Spine-ball-shaped zinc oxide-based SERS biological probe as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological detection and relates to an acanthosphere-shaped zinc oxide-based SERS (Surface Enhanced Raman Scattering) biological probe as well as a preparation method and application thereof. The SERS biological probe comprises acanthosphere ZnO, a dye molecular layer, a polymer layer and a targeted antibody layer, and the average particle size of the acanthosphere-shaped ZnO is 300 to 1500 nm. The preparation method of the acanthosphere-shaped ZnO comprises the following steps: dissolving zinc salt in polyethylene glycol with the molecular weight of 400-600, then adding an ethanol solution containing alkali, carrying out high-temperature reaction, collecting supernate, washing and drying to obtain the acanthosphere-shaped ZnO. According to the invention, the SERS biological probe is constructed by using ZnO in the shape of acanthosphere, so that the SERS signal intensity and SERS stability of the material are improved, the AD biomarker can be sensitively and accurately detected, and the method has huge potential in early screening and evaluation of AD.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, and relates to a spiky zinc oxide-based SERS biological probe, its preparation method, and its application. Background Technology

[0002] In today's society, with the accelerating aging of the population, the incidence of cognitive disorders, such as Alzheimer's disease and vascular dementia, is rising year by year. These diseases not only cause immense physical and mental suffering to patients, gradually leading to a loss of self-care and social abilities, but also impose a heavy economic burden and care pressure on families and society. Accurate and timely assessment of cognitive health is crucial for early detection of cognitive impairment, intervention in disease progression, and improvement of patients' quality of life.

[0003] Alzheimer's disease (AD) is an irreversible neurodegenerative disease, accounting for 60%-70% of all dementia cases and posing a major challenge to global public health. With an aging population, the prevalence of AD continues to rise, and the number of patients worldwide is projected to reach 152 million by 2050. Biomarkers of Alzheimer's disease include β-amyloid (Aβ) plaque deposition, neurofibrillary tangles (NFTs) caused by tau protein hyperphosphorylation, and neuronal synaptic dysfunction. Since changes in biomarkers begin 10-20 years before the onset of clinical symptoms, early and effective detection of Alzheimer's biomarkers is crucial for timely assessment of cognitive health, delaying the onset of the disease, and improving quality of life. Currently, early diagnosis of Alzheimer's disease primarily relies on medical imaging and cerebrospinal fluid analysis. However, the widespread application of current detection technologies is hampered by several limitations. For example, PET imaging (such as Aβ-PET and tau-PET) is expensive and relies on radioactive tracers, making it difficult to popularize; MRI can detect brain atrophy, but lacks molecular specificity and is not sensitive to early Alzheimer's disease (AD). Furthermore, while cerebrospinal fluid testing using ELISA or Simoa to detect Aβ42, Aβ40, and p-tau proteins has high specificity, it also faces challenges such as operational complexity, time consumption, high skill requirements, invasiveness, and low patient acceptance. Therefore, developing more convenient, cost-effective, and patient-friendly methods for early assessment of cognitive health and detection of Alzheimer's disease biomarkers remains a key issue.

[0004] Surface-enhanced Raman scattering (SERS), as an emerging biomolecular analysis technique, has been widely applied in materials science, analytical chemistry, food safety, and biochemical detection due to its high sensitivity, selective enhancement, in-situ detection, non-destructive nature, and unique "fingerprint" spectral characteristics. It is gradually developing into a very promising sensing and analytical tool, particularly showing significant potential for the detection of low-concentration biomarkers (such as Aβ and tau proteins). Therefore, developing a biological probe with excellent SERS performance has become an urgent problem to be solved. Summary of the Invention

[0005] To address the aforementioned problems in the prior art, the present invention aims to provide a spiky zinc oxide-based SERS biological probe, its preparation method, and its application.

[0006] One objective of this invention is achieved through the following technical solution:

[0007] A spiky zinc oxide-based SERS biological probe comprises a spiky ZnO, a dye molecular layer, a polymer layer, and a targeting antibody layer;

[0008] The spiky ZnO includes a ZnO core and a plurality of spiky protrusions extending from the surface of the ZnO core;

[0009] The average particle size of the spiky ZnO is 300–1500 nm, preferably 400–1000 nm.

[0010] A spiky ZnO sphere comprises a ZnO core, with multiple spiky protrusions extending from the surface of the ZnO core to form a spiky sphere appearance. Preferably, each spiky protrusion has a conical or needle-like morphology, and the diameter of each spiky protrusion at the end in contact with the ZnO core ranges from 5 to 100 nm, preferably from 10 to 80 nm, and the length of each spiky protrusion ranges from 40 to 600 nm, preferably from 80 to 400 nm.

[0011] Preferably, the preparation method of the spiky ZnO includes the following steps: zinc salt is dissolved in polyethylene glycol, then an ethanol solution containing alkali is added, the reaction is carried out at 90-180°C for 3-30 hours, the supernatant is collected, and the spiky ZnO is obtained by washing and drying.

[0012] The polyethylene glycol is a liquid with a molecular weight of 400 to 600, preferably 600.

[0013] Preferably, the molar ratio of zinc salt to polyethylene glycol is 1:0.1 to 2, more preferably 1:0.3 to 1, and even more preferably 1:0.5 to 0.9.

[0014] Preferably, the zinc salt is one or more of zinc nitrate, zinc chloride, zinc sulfate, and zinc acetate.

[0015] Preferably, the alkali is one or more selected from sodium hydroxide, potassium hydroxide, calcium hydroxide, and magnesium hydroxide.

[0016] Preferably, the molar ratio of zinc salt to alkali is 1:6 to 12, and more preferably 1:8 to 10.

[0017] Preferably, the reaction temperature is 100–150°C and the reaction time is 5–25 h.

[0018] The dye molecular layer is formed by dye molecules, which are Raman signal molecules and have signal characteristics in Raman spectroscopy analysis. Preferably, the dye molecules include one or more of the following: rhodamine dyes (such as rhodamine 6G, rhodamine B, rhodamine 123, rhodamine 110), cyanine dyes (such as Cy3, Cy5, Cy7, indocyanine green (ICG)), thiol organic molecules (such as p-mercaptobenzoic acid, 4-mercaptobenzonitrile), azo dyes (such as direct blue 86, acid orange 7, methyl orange), basic dyes (such as methylene blue, crystal violet, basic fuchsin), and cyanide complexes (such as Prussian blue, Prussian blue analogues).

[0019] Preferably, the polymer layer is formed of a polymer compound, which is one or more of polydopamine, bovine serum albumin, and reduced bovine serum albumin.

[0020] Preferably, the targeting antibody layer is formed from AD biomarker antibodies, wherein the AD biomarker antibodies are one or more of anti-Aβ1-42 antibody, anti-total tau protein antibody, and anti-neural light filament chain antibody.

[0021] The dye molecular layer is formed by dye molecules coating the surface of the spiky ZnO, the polymer layer is formed by polymer compounds coating the surface of the dye molecular layer, and the targeting antibody layer is formed by AD biomarker antibodies coupled to the surface of the polymer layer.

[0022] The second objective of this invention is achieved through the following technical solution:

[0023] A method for preparing a spiky, zinc oxide-based SERS biological probe includes the following steps:

[0024] S1, zinc salt is dissolved in polyethylene glycol, and then an ethanol solution containing alkali is added. The mixture is reacted at 90-180°C for 3-30 hours. The supernatant is collected, washed, and dried to obtain spiky ZnO. The polyethylene glycol is a liquid with a molecular weight of 400-600.

[0025] S2. The spiky ZnO was incubated with dye molecules. After incubation, the mixture was centrifuged and washed to obtain a layer of spiky ZnO-dye molecules.

[0026] S3. The spiky ZnO-dye molecular layer and the compound that forms the polymer layer are stirred and reacted. After the stirring reaction is completed, the mixture is centrifuged and washed to obtain the spiky ZnO-dye molecular layer-polymer layer.

[0027] S4. The spiked ZnO-dye molecular layer-polymer layer was incubated with AD biomarker antibody. After incubation, the spiked ZnO-based SERS bioprobe was obtained by centrifugation and washing.

[0028] In steps S2-S4, the solid raw materials need to be dispersed or dissolved in a solvent to form a suspension or solution before the reaction. For example, in step S2, the spiky ZnO is dispersed in a solvent such as ethanol or water to form a suspension, and the dye molecules are dissolved in a solvent such as ethanol or water to form a solution.

[0029] Preferably, in step S2, the incubation is carried out at 10–45°C for 3–12 hours.

[0030] Preferably, the stirring speed of the stirring reaction in step S3 is 100-1000 rpm, and the reaction time is 0.5-3 hours.

[0031] The compounds that form the polymer layer are one or more of dopamine hydrochloride, bovine serum albumin, and reduced bovine serum albumin.

[0032] When the polymer layer is polydopamine, the compound that forms the polymer layer is dopamine hydrochloride. Dopamine hydrochloride forms a polymer layer on the spiky ZnO-dye molecular layer under alkaline conditions.

[0033] Preferably, in step S4, the incubation with the AD biomarker antibody is carried out at 3–8°C for 3–12 hours.

[0034] The third objective of this invention is achieved through the following technical solution:

[0035] Application of a spiky zinc oxide-based SERS bioprobe in the detection of AD biomarkers not for diagnostic purposes.

[0036] Preferably, the application includes the following steps:

[0037] 1) Preparation of standard curve

[0038] AD biomarkers with different concentration gradients were prepared, and AD biomarker solutions of different concentrations were co-incubated with spiky zinc oxide-based SERS bioprobes. After incubation, the products were collected by centrifugation, dropped onto a silicon wafer, and Raman spectral data were collected. The SERS signal intensity with high SERS enhancement Raman shift was selected as the ordinate and the biomarker concentration as the abscissa to obtain a standard curve and a linear equation.

[0039] 2) Sample testing

[0040] The sample was co-incubated with a spiky zinc oxide-based SERS bioprobe. After incubation, the product was collected by centrifugation, dropped onto a silicon wafer, and Raman spectral data were acquired. The SERS signal intensity at the same Raman shift was obtained, and the concentration of AD biomarkers in the sample was calculated based on a linear equation.

[0041] AD biomarkers are one or more of Aβ1-42, total tau protein, and neural light filament chains. Aβ1-42 is preferred.

[0042] Raman spectral data are available in the spectral range of 600–1800 cm⁻¹. -1 SERS spectral data within.

[0043] The Raman shift enhanced by high SERS is 1350–1400 cm. -1 1610~1640cm -1 A certain displacement in.

[0044] In the aforementioned application, the detection limit of the spiky zinc oxide-based SERS bioprobe for AD biomarkers is ≥10. -12 mol / L.

[0045] The test is not intended for disease diagnosis. For example, it can be used to assess the cognitive health of the brain. By detecting the concentration of AD biomarkers in the blood, the cognitive health of the brain can be accurately and timely assessed, which is beneficial for early detection of cognitive impairment, intervention in disease progression, and improvement of patients' quality of life.

[0046] Compared with the prior art, the present invention has the following beneficial effects:

[0047] 1. By controlling the characteristic parameters of polyethylene glycol and the ratio of zinc salt, polyethylene glycol and alkali during the preparation process, this invention successfully prepared a ZnO with an average particle size of 300-1500 nm and a spiky spherical morphology.

[0048] 2. This invention prepares ZnO with a spiky spheroid morphology. After the formation of the spiky spheroid ZnO, it combines with dye molecules, polymer compounds, and AD antibodies to construct a zinc oxide-based SERS biological probe. It has a high specific surface area, which enhances the photoinduced charge transfer effect of the probe and promotes the improvement of the material's SERS performance, especially the SERS stability. This is of great significance for improving the accuracy of AD marker detection.

[0049] 3. The zinc oxide-based SERS bioprobe of the present invention detects AD biomarkers at concentration gradients and constructs a curve relating SERS signal intensity to AD biomarker concentration, thereby achieving semi-quantitative detection of AD biomarkers.

[0050] 4. The detection limit of the spiky zinc oxide-based SERS bioprobe of this invention for AD biomarkers is as low as 10. - 12 It has a very high sensitivity at mol / L.

[0051] 5. This invention utilizes zinc oxide-based biological probes to establish a relationship curve between SERS signal intensity and AD biomarker concentration, which can effectively detect AD biomarkers and is beneficial for detecting AD biomarkers in complex biological samples. It has great potential in the early screening and assessment of AD. Attached Figure Description

[0052] Figure 1 SEM image of the spiky ZnO prepared in Example 1;

[0053] Figure 2 HAADF image of the spiky ZnO prepared in Example 1;

[0054] Figure 3 SEM image of the spiky ZnO prepared in Example 2;

[0055] Figure 4 SEM image of ZnO prepared in Comparative Example 1;

[0056] Figure 5 SEM image of ZnO prepared in Comparative Example 2;

[0057] Figure 6 This is a comparison of the Raman spectra of ZnO from Example 1 and commercially available ZnO;

[0058] Figure 7 The Raman spectra of ZnO from Example 1 and ZnO from Comparative Example 2 are compared.

[0059] Figure 8 The images show the SERS spectra of ZnO-MB, where (A) represents the SERS spectra at different MB concentrations, and (B) represents 10 SERS spectra at MB concentrations of 10... -6 The SERS spectra of M, (C) show the relative standard deviation distribution of 10 spectra at the characteristic peak of 1393, and (D) show the distribution of the relative standard deviation of 10 spectra at the characteristic peak of 1622 cm⁻¹. -1 The distribution of relative standard deviation at a given location;

[0060] Figure 9 The image shows the results of the fluorescence colocalization experiment of the zinc oxide-based SERS bioprobe for the specific adsorption of Aβ1-42 in Example 4, where A: ten fluorescence display areas under a laser confocal microscope, and B: Raman spectral data collected from the fluorescence display areas.

[0061] Figure 10To construct a curve showing the relationship between SERS signal intensity and AD biomarker concentration, where A represents the detection limit of the zinc oxide-based SERS bioprobe for Aβ1-42, and B represents the relationship between SERS signal intensity and AD biomarker concentration. Detailed Implementation

[0062] The technical solution of the present invention will be further described and illustrated below with reference to specific embodiments and accompanying drawings. It should be understood that the specific embodiments described herein are only for the purpose of helping to understand the present invention and are not intended to limit the specific scope of the present invention. Furthermore, the accompanying drawings used herein are merely for better illustrating the content disclosed in the present invention and do not limit the scope of protection. Unless otherwise specified, the raw materials used in the embodiments of the present invention are all commonly used in the art, and the methods used in the embodiments are all conventional methods in the art.

[0063] The following raw materials are used in the following embodiments:

[0064] Aβ1-42 was purchased from Suzhou Modifu Company. It appears as a white, flocculent lyophilized powder with a molecular weight of 4500–4600 Da.

[0065] Polyethylene glycol 600 was purchased from Maclean's Reagent Company. It is a transparent liquid with a molecular weight of 600 and a density of 1.13 g / cm³. 3 ;

[0066] Polyethylene glycol 200, polyethylene glycol 400, and polyethylene glycol 4000 were purchased from Maclean's Reagent Company.

[0067] Commercially available ZnO was purchased from Maclean's Reagents Company, with a particle size of 200 nm.

[0068] Example 1

[0069] This embodiment prepares spiky zinc oxide, which is obtained by the following steps:

[0070] Weigh 2975 mg Zn(NO3)2·6H2O and dissolve it in 4 mL of PEG 600 solution. Then weigh 4 g NaOH and dissolve it in 50 mL of anhydrous ethanol. Add the two solutions to a hydrothermal reactor and react at 120 °C for 12 hours. Collect the supernatant and wash it repeatedly with deionized water and anhydrous ethanol to remove residual polymer and inorganic ions. Dry the resulting ethanol suspension in an oven at 50 °C for 3 hours to obtain spiky ZnO.

[0071] Figure 1The SEM image of the spiky ZnO prepared in Example 1 shows that the obtained ZnO has a spiky morphology with an average particle size of 850 nm. "Particle size" refers to the maximum distance between any two points on the particle's outline observed using SEM equipment; the "average particle size" value is calculated using the SEM equipment as the average particle size observed in ten fields of view.

[0072] like Figure 2 As shown, the spiky ZnO comprises a ZnO core, with multiple spiky protrusions extending from the surface of the ZnO core to form a spiky appearance. Each spiky protrusion has a needle-like morphology, and the diameter of each spiky protrusion at the end in contact with the ZnO core ranges from 5 to 100 nm, while the length of each spiky protrusion ranges from 99 to 400 nm.

[0073] Example 2

[0074] This embodiment prepares spiky zinc oxide, which is obtained by the following steps:

[0075] Weigh 2975 mg Zn(NO3)2·6H2O and dissolve it in 4 mL of PEG 400 solution. Then weigh 4 g NaOH and dissolve it in 50 mL of anhydrous ethanol. Add the two solutions to a hydrothermal reactor and react at 120 °C for 12 hours. Collect the supernatant and wash it repeatedly with deionized water and anhydrous ethanol to remove residual polymer and inorganic ions. Dry the resulting ethanol suspension in an oven at 50 °C for 3 hours to obtain spiky ZnO.

[0076] Figure 3 The SEM image of the spiky ZnO prepared in Example 2 shows that the obtained ZnO has a spiky morphology. However, the number of spiky protrusions in this ZnO is relatively small, and the size distribution is wide at high magnification, indicating poor uniformity.

[0077] Comparative Example 1

[0078] Zinc oxide in Comparative Example 1 was prepared by the following steps:

[0079] Weigh 2975 mg of Zn(NO3)2·6H2O and dissolve it in 4 mL of PEG 200 solution. Then weigh 4 g of NaOH and dissolve it in 50 mL of anhydrous ethanol. Add the two solutions to a hydrothermal reactor and react at 120 °C for 12 hours. Collect the supernatant and wash it repeatedly with deionized water and anhydrous ethanol to remove residual polymer and inorganic ions. Dry the resulting ethanol suspension in an oven at 50 °C for 3 hours to obtain ZnO.

[0080] Figure 4The image shows a SEM image of the ZnO prepared in Comparative Example 1. This ZnO exhibits a small number of spikes, and the spikes are short in length, failing to form a structure resembling... Figure 1 The needle-like tip shown.

[0081] Comparative Example 2

[0082] Zinc oxide in Comparative Example 2 was prepared by the following steps:

[0083] Weigh 4000 mg of PEG 4000 and dissolve it in 4 mL of anhydrous ethanol. Weigh 2975 mg of Zn(NO3)2·6H2O and dissolve it in 4 mL of PEG 4000 solution. Then weigh 4 g of NaOH and dissolve it in 50 mL of anhydrous ethanol. Add the above two solutions to a hydrothermal reactor and react at 120 °C for 12 hours. Collect the supernatant and wash it repeatedly with deionized water and anhydrous ethanol to remove residual polymer and inorganic ions. Dry the resulting ethanol suspension in an oven at 50 °C for 3 hours to obtain ZnO.

[0084] Figure 5 The image shows a SEM image of ZnO prepared in Comparative Example 2. The average particle size of ZnO is relatively large, at 1.3 μm, and the spiky protrusions of this ZnO are not obvious and the length of the spiky protrusions is relatively short.

[0085] Example 3: SERS detection performance of spiky ZnO for Raman signal molecules

[0086] 2 mg of commercially available ZnO, ZnO from Example 1, and ZnO from Comparative Example 2 were dispersed in 10 mL of anhydrous ethanol to obtain ZnO suspensions; methylene blue (MB) powder, a Raman signaling molecule, was dissolved in ethanol to form 2 × 10⁻⁶ ppm. -3 2 mL of MB ethanol solution and 2 mL of ZnO ethanol suspension were incubated in the dark at 25 °C for 8 hours to form MB-coated ZnO. The precipitate was then collected and repeatedly washed with anhydrous ethanol to remove excess MB, resulting in an ethanol suspension. The suspension was then dropped onto a clean silicon wafer (5 × 5 mm) using a micropipette and dried at room temperature before Raman detection. The SERS spectrum was collected by a confocal micro Raman spectrometer under excitation light at 633 nm.

[0087] Figure 6 This is a comparison of the Raman spectra of ZnO from Example 1 and commercially available ZnO. Figure 7 The image shows a comparison of the Raman spectra of ZnO from Example 1 and ZnO from Comparative Example 2. It can be seen that the spiky ZnO prepared in Example 1 has a stronger Raman signal intensity, while the ZnO prepared from polyethylene glycol with a larger molecular weight in Comparative Example 2 has a larger particle size and less obvious protrusions, resulting in a weaker Raman signal.

[0088] Methylene blue (MB) powder, a Raman signaling molecule, was dissolved in ethanol and diluted to different concentrations. The resulting solutions were incubated with the ZnO ethanol suspension formed in Example 1 in the dark at 25°C for 8 hours to form ZnO-MB. The precipitate was then collected and repeatedly washed with anhydrous ethanol to remove excess MB, resulting in an ethanol suspension. The suspension was then dropped onto a clean silicon wafer (5×5 mm) using a micropipette and dried at room temperature before Raman detection. The SERS spectrum was collected by a confocal micro Raman spectrometer under excitation light at 633 nm.

[0089] from Figure 8 As shown in (A), the LOD value of the spiky ZnO substrate for SERS detection of MB reached 10. -9 The lowest concentration of ZnO-MB was determined. To verify the reproducibility of the SERS spectra, the concentrations at 10 mol / L were tested. -6 The relative standard deviation (RSD) of SERS intensity in a mol / L Raman spectrum is an indicator used to describe the dispersion of statistical data. A smaller RSD value means smaller differences between data points, and vice versa. Therefore, we calculate the RSD value to evaluate the stability of SERS spectra. The formula for calculating the RSD value is as follows: RSD = σ / μ, where σ is the standard deviation of SERS intensity, and μ is the average SERS intensity. Figure 8 As can be seen from (B), (C), and (D), the 10 spectra shown have characteristic peaks at 1393 and 1622 cm⁻¹. -1 The sample exhibited good stability, with relative standard deviations of intensity of 13.35% and 12.57%. These results indicate that the spiky ZnO possesses good SERS stability and sensitivity.

[0090] Example 4: Preparation of zinc oxide-based SERS biological probes

[0091] 2 mg of ZnO from Example 1 was dispersed in 10 mL of anhydrous ethanol to obtain a ZnO suspension; methylene blue powder was dissolved in ethanol to form a 2 × 10⁻⁶ solution. -3A mol / L solution was used to incubate 2 mL of MB ethanol solution and 2 mL of ZnO ethanol suspension in the dark at 25 °C for 8 hours to form ZnO-MB. The precipitate was then collected and repeatedly washed with anhydrous ethanol to remove excess MB, yielding 10 mL of ethanol suspension. 5 mL of the ZnO-MB ethanol suspension was added to a mixture of 13 mL of deionized water and 8 mL of anhydrous ethanol, followed by the addition of 50 μL of ammonia. The mixture was stirred at 200 rpm for 10 minutes, then 2 mL of 5 mg / mL dopamine hydrochloride solution was added. The mixture was stirred at 200 rpm for 1.5 hours until the solution turned light black, indicating the formation of polydopamine (PDA). The reaction solution was then centrifuged and washed with anhydrous ethanol to obtain 5 mL of ZnO-MB-PDA ethanol suspension. Take 1 mL of ZnO-MB-PDA ethanol suspension and add 1 mL of Tris-HCl solution. After mixing well, add 5 μL of Aβ1-42 antibody and incubate in a shaker at 90 rpm and 5 °C for 8 hours. Centrifuge the reaction solution and wash with Tris-HCl solution to remove excess Aβ1-42 antibody to obtain 5 mL of zinc oxide-based SERS biological probe solution.

[0092] Example 5: Verification of the specific adsorption of zinc oxide-based SERS bioprobes for Aβ1-42

[0093] 10 μL of fluorescent Aβ1-42 (1 mg / mL) was added to 1 mL of the zinc oxide-based SERS bioprobe solution from Example 4. The solution was incubated for 8 hours at 90 rpm and 5°C in a shaker. After incubation, the supernatant was removed by centrifugation, and the precipitate was spread onto a confocal microscopy panel. The fluorescence display area was observed and photographed using a laser confocal microscope, and Raman spectroscopy data were acquired in the fluorescence display area using a laser confocal Raman spectrometer. Figure 9 (A) shows the fluorescence display area observed by laser confocal microscopy, with ten points selected for imaging. Figure 9 (B) shows the Raman spectral data acquired in the fluorescence display area.

[0094] from Figure 9 (B) shows that the ten spectra exhibit good repeatability, and the fluorescence colocalization experiment demonstrates that the zinc oxide-based SERS bioprobe can specifically adsorb Aβ1-42.

[0095] Example 6: Constructing the relationship curve between SERS signal intensity and AD biomarker concentration

[0096] Configured concentration gradient of AD biomarker Aβ1-42 (10 -4 ~10 -12M), 100 μL of Aβ1-42 solution and 100 μL of zinc oxide-based SERS bioprobe solution from Example 4 were incubated in a shaker at 90 rpm and 5 °C for 8 hours. After incubation, excess unbound Aβ1-42 was removed by centrifugation, and the precipitate was spread on a clean silicon wafer. Raman spectral data were collected as experimental group data. Blank SERS bioprobes were dropped onto a clean silicon wafer, and Raman spectral data were collected as blank group data.

[0097] Select Raman characteristic peak (1622 cm⁻¹) -1 As a quantitative indicator, baseline correction was performed using Raman spectral data of the blank probe to eliminate background interference; subsequently, the Raman spectral data of each concentration sample at 1622 cm⁻¹ were calculated. -1 The peak intensity at the specified location was determined, and finally, a linear regression analysis was performed with Aβ1-42 concentration (log X) as the x-axis and SERS signal intensity (I) as the y-axis. The fitted linear equation was (I = 1503logX + 57, R0). 2 =0.985), such as Figure 10 As shown, this equation can be used to calculate the concentration of unknown samples and determine the detection limit of the method to be up to 10. -12 M.

[0098] All aspects, embodiments, and features of this invention should be considered illustrative in all respects and not limiting of the invention; the scope of the invention is defined only by the claims. Other embodiments, modifications, and uses will become apparent to those skilled in the art without departing from the spirit and scope of the invention as claimed.

[0099] In the preparation method of this invention, the order of the steps is not limited to the listed order. For those skilled in the art, variations in the order of the steps without creative effort are also within the scope of protection of this invention. Furthermore, two or more steps or actions can be performed simultaneously.

[0100] Finally, it should be noted that the specific embodiments described herein are merely illustrative examples of the invention and are not intended to limit the implementation of the invention. Those skilled in the art can make various modifications or additions to the described specific embodiments or use similar methods to replace them; it is neither necessary nor possible to exemplify all embodiments here. However, these obvious variations or modifications derived from the essential spirit of the invention still fall within the scope of protection of the invention, and interpreting them as any additional limitation would contradict the spirit of the invention.

Claims

1. A spiky, zinc oxide-based SERS biological probe, characterized in that, It includes spiky ZnO, a dye molecular layer, a polymer layer, and a targeting antibody layer; The spiky ZnO includes a ZnO core and a plurality of spiky protrusions extending from the surface of the ZnO core; The average particle size of the spiky ZnO is 300–1500 nm.

2. The spiky zinc oxide-based SERS biological probe according to claim 1, characterized in that, The preparation method of the spiky ZnO includes the following steps: zinc salt is dissolved in polyethylene glycol, then an ethanol solution containing alkali is added, the reaction is carried out at 90-180℃ for 3-30 hours, the supernatant is collected, and the spiky ZnO is obtained by washing and drying. The polyethylene glycol is a liquid with a molecular weight of 400-600.

3. The spiky zinc oxide-based SERS biological probe according to claim 1, characterized in that, The molar ratio of zinc salt to polyethylene glycol is 1:0.1 to 2; And / or, the zinc salt is one or more of zinc nitrate, zinc chloride, zinc sulfate, and zinc acetate; And / or, the base is one or more of sodium hydroxide, potassium hydroxide, calcium hydroxide, and magnesium hydroxide; And / or, the molar ratio of zinc salt to alkali is 1:6 to 12; And / or, the reaction temperature is 100–150℃, and the time is 5–25h.

4. The spiky zinc oxide-based SERS biological probe according to claim 1, characterized in that, The molecular weight of the polyethylene glycol is 600; The molar ratio of zinc salt to polyethylene glycol is 1:0.3 to 1.

5. The spiky zinc oxide-based SERS biological probe according to claim 1, characterized in that, The dye molecule layer is formed by dye molecules, which are Raman signal molecules; And / or, the polymer layer is formed of a polymer compound, which is one or more of polydopamine, bovine serum albumin, and reduced bovine serum albumin; And / or, the targeting antibody layer is formed from AD biomarker antibodies, wherein the AD biomarker antibodies are one or more of anti-Aβ1-42 antibody, anti-total tau protein antibody, and anti-neural light filament chain antibody.

6. A method for preparing a spiky, zinc oxide-based SERS biological probe, characterized in that, Includes the following steps: S1, zinc salt is dissolved in polyethylene glycol, and then an ethanol solution containing alkali is added. The mixture is reacted at 90-180°C for 3-30 hours. The supernatant is collected, washed, and dried to obtain spiky ZnO. The polyethylene glycol is a liquid with a molecular weight of 400-600. S2. The spiky ZnO was incubated with dye molecules. After incubation, the mixture was centrifuged and washed to obtain a layer of spiky ZnO-dye molecules. S3. The spiky ZnO-dye molecular layer and the compound that forms the polymer layer are stirred and reacted. After the stirring reaction is completed, the mixture is centrifuged and washed to obtain the spiky ZnO-dye molecular layer-polymer layer. S4. The spiked ZnO-dye molecular layer-polymer layer was incubated with AD biomarker antibody. After incubation, the spiked ZnO-based SERS bioprobe was obtained by centrifugation and washing.

7. The preparation method according to claim 6, characterized in that, In step S2, the incubation is carried out at 10–45°C for 3–12 hours. And / or, the stirring speed of the stirring reaction in step S3 is 100-1000 rpm, and the reaction time is 0.5-3 hours; And / or, in step S4, incubation with AD biomarker antibodies is performed at 3–8°C for 3–12 hours.

8. The preparation method according to claim 6, characterized in that, The compounds that form the polymer layer are one or more of dopamine hydrochloride, bovine serum albumin, and reduced bovine serum albumin; When the polymer layer is polydopamine, the compound that forms the polymer layer is dopamine hydrochloride. Dopamine hydrochloride forms a polymer layer on the spiky ZnO-dye molecular layer under alkaline conditions.

9. The application of a spiky zinc oxide-based SERS bioprobe as described in any one of claims 1-5, or a spiky zinc oxide-based SERS bioprobe prepared by any one of claims 6-8, in the detection of AD biomarkers not for diagnostic purposes.

10. The application according to claim 9, characterized in that, The detection limit of the spiky zinc oxide-based SERS bioprobe for AD biomarkers is ≥10. -12 mol / L.

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