A biomimetic nano-immune base material, a preparation method and application thereof
By depositing AgNPs and ZIF-67 nanomaterials on the surface of PDMS and combining them with gold nanorod immune probes, a biomimetic nanoimmune substrate material was constructed, which solved the sensitivity and stability problems of PSA detection in traditional detection technologies, and enabled early screening and efficient detection of cancers such as prostate cancer.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- NINGBO FIRST HOSPITAL
- Filing Date
- 2024-05-11
- Publication Date
- 2026-05-22
AI Technical Summary
Traditional detection techniques are insufficient for the efficient and accurate detection of trace amounts of prostate-specific antigen (PSA) in serum. Existing SERS substrate materials have shortcomings in terms of electromagnetic hotspot formation and chemical stability, which affect the detection sensitivity and reliability.
By using biomimetic nano-immune substrate materials, AgNPs and ZIF-67 nanomaterials were deposited on the surface of PDMS and combined with gold nanorod immune probes to construct a SERS substrate with synergistic enhancement. MOFs were used to promote charge transfer and enhance Raman signal, and the intrinsic Raman signal was provided by the PDMS substrate as an internal reference.
It improves detection sensitivity and efficiency, enabling early screening of cancers such as prostate cancer, enhances the accuracy of test results, and extends the lifespan of SERS substrates.
Smart Images
Figure CN120731366B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the fields of materials engineering and nanotechnology, and in particular to a biomimetic nano-immune substrate material, its preparation method, and its application. Background Technology
[0002] Prostate cancer is the most common malignant tumor of the male genitourinary system, and its incidence is showing an increasing trend year by year. As an important screening indicator for prostate cancer, the serum level of prostate-specific antigen (PSA) is a key method for assessing the course of prostate cancer. However, traditional detection techniques struggle to achieve efficient and accurate detection of trace PSA in serum. Surface-enhanced Raman scattering (SERS), as a novel spectroscopic detection technique, exhibits unique advantages in detection sensitivity, result reproducibility, and ease of operation. In particular, based on electromagnetic and chemical enhancement mechanisms, the intensity of SERS spectra can be greatly enhanced, making it highly suitable for the detection of trace target molecules in biological samples. Typically, the detection sensitivity of trace molecules depends on the density and intensity of electromagnetic "hot spots." Currently, the mainstream active materials for SERS are noble metal nanomaterials. Their surface morphology and structure are crucial to the formation of electromagnetic "hot spots." 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 regions. Over the past decade, numerous 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, polydimethylsiloxane (PDMS) has the advantage that the biological structures imprinted on it tend to maintain good integrity without large-scale defects. On the other hand, metal-organic frameworks (MOFs) with high 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) within organic ligands and target molecules. Furthermore, MOFs exhibit good chemical stability, preventing the oxidation of noble metals and extending the lifespan of SERS substrates. Therefore, studying the construction of SERS substrates with both synergistic enhancement and molecule anchoring capabilities on MOF-modified biomimetic substrates is of great significance for improving the detection efficiency of PSA. Summary of the Invention
[0003] One advantage of this invention is that it provides a biomimetic nano-immune substrate material, its preparation method, and its application, which can improve the sensitivity and efficiency of detection and enable early cancer screening.
[0004] Another advantage of this invention is that it provides a biomimetic nano-immune substrate material, its preparation method and application. The biomimetic nano-immune substrate material has a complete surface nanostructure with periodic repeatability, which is beneficial for outputting repeatable SERS signals. In addition, 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 immunoassay results.
[0005] Another advantage of this invention is that it provides a biomimetic nano-immune substrate material, its preparation method, and its application. The modification of MOFs material on the surface of the biomimetic immune substrate enhances the Raman signal by promoting charge transfer (CT) within organic ligands and target molecules, which is beneficial to increasing the adsorption efficiency of the target molecules, significantly improving the collection efficiency of PSA in the blood, thereby improving the detection sensitivity, enabling early screening of prostate cancer, and facilitating the assessment of the course of prostate cancer so as to formulate a treatment plan that is beneficial to the patient's health as early as possible.
[0006] Another advantage of this invention is that it provides a biomimetic nano-immune substrate material, its preparation method and application. MOFs exhibit good chemical stability, which can prevent the oxidation of noble metals and extend the service life of SERS substrates. Constructing an SERS substrate with both synergistic enhancement and molecular anchoring ability on a MOF-modified biomimetic substrate is of great significance for improving the detection efficiency of PSA.
[0007] Another advantage of this invention is that it provides a biomimetic nano-immune substrate material, its preparation method and application. The preparation method is simple, easy to use, and suitable for clinical application.
[0008] According to one aspect of the present invention, a method for preparing a biomimetic nano-immune substrate material is provided, comprising the following steps:
[0009] Preparation of (S10)ZIF-67 nanomaterials;
[0010] Preparation of (S20)PDMS@AgNPs@ZIF-67 biomimetic polymeric material;
[0011] Preparation of (S30)PDMS@AgNPs@ZIF-67 biomimetic immune substrate material; and
[0012] (S40) Synthesis of gold nanorod immunoprobes.
[0013] The step (S20) includes the following steps: (S201) placing a canna leaf in a petri dish; (S202) mixing PDMS gel with a curing agent and removing air bubbles by ultrasonication in a water bath; (S203) pouring the mixture onto the canna leaf in the petri dish, heating to obtain a cured PDMS film, and peeling the PDMS film off 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) dropping the ZIF-67 ethanol solution onto the surface of the PDMS@AgNPs substrate and drying to obtain a PDMS@AgNPs@ZIF-67 biomimetic polymer material.
[0014] In step (S202), the mass ratio of PDMS gel to curing agent is 10:1. In step (S203), the thickness of the cured PDMS film is 1 mm. In step (S204), the magnetron sputtering power is 40 W, and silver nanoparticles are deposited on the biomimetic PDMS film for 30-60 s by magnetron sputtering.
[0015] In step (S201), the canna leaves are cut into small pieces of 4cm×4cm. In step (S203), the PDMS film is peeled off from the surface of the canna leaves and cut into 5mm×5mm samples for later use.
[0016] The step (S30) includes the following steps: (S301) coating the PDMS@AgNPs@ZIF-67 biomimetic polymer material with a PBS solution containing antibodies, incubating, and immobilizing the captured antibodies; (S302) washing the substrate sequentially with TBS, PBS solution, and deionized water to remove unreacted antibodies, and then adding a PBS buffer solution containing bovine serum albumin and reacting at room temperature; and (S303) washing the free BSA sequentially with TBS, PBS solution, and deionized water to obtain the PDMS@AgNPs@ZIF-67 biomimetic immune substrate material, which is stored at 4°C for later use.
[0017] In step (S301), a PBS solution containing 0.2 mg / mL PSA antibody is coated onto 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 is added and reacted at room temperature for 1 hour to block non-specific binding sites.
[0018] The step (S40) includes the following steps: (S401) adding frozen NaBH4 to an aqueous solution of CTAB and HAuCl4, stirring to prepare Au seeds, and incubating at 25°C; (S402) mixing CTAB solution with AgNO3 to prepare AuNRs growth medium; (S403) adding ascorbic acid, HAuCl4 and the incubation seed medium, stirring, and letting stand 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 centrifuged solution, and centrifuging to remove excess MB; (S405) dissolving 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 immune probes, dissolving in PBS aqueous solution, and storing at 4°C.
[0019] In step (S401), 0.4-0.8 mL of frozen NaBH4 (0.01 M) is rapidly added to 20 mL of an aqueous solution of CTAB (0.05 M) and HAuCl4 (0.25 M), and Au seeds are prepared under vigorous stirring. The resulting seed solution is incubated at 25°C for 2-4 hours. In step (S403), 0.1 M of 0.35-0.55 mL of ascorbic acid and 10 mL of... -2 Add 3-7 mL of HAuCl4 and 160 μL of incubation seed solution to M, stir gently, and let stand overnight to obtain gold nanorod solution; in step (S405), add 10-30 μL of PSA antibody at 0.2 mg / m to MB-modified Au NRs solution and incubate 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 two solutions and allowing them to stand 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 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 min and then allowed to stand overnight. In step (S203), the mixture is washed 2-4 times with ethanol and dried at 80°C for 2-6 h to obtain ZIF-67 powder.
[0022] According to another aspect of the present invention, the present invention also provides a biomimetic nano-immune substrate material, comprising 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 nanoimmune substrate material, wherein the biomimetic nanoimmune substrate material is suitable for use in the immunodetection of specific antigens for prostate cancer, colorectal cancer, ovarian cancer, or pancreatic cancer.
[0024] In the application detection process, buffer solutions containing different concentrations of the target cancer biomarker antigen were dropped onto the PDMS@AgNPs@ZIF-67 biomimetic immune substrate material and incubated at 37°C for 2-4 hours. After washing with TBS, PBS solution, and deionized water to remove excess unreacted antigen, the gold nanorod immunoprobe solution was dropped onto the PDMS@AgNPs@ZIF-67 biomimetic immune substrate adsorbed with the target antigen and incubated at 37°C for 2-4 hours. After washing to remove excess unreacted gold nanorod immunoprobe, the complex of the gold nanorod immunoprobe and PDMS@AgNPs@ZIF-67 biomimetic immune substrate obtained after the above immune reaction was measured using Raman spectroscopy. The concentration of the target antigen was calculated based on the linear relationship between antigen concentration and Raman characteristic peak intensity. Attached Figure Description
[0025] Figure 1 This is a scanning electron microscope image of the PDMS@AgNPs@ZIF-67 biomimetic substrate prepared in Example 1 of this invention.
[0026] Figure 2 This is a scanning electron microscope image of the gold nanorods prepared in Example 1 of the present invention.
[0027] Figure 3 The Raman spectra of the PDMS@AgNPs@ZIF-67 biomimetic immune substrate and gold nanorod immune probe prepared in Example 1 of this invention were obtained by Raman detection of the substrate after immunization with different concentrations of the antigen to be tested.
[0028] Figure 4 The Raman spectrum of the PDMS@AgNPs@ZIF-67 biomimetic immune substrate and gold nanorod immune probe prepared in Example 1 of this invention showed a mid-frequency shift of 1264 cm⁻¹ for the immunodetection of prostate-specific antigen (PSA). -1 The graph shows the change in characteristic peak intensity with the concentration of the analyte antigen and the result of internal standard correction.
[0029] Figure 5 This is a scanning electron microscope image of the PDMS@AgNPs@ZIF-67 biomimetic substrate prepared in Example 2 of the present invention.
[0030] Figure 6 The Raman spectra of the PDMS@AgNPs@ZIF-67 biomimetic immune substrate and gold nanorod immune probe prepared in Example 2 of this invention were obtained by Raman detection of the substrate after immunization with different concentrations of the antigen to be tested.
[0031] Figure 7 The Raman spectrum of the PDMS@AgNPs@ZIF-67 biomimetic immune substrate and gold nanorod immune probe prepared in Example 2 of this invention showed a mid-frequency shift of 1264 cm⁻¹ for the immunodetection of prostate-specific antigen (PSA). -1 The graph shows the change in characteristic peak intensity with the concentration of the analyte antigen and the results of internal standard correction.
[0032] Figure 8 This is a scanning electron microscope image of the PDMS@AgNPs@ZIF-67 biomimetic substrate prepared in Example 3 of the present invention.
[0033] Figure 9 The Raman spectra of the PDMS@AgNPs@ZIF-67 biomimetic immune substrate and gold nanorod immune probe prepared in Example 3 of this invention were obtained by Raman detection of the substrate after immunization with different concentrations of the antigen to be tested.
[0034] Figure 10 The Raman spectrum of the PDMS@AgNPs@ZIF-67 biomimetic immune substrate and gold nanorod immune probe prepared in Example 3 of this invention showed a mid-frequency shift of 1264 cm⁻¹ for the immunodetection of prostate-specific antigen (PSA). -1 The graph shows the change in characteristic peak intensity with the concentration of the analyte antigen and the results of internal standard correction. Detailed Implementation
[0035] The following description is intended to disclose the present invention and enable those skilled in the art to implement it. The preferred embodiments described below are merely examples, and other obvious variations will occur to those skilled in the art. The basic principles of the invention defined in the following description can be applied to other embodiments, modifications, improvements, equivalents, and other technical solutions that do not depart from the spirit and scope of the invention.
[0036] To improve the detection efficiency of PSA and better measure the course of prostate cancer, this invention provides a biomimetic nano-immune substrate material and its preparation method for use in the detection of prostate cancer, thereby improving the detection sensitivity of prostate cancer and achieving early screening of prostate cancer.
[0037] The preparation method of the biomimetic nano-immune substrate material includes the following steps:
[0038] Preparation of (S10)ZIF-67 nanomaterials;
[0039] Preparation of (S20)PDMS@AgNPs@ZIF-67 biomimetic polymeric material;
[0040] Preparation of (S30)PDMS@AgNPs@ZIF-67 biomimetic immune substrate material; and
[0041] (S40) Synthesis of gold nanorod immunoprobes.
[0042] The step (S20) includes the following steps: (S201) cutting canna leaves into small pieces and attaching them to a petri dish; (S202) stirring the PDMS gel and curing agent mixture in a test tube and removing air bubbles by ultrasonication in a water bath; (S203) pouring the mixture onto the canna leaves in the petri dish, heating to obtain a cured PDMS film, and peeling the PDMS film off 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) dropping the ZIF-67 ethanol solution onto the surface of the PDMS@AgNPs substrate and drying to obtain a PDMS@AgNPs@ZIF-67 biomimetic polymer material.
[0043] The step (S30) includes the following steps: (S301) coating the PDMS@AgNPs@ZIF-67 biomimetic polymer material with a PBS solution containing antibodies, incubating, and immobilizing the captured antibodies; (S302) washing the substrate sequentially with TBS, PBS solution and deionized water to remove unreacted antibodies, and then adding a PBS buffer solution containing bovine serum albumin and reacting at room temperature.
[0044] The step (S40) includes the following steps: (S401) adding frozen NaBH4 to an aqueous solution of CTAB and HAuCl4, stirring to prepare Au seeds, and incubating at 25°C; (S402) mixing CTAB solution with AgNO3 to prepare AuNRs growth medium; (S403) adding ascorbic acid, HAuCl4 and the incubation seed medium, stirring, and letting stand 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 centrifuged solution, and centrifuging to remove excess MB; (S405) dissolving 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 immune probes, dissolving in PBS aqueous solution, and storing at 4°C.
[0045] The application process also includes a detection step (S50): a buffer solution containing different concentrations of the target cancer biomarker antigen is dropped onto the PDMS@AgNPs@ZIF-67 biomimetic immune substrate material, incubated at 37°C for 2-4 hours, and after washing with TBS, PBS solution and deionized water to remove excess unreacted target antigen, the gold nanorod immunoprobe solution is dropped onto the PDMS@AgNPs@ZIF-67 biomimetic immune substrate adsorbed with the target antigen, and incubated at 37°C for 2-4 hours. After washing to remove excess unreacted gold nanorod immunoprobe, the complex of the gold nanorod immunoprobe and PDMS@AgNPs@ZIF-67 biomimetic immune substrate obtained after the above immune reaction is spectrally measured using a Raman spectrometer. The concentration of the target antigen is calculated based on the linear relationship between the antigen concentration and the Raman characteristic peak intensity.
[0046] All raw materials used were commercially available products. The BWS415 Raman spectrometer 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 it is not limited to PSA; it can also be carcinoembryonic antigen (CEA), alpha-fetoprotein antigen (AFP), ferritin antigen, and carbohydrate antigen (CA199), etc.
[0047] Example 1
[0048] A method for preparing a biomimetic nano-immune substrate material includes 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 methanol and ethanol in equal proportions. Then, under magnetic stirring, one solution was slowly added dropwise to the other solution using a dropper. After the addition was complete, the mixture was stirred for 30 min and allowed to stand overnight. After sufficient reaction time, the mixture was washed twice by centrifugation at 8000 rpm using ethanol as the solvent. The solid after centrifugation was then dried at 80 °C for 2 h to obtain ZIF-67 powder, which was stored at room temperature under dry conditions.
[0051] (2) Preparation of PDMS@AgNPs@ZIF-67 biomimetic polymeric material
[0052] Canna leaves collected from the riverbank were cut into 4cm x 4cm pieces and attached to petri dishes using ultra-thin transparent double-sided tape. A mixture of PDMS gel and curing agent at a mass ratio of 10:1 was then manually stirred in a test tube for 2 minutes, followed by ultrasonic degassing in a water bath for 3 minutes. The mixture was poured over the canna leaves in the petri dish and heated at 80°C for 4 hours to obtain a cured PDMS film (approximately 1mm thick). The PDMS film was then carefully peeled from the surface of the canna leaves and cut into 5mm x 5mm samples for later use. Subsequently, silver nanoparticles were deposited on the biomimetic PDMS film for 30 seconds using magnetron sputtering (40W). Finally, the ZIF-67 powder obtained in step (1) was dissolved in anhydrous ethanol and sonicated for 2 minutes to mix it evenly, resulting in 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 material
[0054] 20 μL of PBS solution containing 0.2 mg / mL PSA antibody was spread onto PDMS@AgNPs@ZIF-67 biomimetic material and incubated overnight at 0°C to immobilize the capture antibody. The substrate was then washed sequentially with TBS, PBS, and deionized water to remove unreacted antibody. 10 μL of PBS buffer solution containing bovine serum albumin was then added, and the mixture was reacted at room temperature for 1 h to block non-specific binding sites. Free BSA was then washed sequentially with TBS, PBS, and deionized water. Finally, the immunomodulatory substrate 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 rapidly added to 20 mL of an aqueous solution of CTAB (0.05 M) and HAuCl4 (0.25 M), and Au seeds were prepared under vigorous stirring. The resulting seed solution was incubated at 25 °C for 2 h. Subsequently, 85 mL of CTAB solution (0.1 M) and 0.5 L of AgNO3 (10 mL) were added to prepare Au seeds. -2 The Au NRs growth medium was prepared by mixing M) and then ascorbic acid (0.35, 0.1M) and HAuCl4 (3 mL, 10) in sequence. -2 Mix M) and incubation seed solution (160 μL), stir gently, and let stand overnight to obtain gold nanorod solution.
[0057] Subsequently, 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) was added to the Au centrifuged solution. -5 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 the mixture was 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 reproducible immunoassay application of cancer biomarkers based on the aforementioned PDMS@AgNPs@ZIF-67 biomimetic immunomodulator material and Au NRs immunoprobes includes the following steps:
[0059] (1) Immunological detection of cancer markers
[0060] 20 μL of buffer solution containing different concentrations of the target cancer biomarker antigen was added dropwise to the prepared PDMS@AgNPs@ZIF-67 biomimetic immune substrate material, and then incubated at 37°C for 2 h. After washing with TBS, PBS solution and deionized water to remove excess unreacted antigen, 20 μL of gold nanorod immunoprobe solution was added dropwise to the PDMS@AgNPs@ZIF-67 biomimetic immune substrate adsorbed with the target antigen, and incubated at 37°C for 2 h. After washing to remove excess unreacted gold nanorod immunoprobe, the complex of the gold nanorod immunoprobe and PDMS@AgNPs@ZIF-67 biomimetic immune substrate obtained after the above immune reaction was measured by Raman spectroscopy. The concentration of the target antigen was calculated based on the linear relationship between antigen concentration and Raman characteristic peak intensity.
[0061] Figure 1 The image shows a scanning electron microscope (SEM) image of the PDMS@AgNPs@ZIF-67 biomimetic substrate prepared in this embodiment. From... Figure 1 As can be seen, ZIF-67 and Ag are uniformly coated on the surface of a PDMS film with a clear cell-inspired periodic micro / nano structure, which is covered by a thin Ag film, on which ZIF-67 nanoparticles are uniformly distributed.
[0062] Figure 2 This shows a scanning electron microscope image of the gold nanorods prepared in this embodiment. Figure 2 It can be seen that the prepared gold nanorods have relatively uniform size and an aspect ratio of 2-3.
[0063] Figure 3 The Raman spectra of the substrate prepared in this embodiment using the PDMS@AgNPs@ZIF-67 biomimetic immune substrate and gold nanorod immune probe were obtained by Raman detection after reacting with different concentrations of the target antigen (from 1 μg / mL to 1 picogram / mL). 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 until the concentration of the antigen to be tested decreases to 1 picogram per milliliter, at which point the Raman characteristic peak of the labeled molecule is still very obvious relative to the background signal.
[0064] Figure 4 The Raman spectrum of the PDMS@AgNPs@ZIF-67 biomimetic immune substrate and gold nanorod immune probe prepared in this embodiment showed a mid-frequency shift of 1621 cm⁻¹ for the immunodetection of prostate-specific antigen (PSA). -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 as the analyte concentration increases from 1 μg / mL to 1 picogram / mL. The fitting results show that this trend conforms to the linear equation Y = 24041.8 + 2503.5X, with a goodness of fit of 0.981. 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 0.998, and the detection limit is 210 femtograms per milliliter.
[0065] Example 2
[0066] A method for preparing a biomimetic nano-immune substrate material includes 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 methanol and ethanol in equal proportions. Then, under magnetic stirring, one solution was slowly added dropwise to the other solution using a dropper. After the addition was complete, the mixture was stirred for 30 min and allowed to stand overnight. After sufficient reaction time, the mixture was washed three times by centrifugation at 8000 rpm using ethanol as the solvent. The solid after centrifugation was then dried at 80 °C for 3 h to obtain ZIF-67 powder, which was stored at room temperature under dry conditions.
[0069] (2) Preparation of PDMS@AgNPs@ZIF-67 biomimetic polymeric material
[0070] Canna leaves collected from the riverbank were cut into 4cm x 4cm pieces and attached to petri dishes using ultra-thin transparent double-sided tape. A mixture of PDMS gel and curing agent at a mass ratio of 10:1 was then manually stirred in a test tube for 3 minutes, followed by ultrasonic removal of air bubbles in a water bath for 5 minutes. The mixture was poured onto the canna leaves in the petri dishes and heated at 80°C for 6 hours to obtain a cured PDMS film (approximately 1mm thick). The PDMS film was then carefully peeled from the surface of the canna leaves and cut into 5mm x 5mm samples for subsequent use. Silver nanoparticles were then deposited on the biomimetic PDMS film for 45 seconds using magnetron sputtering (40W). Finally, the ZIF-67 powder obtained in step (1) was dissolved in anhydrous ethanol and sonicated for 3 minutes to mix it evenly, resulting in 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 material
[0072] 30 μL of PBS solution containing 0.2 mg / mL LSA antibody was spread onto PDMS@AgNPs@ZIF-67 biomimetic material and incubated overnight at 0°C to immobilize the capture antibody. The substrate was then washed sequentially with TBS, PBS, and deionized water to remove unreacted antibody. 35 μL of PBS buffer solution containing bovine serum albumin was then added, and the mixture was reacted at room temperature for 1 h to block non-specific binding sites. Free BSA was then washed sequentially with TBS, PBS, and deionized water. Finally, the immunomodulatory substrate was stored at 4°C for subsequent detection.
[0073] (4) Synthesis of gold nanorod immunoprobes
[0074] First, 0.6 mL of frozen NaBH4 (0.01 M) was rapidly added to 20 mL of an aqueous solution of CTAB (0.05 M) and HAuCl4 (0.25 M), and Au seeds were prepared under vigorous stirring. The resulting seed solution was incubated at 25 °C for 2 h. Subsequently, 95 mL of CTAB solution (0.1 M) and 1.0 mL of AgNO3 (10 M) were added to prepare Au seeds. -2 To prepare the Au NRs growth medium, mix M) and ascorbic acid (0.45 mL, 0.1 M), and HAuCl4 (3 mL, 10 M) sequentially. -2 Mix M) and incubation seed solution (160 μL), stir gently, and let stand overnight to obtain gold nanorod solution.
[0075] Subsequently, 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) was added to the Au centrifuged solution. -5 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 the mixture was 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 reproducible immunoassay application of cancer biomarkers based on the aforementioned PDMS@AgNPs@ZIF-67 biomimetic immunomodulator material and Au NRs immunoprobes includes the following steps:
[0077] (1) Immunological detection of cancer markers
[0078] 20 μL of buffer solution containing different concentrations of the target cancer biomarker antigen was added dropwise to the prepared PDMS@AgNPs@ZIF-67 biomimetic immune substrate material, and then incubated at 37°C for 2 h. After washing with TBS, PBS solution and deionized water to remove excess unreacted antigen, 20 μL of gold nanorod immunoprobe solution was added dropwise to the PDMS@AgNPs@ZIF-67 biomimetic immune substrate adsorbed with the target antigen, and incubated at 37°C for 2 h. After washing to remove excess unreacted gold nanorod immunoprobe, the complex of the gold nanorod immunoprobe and PDMS@AgNPs@ZIF-67 biomimetic immune substrate obtained after the above immune reaction was measured by Raman spectroscopy. The concentration of the target antigen was calculated based on the linear relationship between antigen concentration and Raman characteristic peak intensity.
[0079] Figure 5 The image shows a scanning electron microscope (SEM) image of the PDMS@AgNPs@ZIF-67 biomimetic substrate prepared in this embodiment. From... Figure 5 As can be seen, ZIF-67 and Ag are uniformly coated on the surface of a PDMS film with a clear cell-inspired periodic micro / nano structure, which is covered by a thin Ag film, on which ZIF-67 nanoparticles are uniformly distributed.
[0080] Figure 6 The Raman spectra of the PDMS@AgNPs@ZIF-67 biomimetic immune substrate and gold nanorod immune probe prepared in this embodiment were obtained by Raman detection of the substrate after immunization with different concentrations of the target antigen (from 100 nanograms per milliliter to 1 picogram per milliliter). 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 until the concentration of the antigen to be tested decreases to 1 picogram per milliliter, at which point the Raman characteristic peak of the labeled molecule is still very obvious relative to the background signal.
[0081] Figure 7 The Raman spectrum of the PDMS@AgNPs@ZIF-67 biomimetic immune substrate and gold nanorod immune probe prepared in this embodiment showed a mid-frequency shift of 1621 cm⁻¹ for the immunodetection of prostate-specific antigen (PSA). -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 = 18830.1 + 1865.2X, with a goodness of fit of 0.984. 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 = 18.0 + 1.7X, the goodness of fit is improved to 0.995, and the detection limit is 38 femtograms per milliliter.
[0082] Example 3
[0083] A method for preparing a biomimetic nano-immune substrate material includes 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 methanol and ethanol in equal proportions. Then, under magnetic stirring, one solution was slowly added dropwise to the other solution using a dropper. After the addition was complete, the mixture was stirred for 40 min and allowed to stand overnight. After sufficient reaction time, the mixture was washed four times by centrifugation at 8000 rpm using ethanol as the solvent. The solid after centrifugation was then dried at 80 °C for 6 h to obtain ZIF-67 powder, which was stored at room temperature under dry conditions.
[0086] (2) Preparation of PDMS@AgNPs@ZIF-67 biomimetic polymeric material
[0087] Canna leaves collected from the riverbank were cut into 4cm x 4cm pieces and attached to petri dishes using ultra-thin transparent double-sided tape. A mixture of PDMS gel and curing agent at a mass ratio of 10:1 was then manually stirred in a test tube for 5 minutes, followed by ultrasonic degassing in a water bath for 8 minutes. The mixture was poured over the canna leaves in the petri dish and heated at 80°C for 8 hours to obtain a cured PDMS film (approximately 1mm thick). The PDMS film was then carefully peeled from the surface of the canna leaves and cut into 5mm x 5mm samples for later use. Subsequently, silver nanoparticles were deposited on the biomimetic PDMS film for 60 seconds using magnetron sputtering (40W). Finally, the ZIF-67 powder obtained in step (1) was dissolved in anhydrous ethanol and sonicated for 4 min to mix it evenly, resulting in 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 material
[0089] 40 μL of PBS solution containing 0.2 mg / mL PSA antibody was spread onto PDMS@AgNPs@ZIF-67 biomimetic material and incubated overnight at 0°C to immobilize the capture antibody. The substrate was then washed sequentially with TBS, PBS, and deionized water to remove unreacted antibody. Next, 60 μL of PBS buffer solution containing bovine serum albumin was added, and the mixture was reacted at room temperature for 1 h to block non-specific binding sites. Free BSA was then washed sequentially with TBS, PBS, and deionized water. Finally, the immunomodulatory substrate was stored at 4°C for subsequent detection.
[0090] (4) Synthesis of gold nanorod immunoprobes
[0091] First, 0.8 mL of frozen NaBH4 (0.01 M) was rapidly added to 20 mL of an aqueous solution of CTAB (0.05 M) and HAuCl4 (0.25 M), and Au seeds were prepared under vigorous stirring. The resulting seed solution was incubated at 25 °C for 2–4 h. Subsequently, 105 mL of CTAB solution (0.1 M) and 1.5 mL of AgNO3 (10 M) were added to prepare Au seeds. -2 To prepare the Au NRs growth medium, mix M) and ascorbic acid (0.55 mL, 0.1 M), and HAuCl4 (7 mL, 10 M) sequentially. -2 Mix M) and incubation seed solution (160 μL), stir gently, and let stand overnight to obtain gold nanorod solution.
[0092] Subsequently, 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) was added to the Au centrifuged solution. -5 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 the mixture was incubated at 4 °C for 2–4 h. 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 h, 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 application of cancer biomarker immunoassay based on the above-mentioned PDMS@AgNPs@ZIF-67 biomimetic immunomodulator material and Au NRs immunoassay probes includes the following steps:
[0094] (1) Immunological detection of cancer markers
[0095] 20 μL of buffer solution containing different concentrations of the target cancer biomarker antigen was added dropwise to the prepared PDMS@AgNPs@ZIF-67 biomimetic immune substrate material, and then incubated at 37°C for 4 h. After washing with TBS, PBS solution and deionized water to remove excess unreacted antigen, 20 μL of gold nanorod immunoprobe solution was added dropwise to the PDMS@AgNPs@ZIF-67 biomimetic immune substrate adsorbed with the target antigen, and incubated at 37°C for 4 h. After washing to remove excess unreacted gold nanorod immunoprobe, the complex of the gold nanorod immunoprobe and PDMS@AgNPs@ZIF-67 biomimetic immune substrate obtained after the above immune reaction was measured by Raman spectroscopy. The concentration of the target antigen was calculated based on the linear relationship between antigen concentration and Raman characteristic peak intensity.
[0096] Figure 8 The image shows a scanning electron microscope (SEM) image of the PDMS@AgNPs@ZIF-67 biomimetic substrate prepared in this embodiment. From... Figure 8 As can be seen, ZIF-67 and Ag are uniformly coated on the surface of the PDMS film with a periodic micro / nano structure.
[0097] Figure 9 The Raman spectra of the PDMS@AgNPs@ZIF-67 biomimetic immune substrate and gold nanorod immune probe prepared in this embodiment were obtained by Raman detection of the substrate after immunization with different concentrations of the target antigen (from 100 nanograms per milliliter to 1 picogram per milliliter). 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 until the concentration of the antigen to be tested decreases to 1 picogram per milliliter, at which point the Raman characteristic peak of the labeled molecule is still very obvious relative to the background signal.
[0098] Figure 10 The Raman spectrum of the PDMS@AgNPs@ZIF-67 biomimetic immune substrate and gold nanorod immune probe prepared in this embodiment showed a mid-frequency shift of 1621 cm⁻¹ for the immunodetection of prostate-specific antigen (PSA). -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.
[0099] 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.
[0100] 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-immunoassay substrate material for prostate-specific antigen immunoassay, characterized in that, Includes the following steps: (S10) Cobalt nitrate hexahydrate and 2-methylimidazole were dissolved in a mixture of methanol and ethanol in equal proportions, stirred and allowed to stand for reaction, washed with ethanol and dried to obtain ZIF-67 nanomaterials. (S20) Using canna leaves as a biological template, PDMS gel was cast and solidified, and then peeled off to obtain a PDMS biomimetic membrane with periodic cell-level micro-nano structures; silver nanoparticles were deposited on the PDMS membrane by magnetron sputtering to obtain a PDMS@AgNPs substrate. ZIF-67 powder was dissolved in anhydrous ethanol to prepare a solution, which was then dropped onto the surface of PDMS@AgNPs and dried to obtain PDMS@AgNPs@ZIF-67 biomimetic polymer material. (S30) Coat the surface of the biomimetic polymer material with PBS solution containing antibodies, incubate and fix the captured antibodies, and obtain PDMS@AgNPs@ZIF-67 biomimetic immune substrate after blocking and washing; as well as (S40) Prepare MB-labeled gold nanorods and modify them with antibodies to obtain gold nanorod immune probes.
2. The method for preparing the biomimetic nano-immune substrate material for prostate-specific antigen immunodetection according to claim 1, wherein step (S20) includes the following steps: (S201) Place the canna leaves in the petri dish; (S202) Mix PDMS gel with curing agent and remove air bubbles by ultrasonic bath in water bath; (S203) Pour the mixture onto the canna leaves in a petri dish, heat to obtain a solidified PDMS film, and peel the PDMS film off the surface of the canna leaves; (S204) Deposit silver nanoparticles on the biomimetic PDMS film by magnetron sputtering to obtain PDMS@AgNPs substrate; (S205) Dissolve the ZIF-67 powder obtained in step (S10) in anhydrous ethanol to obtain a ZIF-67 nanomaterial solution; and (S206) Drop the ZIF-67 ethanol solution onto the surface of the PDMS@AgNPs substrate, dry, and obtain PDMS@AgNPs@ZIF-67 biomimetic polymer material.
3. The method for preparing biomimetic nano-immune substrate material for prostate-specific antigen immunodetection according to claim 2, wherein in step (S202), the mass ratio of PDMS gel to curing agent is 10:1; in step (S203), the thickness of the cured PDMS film is 1 mm; and in step (S204), the magnetron sputtering power is 40 W, and silver nanoparticles are deposited on the biomimetic PDMS film for 30-60 s by magnetron sputtering.
4. The method for preparing biomimetic nano-immune substrate material for prostate-specific antigen immunodetection according to claim 3, wherein in step (S201), the canna leaf is cut into small pieces of 4cm×4cm, and in step (S203), the PDMS film is peeled off from the surface of the canna leaf and cut into 5mm×5mm samples for later use.
5. The method for preparing the biomimetic nano-immune substrate material for prostate-specific antigen immunodetection according to claim 2, wherein step (S30) includes the following steps: (S301) Coat PBS solution containing antibodies onto PDMS@AgNPs@ZIF-67 biomimetic polymer material, incubate, and immobilize the captured antibodies; (S302) The substrate was washed with TBS, PBS solution and deionized water in sequence to remove unreacted antibodies, and then PBS buffer solution containing bovine serum albumin was added dropwise and reacted at room temperature; and (S303) the free BSA was washed with TBS, PBS solution and deionized water in sequence to obtain PDMS@AgNPs@ZIF-67 biomimetic immune substrate, which was stored at 4℃ for later use.
6. The method for preparing the biomimetic nano-immune substrate material for prostate-specific antigen immunodetection according to claim 5, wherein in step (S301), a PBS solution containing 0.2 mg / mL PSA antibody is coated onto 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 is added and reacted at room temperature for 1 h to block non-specific binding sites.
7. The method for preparing a biomimetic nano-immune substrate material for prostate-specific antigen immunodetection according to any one of claims 1 to 6, wherein step (S40) includes the following steps: (S401) Frozen NaBH4 was added to an aqueous solution of CTAB and HAuCl4, stirred to prepare Au seeds, and incubated at 25°C; (S402) CTAB solution was mixed with AgNO3 to prepare Au NRs growth solution; (S403) Ascorbic acid, HAuCl4 and seed incubation solution were added, stirred and allowed to stand overnight to obtain gold nanorod solution; (S404) Centrifuge the synthesized Au NRs aqueous solution to remove excess CTAB, and add MB solution to the Au centrifuged liquid, then centrifuge to remove excess MB; (S405) Dissolve the MB-modified Au NRs in PBS solution, then add the antibody, incubate at 4°C, and centrifuge to remove unbound antibody; (S406) Add PBS buffer solution containing bovine serum albumin, incubate at room temperature, centrifuge to remove excess bovine serum albumin, obtain Au NRs immune probes, dissolve in PBS aqueous solution, and store at 4°C.
8. The method for preparing biomimetic nano-immune substrate material for prostate-specific antigen immunodetection according to claim 7, wherein in step (S401), 0.4-0.8 mL of frozen 0.01 M NaBH4 is added to 20 mL of an aqueous solution containing 0.05 M CTAB and 0.25 M HAuCl4, and Au seeds are prepared under vigorous stirring; the resulting seed solution is incubated at 25°C for 2-4 h; in step (S403), 0.1 M 0.35-0.55 mL of ascorbic acid and 10 mL of... −2 Add 3-7 mL of HAuCl4 and 160 μL of incubation seed solution to M, stir, and let stand overnight to obtain gold nanorod solution; in step (S405), add 10-30 μL of PSA antibody at 0.2 mg / mL to MB-modified Au NRs solution and incubate at 4℃ for 2-4 h.
9. The preparation method of the biomimetic nano-immune substrate material for prostate-specific antigen immunodetection according to claim 8, wherein in step (S10), 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, mixed and stirred for 20-40 min, allowed to stand overnight, washed 2-4 times with ethanol, and dried at 80°C for 2-6 h to obtain ZIF-67 powder.
10. A biomimetic nano-immunoassay substrate material for prostate-specific antigen immunoassay prepared by the preparation method according to any one of claims 1 to 9, characterized in that, It includes 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 PDMS@AgNPs@ZIF-67 biomimetic polymer material.