MB-modified Au NRs in a PBS solution, then adding an antibody, incubating at 4°C, and removing unbound antibodies by centrifugation; (S406) adding a PBS buffer solution containing bovine serum albumin, incubating at room temperature, and removing excess bovine serum albumin by centrifugation to obtain an Au NRs immunoprobe, which was dissolved in a PBS aqueous solution and stored at 4°C.

8. The method for preparing a biomimetic nanoimmune substrate material according to claim 7, wherein in the step (S401), 0.4-0.8 mL of frozen 0.01 M NaBH4 () is added to a mixed aqueous solution of 20 mL, 0.05 M CTAB and 0.25 M HAuCl4, and Au seeds are prepared 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 of ascorbic acid, 10 -2 3-7 mL of HAuCl4 and 160 μL of incubation seed solution were stirred and allowed to stand overnight to obtain a gold nanorod solution; in the step (S405), 10-30 μL PSA antibody at 0.2 mg / mL was added to the MB-modified Au NRs solution and incubated at 4°C for 2-4 h.

9. The method for preparing the biomimetic nano-immune substrate material according to claim 8, wherein the step (S10) comprises the following steps: (S101) adding cobalt nitrate hexahydrate and 2-methylimidazole to a mixed solution of methanol and ethanol, respectively; (S102) mixing the two solutions and stirring them, and allowing them to react; (S103) washing with ethanol and drying to obtain ZIF-67 powder.

10. The method for preparing a biomimetic nanoimmune 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 respectively added to 4-8 mL of a mixed solution of methanol and ethanol in equal proportions, the two solutions are mixed and stirred for 20-40 minutes, and allowed to stand overnight. In the step (S203), the ZIF-67 powder is washed with ethanol 2-4 times and dried at 80°C for 2-6 hours to obtain the ZIF-67 powder.

11. A biomimetic nanoimmune substrate material, characterized in that: The invention comprises a PDMS@AgNPs@ZIF-67 biomimetic immune substrate and a gold nanorod immune probe, wherein the PDMS@AgNPs@ZIF-67 biomimetic immune substrate is prepared from a PDMS@AgNPs@ZIF-67 biomimetic polymer material. 12 . The biomimetic nanoimmune base material according to claim 11 , wherein the biomimetic nanoimmune base material is prepared by the preparation method according to claim 1 .

13. An application of a biomimetic nanoimmune substrate material, characterized in that: The bionic nano-immune substrate material is suitable for application in prostate cancer, colorectal cancer, ovarian cancer or pancreatic cancer specific antigen immunodetection.

14. The use of the biomimetic nanoimmune substrate material according to claim 13, wherein the biomimetic nanoimmune substrate material comprises a PDMS@AgNPs@ZIF-67 biomimetic immune substrate and a gold nanorod immune probe, 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 biomimetic nanoimmune substrate material according to claim 14, wherein in the application detection process, a buffer solution containing different concentrations of the cancer marker antigen to be tested is added dropwise to the PDMS@AgNPs@ZIF-67 biomimetic immune substrate material, incubated at 37°C for 2-4 hours, and rinsed with TBS, PBS solution and deionized water in sequence to remove excess unreacted antigen to be tested. Then, the gold nanorod immune probe solution is added dropwise to the PDMS@AgNPs@ZIF-67 biomimetic immune substrate adsorbed with the antigen to be tested, and incubated at 37°C for 2-4 hours to remove excess unreacted gold nanorod immune probe. The complex of the gold nanorod immune probe and the PDMS@AgNPs@ZIF-67 biomimetic immune substrate obtained after the above immune reaction is spectrally measured using a Raman spectrometer, and the concentration of the antigen to be tested is calculated based on the linear relationship between the antigen concentration and the Raman characteristic peak intensity.

Images