Preparation method and application of nano-enzyme SERS sensor for detecting diabetic retinopathy

Abstract

The invention relates to the technical field of biomedical engineering, and discloses a preparation method of a nano-enzyme SERS (Surface Enhanced Raman Scattering) sensor for detecting diabetic retinopathy, which comprises the following steps: (1) synthesizing a bimetallic gold-palladium nanorod; (2) assembling a gold trioctahedron array; and (3) modifying the surface of the gold-palladium nanorod synthesized in the step (1) with single-stranded DNA1 to prepare a nano-enzyme probe, hybridizing an aptamer of a vascular endothelial growth factor (VEGF) with single-stranded DNA2, modifying the surface of the gold trioctahedron array assembled in the step (2) with the hybridized aptamer, preparing a capture substrate, and constructing the nano-enzyme SERS sensor by the nano-enzyme probe and the capture substrate. In the detection process, the VEGF triggers a competitive recognition mechanism to promote the nano-enzyme probe to be captured by the capture substrate and catalyze tetramethyl benzidine (TMB) to generate oxTMB with a strong SERS signal. The sensor has the advantages of high sensitivity, strong specificity and good accuracy, and can realize rapid quantitative detection of DR biomarkers in serum.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical engineering, and specifically provides a preparation method and application of a nanozyme SERS sensor for detecting diabetic retinopathy. Background Art

[0002] Diabetic retinopathy (DR) is a common and serious ocular complication of diabetes and the main cause of visual impairment in diabetic patients. The global prevalence of diabetes is approximately 10%, and the prevalence of DR is as high as 22%. In the early stage of DR, patients usually have no obvious clinical symptoms, which makes early diagnosis rely on systematic screening. However, currently commonly used diagnostic techniques in clinical practice, such as funduscopy and optical coherence tomography (OCT), are difficult to carry out screening work in large-scale populations due to high time and economic costs.

[0003] With the in-depth research, biomarkers have provided a new approach for DR screening. Vascular endothelial growth factor (VEGF) has been confirmed as a key protein biomarker for DR diagnosis. Its level will be significantly up-regulated in a hyperglycemic and hypoxic environment and is involved in the characteristic pathological changes of DR. However, the content of biomarkers in body fluids such as serum is extremely low, and there are a large number of interfering components, which pose high requirements for the sensitivity and specificity of detection techniques.

[0004] Traditional detection methods are difficult to meet the requirements. Although surface-enhanced Raman scattering (SERS) technology has potential, it has high requirements for the substrate material. At the same time, although noble metal nanozyme materials have various enzyme mimicking activities, their individual use also has limitations. Therefore, it is urgent to develop a method for efficiently detecting DR biomarkers. Summary of the Invention

[0005] (I) Technical Problems to be Solved

[0006] In view of the deficiencies of the prior art, the present invention provides a preparation method and application of a nanozyme SERS sensor for detecting diabetic retinopathy, and solves the problems raised in the above background art.

[0007] (II) Technical Solutions

[0008] To achieve the above object, the present invention provides the following technical solutions: A preparation method of a nanozyme SERS sensor for detecting diabetic retinopathy, comprising the following steps:

[0009] Step 1: Synthesize bimetallic gold-palladium nanorods (Au@PdNRs) by the seed growth method;

[0010] Step 2: Synthesize gold octahedra (AuTOHs) by the seed growth method, and form a neatly arranged and compact AuTOHs array through self-assembly at the oil-water interface;

[0011] Step 3: Modify single-stranded DNA 1 (ssDNA1) on the surface of the Au@PdNRs synthesized in Step 1 to prepare a nanozyme probe. Hybridize the aptamer of vascular endothelial growth factor (VEGF) with single-stranded DNA 2 (ssDNA2) and then modify it on the surface of the AuTOHs array assembled in Step 2 to prepare a capture substrate. The nanozyme probe and the capture substrate jointly construct a nanozyme SERS sensor.

[0012] Preferably, the specific implementation of Step 1 is as follows:

[0013] (1) First, mix 0.196 mL of chloroauric acid (HAuCl4) solution (25.4 mM) with 9.78 mL of deionized water. Subsequently, add 9.9 mL of cetyltrimethylammonium bromide (CTAB) solution (0.2 M) and mix well. Then, take 2 mL of freshly prepared sodium borohydride (NaBH4) solution (0.0055 M) and inject it into the above mixed solution under stirring at 1100 rpm. Stir continuously for 120 seconds, and the solution color changes from yellow to light brown. Let it stand for 1 hour to complete the preparation of gold seeds. Subsequently, take 50 mL of deionized water preheated to 50 °C, dissolve CTAB (0.14 M) and sodium oleate (0.026 M). After the solution cools to 30 °C, add 500 mL of silver nitrate (AgNO3) solution (3.95 mM), 500 mL of HAuCl4 solution (1.0 mM), 4.2 mL of 37% hydrochloric acid solution, and 2.5 mL of ascorbic acid (AA) solution (12 mM) in sequence. Under stirring at 800 rpm, react for 30 minutes. Finally, centrifuge the product at 9000 rpm for 15 minutes, and disperse the precipitate in a 7 mM CTAB solution to obtain gold nanorods (AuNRs).

[0014] (2) Synthesize Au@PdNRs by the seed growth method. Dissolve 0.0354 g of PdCl2 powder in 2.4 mL of HCl (0.2 M), continuously dissolve it in a 60 °C water bath for 60 minutes, and further dilute it with deionized water to 20 mL to prepare a chloropalladic acid (H2PdCl4) (10 mM) solution. Add 4 mL of H2PdCl4 (10 mM) solution and 100 mM AA to 70 mL of CTAB (7 mM), then add 80 mL of deionized water, and mix under vigorous stirring for 5 minutes to prepare the growth solution. Then, add the AuNRs solution (seed) prepared in Step (1) to the above growth solution, and continuously stir at 25 °C for 3 hours to complete the growth.

[0015] Preferably, the volume ratio of the AuNRs solution to the growth solution in Step (2) is 1:10.

[0016] Preferably, the specific implementation of Step 2 is as follows:

[0017] (a) AuTOHs were synthesized by the seed growth method. First, 19.5 mL of CTAB (0.1 M) solution, 0.3 mL of HAuCl4 (0.01 M), and 0.2 mL of H2PtCl6 (0.1 M) were uniformly mixed, and 1.8 mL of freshly prepared ice-cold NaBH4 (0.01 M) solution was added. The mixture was stirred at a low speed of 300 rpm until the solution turned light brown and a small amount of foam was generated. 40 μL of the reaction solution was mixed with 20 mL of deionized water to obtain a seed dilution solution for the next step. The seed dilution solution must be used within 30 minutes to avoid crystallization. Subsequently, 1 mL of HAuCl4 (0.01 M), 10 μL of H2PtCl6 (0.01 M), and 0.5 mL of AA (0.1 M) solution were successively added to 10 mL of cetyltrimethylammonium chloride (CTAC) (25 mM) solution and mixed well. Then, a certain volume of the seed dilution solution was added. After gently shaking and mixing, the mixture was placed in an oven at 45 °C and allowed to react statically for 7 minutes. The solution finally turned dark red, and AuTOHs were obtained.

[0018] (b) The AuTOHs solution prepared in step (a) was mixed with n-hexane to create an oil-water liquid-liquid interface. An equal volume of absolute ethanol was quickly added to the reaction system. The absolute ethanol drove the self-assembly of nanoparticles at the oil-water interface to form a closely arranged monolayer AuTOHs array. After about 2 minutes, a clean silicon wafer hydrophilized with piranha solution was contacted with the array parallelly and slowly. The monolayer AuTOHs array was transferred to the surface of the silicon wafer and dried in a desiccator for 15 minutes to obtain the AuTOHs array.

[0019] Preferably, in step (a), the volume of the seed dilution solution is 1 mL, and in step (b), the volume ratio of the AuTOHs solution to n-hexane is 1:1.

[0020] Preferably, the specific implementation manner of step three is:

[0021] (A) First, ssDNA1 (0.1 mM) activated by 40 mL of TCEP was added to 1 mL of the Au@PdNRs solution prepared in step one, and the mixture was incubated at 37 °C. ssDNA1 was modified onto the surface of Au@PdNRs through the thiol group at the 5ˊ end. Then, 15 μL of 1 wt% bovine serum albumin (BSA) solution was added to the mixture to block the non-specific binding sites on the particle surface, and the reaction continued for 1.5 hours. Finally, the product was centrifuged at a speed of 12,000 rpm for 7 minutes to remove the excess reagents and nucleic acid chains, thereby obtaining a nanozyme probe (ssDNA1-functionalized Au@PdNRs). The nucleotide sequence of ssDNA1 is 5ˊ-SH-AATCTACCCGGCCCGT-3ˊ.

[0022] (B) Pre-hybridize ssDNA2 and aptamer with phosphate buffer (8 mM) containing magnesium chloride (MgCl2) (0.8 mM) and sodium chloride (NaCl) (20 mM). After incubating for 1.5 hours, mix 800 μL of ssDNA2 (0.1 mM) with 80 μL of TCEP (1 M) solution and react at room temperature for 60 minutes to activate the terminal thiol groups of the nucleic acid duplex. Drop the activated nucleic acid duplex onto the surface of the Au TOHs array and incubate at 37 °C. The nucleic acid duplex is modified onto the surface of the AuTOHs array through the 5ˊ-terminal thiol group of ssDNA2. Finally, wash repeatedly with phosphate PBS buffer to finally obtain the capture substrate (Au TOHs array functionalized with ssDNA2 and aptamer). The nucleotide sequences of the ssDNA2 and aptamer are 5ˊ-SH-AAACGGGCCGGGTAGA-3ˊ and 5ˊ-TTTTTTTTTTGTGGGGGTGGACGGG CCGGGTAGA-3ˊ.

[0023] (C) Drop 200 mL of the nanozyme probe prepared in step (A) onto the capture substrate assembled in step (B) to construct a nanozyme SERS sensor for detecting DR biomarkers.

[0024] Preferably, the optimal incubation time for Au@PdNRs and TCEP-activated ssDNA1 in step (A) at 37 °C is 2 hours to allow ssDNA1 to fully bind to Au@PdNRs. In step (B), when the activated nucleic acid duplex is dropped onto the surface of the Au TOHs array, the optimal incubation time at 37 °C is 2 hours to allow the duplex to fully bind to the Au TOHs array.

[0025] Preferably, based on the preparation method of the nanozyme SERS sensor for detecting diabetic retinopathy, a nanozyme SERS sensor for detecting diabetic retinopathy is prepared.

[0026] The present invention further provides the application of the nanozyme SERS sensor for detecting diabetic retinopathy, including the following steps:

[0027] The first step: Add VEGF to the serum to prepare VEGF solutions with different concentrations. Drop 200 mL of the VEGF solutions with different concentrations onto the nanozyme SERS sensor for detecting DR biomarkers prepared as claimed in claim 8, perform a hybridization reaction at a specific incubation temperature, and wash away the excess nanozyme probe with PBS buffer. Then, adjust a mixture of 500 mL of TMB and H2O2 with sodium acetate buffer and drop it onto the nanozyme SERS sensor, and perform SERS detection at room temperature to obtain the signal of oxTMB; according to oxTMB at 1062 cm -1Perform a fitting curve between the logarithm of the VEGF concentration and the SERS signal intensity at the characteristic peak;

[0028] The second step: Collect 30 samples of healthy human serum and DR patient serum each. Drop 200 mL of the clinical sample onto the nanozyme SERS sensor for detecting DR biomarkers prepared as claimed in claim 8, and carry out a hybridization reaction at a specific incubation temperature. Wash away the excess nanozyme probes with PBS buffer. Then, adjust a mixture of 500 mL of TMB and H2O2 with sodium acetate buffer and drop it onto the platform, and perform SERS testing at room temperature.

[0029] The third step: Substitute the intensity of the characteristic peak at 1062 cm -1 obtained from the detection of the clinical sample in the second step into the working curve determined in the first step to measure the concentration of VEGF in the clinical sample.

[0030] Preferably, in the first step, the optimal incubation temperature is 30 °C, the optimal pH is 4.5, the optimal incubation time is 14 minutes, and the optimal concentrations of TMB and H2O2 dropped onto the surface of the nanozyme SERS sensor are 1.0 mM and 0.6 mM respectively.

[0031] (III) Beneficial effects

[0032] Compared with the prior art, the present invention provides a preparation method and application of a nanozyme SERS sensor for detecting diabetic retinopathy, having the following beneficial effects:

[0033] 1. The preparation method and application of the nanozyme SERS sensor for detecting diabetic retinopathy, through the successfully prepared nanozyme SERS sensor, combine the peroxidase (POD) catalytic activity and SERS enhancement ability of Au@PdNRs, and the high electromagnetic field "hot spot" characteristics of the AuTOHs array, realizing ultrasensitive and highly specific detection of VEGF in serum. The detection limit is as low as 0.11 pg / mL, and the detection time is shortened to 14 minutes, greatly improving the detection efficiency. In a complex serum environment, the detection results are highly consistent with ELISA, showing good clinical applicability, and are expected to be widely used in the early screening of DR, providing an economical and convenient new method for the diagnosis of DR in a large population, and having important clinical significance. Description of the drawings

[0034] Figure 1 A, B, and C shown in the figure are respectively the schematic diagram of the preparation and functionalization of Au@PdNRs of the present invention, the schematic diagram of the assembly and functionalization of Au TOHs, and the schematic diagram of the nanozyme SERS sensor for detecting DR biomarkers;

[0035] Figure 2This is the transmission electron microscope (TEM) image of Au@PdNRs of the present invention;

[0036] Figure 3 This is the high-resolution transmission electron microscope (HRTEM) image of Au@PdNRs of the present invention;

[0037] Figure 4 This is the broad particle size distribution diagram of Au@PdNRs of the present invention;

[0038] Figure 5 This is the length particle size distribution diagram of Au@PdNRs of the present invention;

[0039] Figure 6 This is the ultraviolet-visible absorption spectrum of the Au@Pd NRs solution of the present invention and the schematic diagram of the color change of the solution during the catalytic process;

[0040] Figure 7 This is the energy-dispersive X-ray spectroscopy (EDX) image and the schematic diagram of elemental imaging of Au@Pd NRs of the present invention;

[0041] Figure 8 This is the schematic diagram of the Raman spectrum of DTNB and DTNB-labeled Au@PdNRs of the present invention;

[0042] Figure 9 This is the schematic diagram of the Michaelis-Menten equation curve of the Au@PdNRs nanozyme of the present invention;

[0043] Figure 10 This is the schematic diagram of the double-reciprocal curve of the Au@PdNRs nanozyme of the present invention;

[0044] Figure 11 This is the scanning electron microscope (SEM) image of Au TOHs of the present invention;

[0045] Figure 12 This is the TEM image of Au TOHs of the present invention;

[0046] Figure 13 This is the HRTEM image and the selected area electron diffraction (SAED) image of Au TOHs of the present invention;

[0047] Figure 14 This is the ultraviolet-visible absorption spectrum of the Au TOHs solution of the present invention and the bright-field photo;

[0048] Figure 15 This is the SEM image of the Au TOHs array of the present invention;

[0049] Figure 16 This is the AuTOHs array labeled with DTNB (1×10 -8 ) and DTNB (1×10 -2Schematic diagram of the Raman spectrum;

[0050] Figure 17 Schematic diagram of the SERS spectra at 20 randomly selected positions on the surface of the DTNB-labeled Au TOHs array of the present invention;

[0051] Figure 18 For the present invention Figure 4 Signal intensity scatter plot at 1340 cm -1 in the spectrum;

[0052] Figure 19 SERS spectrum of the DTNB-labeled Au TOHs array of the present invention stored at room temperature for 5 days and its line graph at 1340 cm -1 ;

[0053] Figure 20 Schematic diagram of the optimization of the reaction temperature of the present invention;

[0054] Figure 21 Schematic diagram of the optimization of the pH of the present invention;

[0055] Figure 22 Schematic diagram of the optimization of the incubation time of the present invention;

[0056] Figure 23 Schematic diagram of the optimization of the H2O2 concentration of the present invention;

[0057] Figure 24 Schematic diagram of the optimization of the TMB concentration of the present invention;

[0058] Figure 25 Schematic diagram of the SERS spectra of the same concentration of VEGF detected by five different nanozyme SERS sensors of the present invention;

[0059] Figure 26 For the present invention Figure 25 Signal intensity line graph at 1062 cm -1 in;

[0060] Figure 27 Schematic diagram of the SERS spectra of different analytes such as VEGF, BSA, IgG, CEA, PCT and blank detected by the nanozyme SERS sensor of the present invention;

[0061] Figure 28 For the present invention Figure 27 Histogram of the SERS signal intensity at 1601 cm-1 in;

[0062] Figure 29 Schematic diagram of the SERS spectra of different concentrations of VEGF in serum of the present invention;

[0063] Figure 30For the calibration curve schematic diagram of the intensity of the characteristic peak at 1062 cm of the present invention -1 ;

[0064] Figure 31 Image of the first scanning laser fundus examination of the present invention;

[0065] Figure 32 Image of the second scanning laser fundus examination of the present invention;

[0066] Figure 33 Image of the first fundus fluorescein angiography of the present invention;

[0067] Figure 34 Image of the second fundus fluorescein angiography of the present invention;

[0068] Figure 35 Image of the optical coherence tomography of the present invention;

[0069] Figure 36 SERS spectrum schematic diagram of VEGF in the sera of healthy subjects and DR patients of the present invention;

[0070] Figure 37 For the bar chart of the signal intensity at 1062 cm -1 of healthy subjects and DR patients of the present invention. Detailed implementation manners

[0071] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0072] Please refer to Figures 1 - 37 , the present invention provides a technical solution: a preparation method of a nanozyme SERS sensor for detecting diabetic retinopathy, including the following steps:

[0073] Step 1: Synthesize bimetallic gold-palladium nanorods (Au@PdNRs) by the seed growth method;

[0074] Step 2: Synthesize gold octahedra (AuTOHs) by the seed growth method, and form a neatly arranged and compact AuTOHs array through self-assembly at the oil-water interface;

[0075] Step 3: Modify single-stranded DNA1 (ssDNA1) on the surface of the Au@PdNRs synthesized in Step 1 to prepare a nanozyme probe. Hybridize the aptamer of vascular endothelial growth factor (VEGF) with single-stranded DNA2 (ssDNA2) and then modify it on the surface of the AuTOHs array assembled in Step 2 to prepare a capture substrate. The nanozyme probe and the capture substrate jointly construct a nanozyme SERS sensor.

[0076] In the present invention, the specific implementation manner of Step 1 is as follows:

[0077] (1) First, mix 0.196 mL of chloroauric acid (HAuCl4) solution (25.4 mM) with 9.78 mL of deionized water. Subsequently, add 9.9 mL of cetyltrimethylammonium bromide (CTAB) solution (0.2 M) and mix well. Then, take 2 mL of freshly prepared sodium borohydride (NaBH4) solution (0.0055 M) and inject it into the above mixed solution under stirring at 1100 rpm. Stir continuously for 120 seconds, and the color of the solution changes from yellow to light brown. Let it stand for 1 hour to complete the preparation of gold seeds. Subsequently, dissolve CTAB (0.14 M) and sodium oleate (0.026 M) in 50 mL of deionized water preheated to 50 °C. After the solution cools to 30 °C, add 500 mL of silver nitrate (AgNO3) solution (3.95 mM), 500 mL of HAuCl4 solution (1.0 mM), 4.2 mL of 37% hydrochloric acid solution, and 2.5 mL of ascorbic acid (AA) solution (12 mM) in sequence. Under stirring at 800 rpm, react for 30 minutes. Finally, centrifuge the product at 9000 rpm for 15 minutes, and disperse the precipitate thoroughly in a 7 mM CTAB solution to obtain gold nanorods (AuNRs).

[0078] (2) Synthesize Au@PdNRs by the seed growth method. Dissolve 0.0354 g of PdCl2 powder in 2.4 mL of HCl (0.2 M), continuously dissolve it in a 60 °C water bath for 60 minutes, and further dilute it with deionized water to 20 mL to prepare a chloropalladic acid (H2PdCl4) (10 mM) solution. Add 4 mL of H2PdCl4 (10 mM) solution and 100 mM AA to 70 mL of CTAB (7 mM), then add 80 mL of deionized water, and mix under vigorous stirring for 5 minutes to prepare the growth solution. Then, add the AuNRs solution (seed) prepared in Step (1) to the above growth solution and continuously stir at 25 °C for 3 hours to complete the growth.

[0079] In the present invention, the volume ratio of the AuNRs solution to the growth solution in Step (2) is 1:10.

[0080] In the present invention, the specific implementation manner of Step 2 is as follows:

[0081] (a) AuTOHs were synthesized by the seed growth method. First, 19.5 mL of CTAB (0.1 M) solution, 0.3 mL of HAuCl4 (0.01 M), and 0.2 mL of H2PtCl6 (0.1 M) were uniformly mixed. Then, 1.8 mL of freshly prepared ice-cold NaBH4 (0.01 M) solution was added, and the mixture was stirred at a low speed of 300 rpm until the solution turned light brown and a small amount of foam was generated. 40 μL of the reaction solution was mixed with 20 mL of deionized water to obtain the seed dilution solution for the next step. The seed dilution solution must be used within 30 minutes to avoid crystallization. Subsequently, 1 mL of HAuCl4 (0.01 M), 10 μL of H2PtCl6 (0.01 M), and 0.5 mL of AA (0.1 M) solution were successively added to 10 mL of cetyltrimethylammonium chloride (CTAC) (25 mM) solution and mixed evenly. Then, a certain volume of the seed dilution solution was added. After gently mixing by oscillation, the mixture was placed in an oven at 45 °C and allowed to stand and react for 7 minutes. The solution finally turned dark red, and AuTOHs were obtained.

[0082] (b) The AuTOHs solution prepared in step (a) was mixed with n-hexane to generate an oil-water liquid-liquid interface. An equal volume of absolute ethanol was quickly added to the reaction system. The absolute ethanol drove the self-assembly of nanoparticles at the oil-water interface to form a closely arranged monolayer AuTOHs array. After about 2 minutes, a clean silicon wafer hydrophilized with piranha solution was gently and slowly brought into contact with the array. The monolayer AuTOHs array was transferred to the surface of the silicon wafer and dried in a desiccator for 15 minutes to obtain the AuTOHs array.

[0083] In the present invention, the volume of the seed dilution solution in step (a) is 1 mL. The volume ratio of the AuTOHs solution to n-hexane in step (b) is 1:1.

[0084] In the present invention, the specific implementation manner of step three is as follows:

[0085] (A) First, ssDNA1 (0.1 mM) activated by 40 mL of TCEP was added to 1 mL of the Au@PdNRs solution prepared in step one, and the mixture was incubated at 37 °C. ssDNA1 was modified onto the surface of Au@PdNRs through the thiol group at the 5ˊ end. Then, 15 μL of 1 wt% bovine serum albumin (BSA) solution was added to the mixture to block the non-specific binding sites on the particle surface, and the reaction continued for 1.5 hours. Finally, the product was centrifuged at a speed of 12,000 rpm for 7 minutes to remove the excess reagents and nucleic acid chains, thereby obtaining the nanozyme probe (ssDNA1-functionalized Au@PdNRs). The nucleotide sequence of ssDNA1 is 5ˊ-SH-AATCTACCCGGCCCGT-3ˊ.

[0086] (B) Pre-hybridize ssDNA2 and aptamer with phosphate buffer (8 mM) containing magnesium chloride (MgCl2) (0.8 mM) and sodium chloride (NaCl) (20 mM). After incubating for 1.5 hours, mix 800 μL of ssDNA2 (0.1 mM) with 80 μL of TCEP (1 M) solution and react at room temperature for 60 minutes to activate the terminal thiol groups of the nucleic acid duplex. Drop the activated nucleic acid duplex onto the surface of the Au TOHs array and incubate at 37 °C. The nucleic acid duplex is modified onto the surface of the AuTOHs array through the 5ˊ-terminal thiol group of ssDNA2. Finally, wash repeatedly with phosphate PBS buffer to obtain the capture substrate (Au TOHs array functionalized with ssDNA2 and aptamer). The nucleotide sequences of ssDNA2 and aptamer are 5ˊ-SH-AAACGGGCCGGGTAGA-3ˊ and 5ˊ-TTTTTTTTTTGTGGGGGTGGACGGG CCGGGTAGA-3ˊ.

[0087] (C) Drop 200 mL of the nanozyme probe prepared in step (A) onto the capture substrate assembled in step (B) to construct a nanozyme SERS sensor for detecting DR biomarkers.

[0088] In the present invention, the optimal incubation time of Au@PdNRs and TCEP-activated ssDNA1 in step (A) at 37 °C is 2 hours to allow ssDNA1 to fully bind to Au@PdNRs. The optimal incubation time for dropping the activated nucleic acid duplex onto the surface of the Au TOHs array in step (B) at 37 °C is 2 hours to allow the duplex to fully bind to the Au TOHs array.

[0089] In the present invention, a nanozyme SERS sensor for detecting diabetic retinopathy is prepared based on the preparation method of the nanozyme SERS sensor for detecting diabetic retinopathy.

[0090] The present invention further provides the application of the nanozyme SERS sensor for detecting diabetic retinopathy, including the following steps:

[0091] The first step: Add VEGF to serum to prepare VEGF solutions with different concentrations. Drop 200 mL of VEGF solutions with different concentrations onto the nanozyme SERS sensor for detecting DR biomarkers prepared as claimed in claim 8, perform a hybridization reaction at a specific incubation temperature, and wash away the excess nanozyme probe with PBS buffer. Then, adjust a mixture of 500 mL of TMB and H2O2 with sodium acetate buffer and drop it onto the nanozyme SERS sensor, and perform SERS detection at room temperature to obtain the signal of oxTMB; according to oxTMB at 1062 cm -1Make a fitting curve between the logarithm of the VEGF concentration and the SERS signal intensity at the characteristic peak;

[0092] The second step: Collect 30 samples of healthy human serum and DR patient serum each. Drop 200 mL of the clinical sample onto the nanozyme SERS sensor for detecting DR biomarkers prepared as claimed in claim 8, carry out a hybridization reaction at a specific incubation temperature, and wash away the excess nanozyme probe with PBS buffer solution. Then, adjust a mixture of 500 mL of TMB and H2O2 with sodium acetate buffer solution and drop it onto the platform, and conduct SERS testing at room temperature.

[0093] The third step: Substitute the intensity of the characteristic peak at 1062 cm -1 into the working curve determined in the first step to measure the concentration of VEGF in the clinical sample.

[0094] In the present invention, in the first step, the optimal incubation temperature is 30 °C, the optimal pH is 4.5, the optimal incubation time is 14 minutes, and the optimal concentrations of TMB and H2O2 dropped onto the surface of the nanozyme SERS sensor are 1.0 mM and 0.6 mM respectively.

[0095] The instrument and equipment used in the present invention and the test conditions are as follows:

[0096] Under the condition of an acceleration voltage of 10 kV, use the S-4800 type field emission scanning electron microscope of Hitachi, Japan to characterize the morphology of the nanomaterial.

[0097] Use the Tecnai 12 type transmission electron microscope of Philips, Netherlands to characterize the size and structure of the nanomaterial. Use the Tecnai G2 F30 S-TWIN type field emission transmission electron microscope of FEI, USA to take the HRTEM image and SAED image of the nanomaterial.

[0098] Under the condition of an acceleration voltage of 120 kV, use the ultraviolet-visible-near-infrared (UV-Vis-NIR) absorption spectrometer of Agilent to measure the UV-Vis-NIR absorption spectrum of the sample.

[0099] Use the Invia Reflex type laser micro-Raman spectrometer of Renishaw, UK to measure the Raman spectrum of the sample. The test conditions are a laser wavelength of 785 nm, an exposure time of 10 s, and a laser intensity of 50 mW.

[0100] Use the Agilent BioTek Epoch microplate spectrophotometer (microplate reader) of Agilent, USA to measure the catalytic activity of the nanozyme.

[0101] Preparation and Characterization of Example 1 Au@PdNRs:

[0102] (1) First, mix 0.196 mL of HAuCl4 solution (25.4 mM) with 9.78 mL of deionized water. Subsequently, add 9.9 mL of CTAB solution (0.2 M) and mix well. Then, take 2 mL of freshly prepared NaBH4 solution (0.0055 M) and inject it into the above mixed solution under stirring at 1100 rpm. Stir continuously for 120 seconds. The color of the solution changes from yellow to light brown. Let it stand for 1 hour to complete the preparation of gold seeds. Subsequently, take 50 mL of deionized water preheated to 50 °C, dissolve CTAB (0.14 M) and sodium oleate (0.026 M). After the solution cools to 30 °C, add 500 μL of AgNO3 solution (3.95 mM), 500 μL of HAuCl4 solution (1.0 mM), 4.2 μL of 37% hydrochloric acid solution, and 2.5 mL of AA solution (12 mM) in sequence. Under stirring at 800 rpm, react for 30 minutes. Finally, centrifuge the product at 9000 rpm for 15 minutes, and disperse the precipitate fully in 7 mM CTAB solution to obtain gold nanorods (AuNRs).

[0103] (2) Synthesize Au@PdNRs by the seed growth method. Dissolve 0.0354 g of PdCl2 powder in 2.4 mL of HCl (0.2 M), continuously dissolve it in a 60 °C water bath for 60 minutes, and further dilute it with deionized water to 20 mL to prepare H2PdCl4 (10 mM) solution. Add 4 mL of H2PdCl4 (10 mM) solution and 100 mM AA to 70 μL of CTAB (7 mM), then add 80 mL of deionized water, and mix under vigorous stirring for 5 minutes to prepare the growth solution. Then, add 10 mL of the AuNRs solution (seeds) prepared in step (1) to 10 mL of the above growth solution, and continuously stir at 25 °C for 3 hours to complete the growth.

[0104] (3) Characterization of the morphology, structure, and catalytic activity of Au@PdNRs:

[0105] Detect the TEM image, particle size distribution image, SAED image, and EDS spectrum of Au@Pd NRs by transmission electron microscopy. Detect the UV-Vis-NIR absorption spectrum of the Au@PdNRs solution by a UV-Vis-NIR absorption spectrometer. At room temperature, prepare a DTNB solution with a concentration of 1×10 -8 M. Add 10 μL of DSNB solution with a concentration of 1×10 -8 M to 0.1 mL of the prepared Au@PdNRs solution and mix well. Use a laser micro-Raman spectrometer for DTNB (1×10 -2 M) and DTNB (1×10-8 Raman detection was carried out on Au@Pd NRs labeled with M). At a fixed concentration of H2O2 (0.6 mM), the concentration of TMB was changed (0.2 mM - 1.0 mM), and sodium acetate solution (pH = 4.5) was used as a buffer and added into a 96-well plate. An enzyme-linked immunosorbent assay (ELISA) reader was used to measure the reaction process, and the Michaelis-Menten equation curve and double-reciprocal curve of this nanozyme were obtained by fitting.

[0106] As Figure 2 shown, the morphology of Au@Pd NRs is in the shape of regular long bars with good uniformity. As Figure 3 shown, it can be clearly observed that Au@Pd NRs are composed of a Pd shell and an Au NRs core, with a rough surface and a lattice fringe spacing of 0.192 nm. The average length of Au@Pd NRs is 80 nm, the width is 32 nm, and the aspect ratio is about 2.5:1 (as Figure 4 and 5 shown). As Figure 6 shown, Au@Pd NRs have a significant absorption peak at 747 nm. When H2O2 and TMB exist alone, the solution turns colorless. When the nanozyme Au@Pd NRs are added to both of them, the solution changes from colorless to blue, and an obvious absorption peak appears at 646 nm. EDX and elemental imaging show that gold and palladium are the main components of Au@Pd NRs (as Figure 7 shown). As Figure 8 shown, at the characteristic peak of 1062 cm -1 , the signal intensity of Au@Pd NRs labeled with DTNB is significantly stronger than that of DTNB, showing a good enhancement effect. Through the formula EF = (I SERS / C SERS ) / (I Raman / C Raman ) for calculation, where I and C represent the measured signal intensity and the corresponding concentration at 1062 cm -1 . When C SERS and C Raman are set to 10 -2 M and 10 -8 M respectively, its EF is calculated to be 9.9×10 8 . Michaelis-Menten kinetics was used to measure the nanozyme activity (as Figure 9 and Figure 10 shown), and the Vmax of the Au@Pd NRs nanozyme was obtained as 0.663 mM s -1 , and the Km was 0.351 mM

[0107] Example 2 Assembly and Characterization of Au TOHs Array:

[0108] (1) AuTOHs were synthesized by the seed growth method. First, 19.5 mL of CTAB (0.1 M) solution, 0.3 mL of HAuCl4 (0.01 M), and 0.2 mL of H2PtCl6 (0.1 M) were uniformly mixed, and 1.8 mL of freshly prepared ice-cold NaBH4 (0.01 M) solution was added. The mixture was stirred at a low speed of 300 rpm until the solution turned light brown and a small amount of foam was generated. 40 μL of the reaction solution was mixed with 20 mL of deionized water to obtain a seed dilution solution for the next step. The prepared solution must be used within 30 minutes to avoid crystallization. Subsequently, 1 mL of HAuCl4 (0.01 M), 10 μL of H2PtCl6 (0.01 M), and 0.5 mL of AA (0.1 M) solution were successively added to 10 mL of CTAC (25 mM) solution and mixed evenly. Then, 11.6 mL of the seed dilution solution was added. After gently shaking and mixing, the mixture was placed in an oven at 45 °C and allowed to react statically for 7 minutes. The solution finally turned dark red, and AuTOHs were obtained.

[0109] (2) 5 mL of the AuTOHs solution prepared in step (a) was mixed with 5 mL of n-hexane to generate an oil-water liquid-liquid interface. An equal volume of absolute ethanol was quickly added to the reaction system. The absolute ethanol drove the self-assembly of nanoparticles at the oil-water interface to form a closely arranged monolayer AuTOHs array. After about 2 minutes, a clean silicon wafer hydrophilized with piranha solution was contacted with the array parallelly and slowly. The monolayer AuTOHs array was transferred to the surface of the silicon wafer and dried in a desiccator for 15 minutes to obtain the AuTOHs array.

[0110] (3) Characterization of the morphology and structure of the Au TOHs array and its SERS effect:

[0111] The SEM images, TEM images, SAED images, and electron diffraction images of Au TOHs and the Au TOHs array were detected by scanning electron microscopy and transmission electron microscopy. The UV-Vis-NIR absorption spectrum of the Au TOHs solution was detected by a UV-Vis-NIR absorption spectrometer. At room temperature, a DTNB solution with a concentration of 1×10 -8 M was prepared. The Au TOHs array was immersed in a DSNB solution with a concentration of 1×10 -8 M, and after taking it out, it was allowed to stand for 0.5 h. Raman detection was performed on the Au TOHs array labeled with DTNB (1×10 -2 M) and DTNB (1×10 -8 M) by a laser micro-Raman spectrometer. 20 different positions were randomly selected on the surface of the Au TOHs array labeled with (1×10 -8 M), and the SERS spectra were measured. The (1×10 -8M) The labeled Au TOHs array was stored at room temperature for 15 days and measured every 5 days to measure the changes in the SERS spectrum.

[0112] like Figure 11 and 12 As shown in the figure, the SEM and TEM images of Au TOHs are shown, which indicate that Au TOHs with regular morphology, good dispersion and sharp edges were successfully prepared. Figure 13 As shown in the figure, the HRTEM image and SAED image of Au TOHs show that the lattice fringe spacing is 0.223 nm and it is a single crystal. Figure 14 As shown in Figure 2, Au TOHs has an obvious absorption peak near 546nm. After self-assembly at the oil-water interface, an ordered and tightly arranged Au TOHs array is formed. Figure 15 As shown in Figure 2, AuTOHs are evenly distributed, densely, and over a large area on the surface of a clean silicon wafer to form a single layer. -8 M) labeled Au TOHs and DTNB (10 -2 M) for comparison, according to the formula: The value of EF is 1.88×10 9 , indicating that the AuTOHs array has a significant SERS enhancement effect (e.g. Figure 16 As shown). Figure 17 and 18 As shown in the figure, the DTNB-labeled AuTOHs array has high reproducibility, with 20 different positions on its surface at 1340 cm -1 The relative standard deviation (RSD) value of the characteristic peak intensity at 4.22% is 4.22%. Figure 19 As shown in the SERS spectra of the DTNB-labeled AuTOHs array stored at room temperature for 15 days, compared with the initial state, the 1340 cm -1 The decrease in the intensity of the characteristic peak at is only 10.11%, and it decreases with time, indicating that the AuTOHs array has good stability.

[0113] Example 3 Optimization of the preparation of nanozyme SERS sensor:

[0114] (1) The Au@PdNRs were prepared in the same manner as in Example 1.

[0115] (2) Assemble the AuTOHs array in the same manner as in Example 2.

[0116] (3) First, add ssDNA1 (0.1 mM) activated by 40 μL of TCEP to 1 mL of Au@PdNRs solution, and incubate at 37 °C for 2 hours to allow ssDNA1 to fully bind to Au@PdNRs. Then, add 15 μL of 1 wt% BSA solution to the mixture to block the non-specific binding sites on the particle surface, and the reaction continues for 1.5 hours. Finally, centrifuge the product at 12,000 rpm for 7 minutes to remove excess reagents and nucleic acid strands, thereby obtaining the nanozyme probe (ssDNA1-functionalized Au@PdNRs). The nucleotide sequence of the ssDNA1 is 5′-SH-AATCTAC CCGGCCCGT-3′.

[0117] (4) Pre-hybridize ssDNA2 and aptamer using phosphate buffer (8 mM) containing MgCl2 (0.8 mM) and NaCl (20 mM). After incubating for 1.5 hours, mix 800 μL of ssDNA2 (0.1 mM) with 80 μL of TCEP (1 M) solution and react at room temperature for 60 minutes to activate the terminal thiol groups of the nucleic acid duplex. Drop the activated nucleic acid duplex onto the surface of the AuTOHs array and incubate at 37 °C for the optimal time of 2 hours to allow the duplex to fully bind to the AuTOHs array. Finally, wash repeatedly with PBS buffer to ultimately obtain the capture substrate (AuTOHs array functionalized with ssDNA2 and aptamer). The nucleotide sequences of the ssDNA2 and aptamer are 5′-SH-AAACGGGCCGGGTAGA-3′ and 5′-TTTTTTTTTTGTGGGGGTGGACGGG CCGGGTAGA-3′.

[0118] (5) Drop 200 μL of the nanozyme probe prepared in step (A) onto the capture substrate assembled in step (B) to construct a nanozyme SERS sensor for detecting DR biomarkers.

[0119] (6) Add 200 μL of 1 μg / mL VEGF solution dropwise onto the nanozyme SERS sensor, and place it in an incubator for hybridization reaction. Every 5 minutes, take out the nanozyme SERS sensor and wash away the excess nanozyme probes with PBS buffer. Drop 500 μL of the mixture of TMB and H2O2 onto the sensor and perform SERS detection at room temperature to screen for the optimal incubation time. Add 200 μL of 1 μg / mL VEGF solution dropwise onto the nanozyme SERS sensor, place it in an incubator at different temperatures for the optimal reaction time, take out the nanozyme SERS sensor, and wash away the excess nanozyme probes with PBS buffer. Then, add 500 μL of the mixture of TMB and H2O2 with different concentrations and adjust the pH with sodium acetate buffer, and drop it onto the platform for SERS detection. Screen for the optimal incubation temperature, optimal pH, and optimal concentrations of TMB and H2O2.

[0120] (7) During the process of the nanozyme SERS sensor recognizing the target, the reaction pH and the concentration of the substrate will affect the activity of the nanozyme. To obtain highly reliable results, this study optimized the key parameters. As Figure 20 and 21 show, the SERS signal intensity first increases with the increase of temperature and pH value, and then decreases after reaching the peak. The optimal temperature is 30 °C and the pH is 4.5. As Figure 22 shows, the SERS signal intensity continuously increases within 0 to 14 minutes, but the slope continuously decreases and stabilizes after 14 minutes. Therefore, the optimal incubation time is selected as 14 minutes. As Figure 23 and 24 show, when TMB is 1.0 mM and H2O2 is 0.6 mM, the SERS signal intensity is the highest.

[0121] Example 4 Specificity and Repeatability of the Nanozyme SERS Sensor:

[0122] (1) Prepare the nanozyme SERS sensor in the same way as in Example 3.

[0123] (2) Prepare 5 different nanozyme SERS sensors. Add 200 μL of 1 μg / mL VEGF solution dropwise onto these nanozyme SERS sensors, place them in an incubator at the optimal incubation temperature in Example 3 for the optimal reaction time, take out the nanozyme SERS sensors, and wash away the excess nanozyme probes with PBS buffer. Then, add 500 μL of the mixture of TMB and H2O2 with the optimal concentration and optimal pH, and perform SERS detection at room temperature to evaluate the repeatability of the nanozyme SERS sensor. Add 200 μL of 1 μg / mL VEGF, BSA, IgG, CEA, and PCT solutions dropwise onto the nanozyme SERS sensor respectively, and perform SERS detection at room temperature to evaluate the specificity of the SERS sensor.

[0124] (3) As Figure 25 and 26 shown, the SERS spectra of the same concentration of VEGF detected by 5 different nanozyme SERS sensors had an RSD of 2.34%. Some biomolecular solutions (such as BSA, IgG, CEA, PCT, etc.) similar to the target were detected using the nanozyme SERS sensor to test the specificity of this platform. As Figure 27 and 28 shown, the signal intensity of the VEGF solution was significantly stronger than that of the blank group and the solutions of other biomolecules. The results verified that the nanozyme SERS sensor had good specificity and repeatability.

[0125] Example 5 Detection of VEGF in clinical samples by nanozyme SERS sensor:

[0126] (1) Prepare the nanozyme SERS sensor in the same way as in Example 3.

[0127] (2) Disperse VEGF in serum to prepare VEGF solutions with concentrations ranging from 1 pg / mL to 1 μg / mL. Drop 200 μL of VEGF solutions with different concentrations onto the nanozyme SERS sensor, place it in an incubator at the optimal incubation temperature in Example 3 for the optimal reaction time, take out the nanozyme SERS sensor, and wash away the excess nanozyme probe with PBS buffer. Then add 500 μL of the mixed solution of TMB and H2O2 with the optimal concentration and the optimal pH, and perform SERS detection at room temperature. According to the characteristic peak of oxTMB at 1062 cm -1 , a fitting curve was made between the logarithm of the VEGF concentration and the SERS signal intensity.

[0128] (3) Collect 30 samples each of healthy human serum and DR patient serum. Drop 200 μL of the clinical sample onto the nanozyme SERS sensor, place it in an incubator at the optimal incubation temperature in Example 3 for the optimal reaction time, take out the nanozyme SERS sensor, and wash away the excess nanozyme probe with PBS buffer. Then add 500 μL of the mixed solution of TMB and H2O2 with the optimal concentration and the optimal pH, and perform SERS detection at room temperature.

[0129] (4) As Figure 29 shown, as the VEGF concentration increased, the SERS signal intensity of oxTMB gradually increased. Using the signal intensity at 1062 cm -1 and the logarithm of the VEGF concentration, a linear regression equation was plotted: y = 2809.74x + 37410.48 (R 2 = 0.99). According to this equation, the detection limit (LOD) was calculated to be 0.11 pg / mL (as Figure 30 shown). Compared with other methods (Table 1), this method had higher sensitivity.

[0130] Method Target LOD Fluorescence Hb 260 ng / mL Electrochemistry CEA 0.08 ng / mL Fluorescence AFP 26 ng / mL SERS AFP 2 ng / mL This method VEGF 0.11 pg / mL

[0131] Table 1 Comparison of this method with other biomarker detection methods

[0132] All the subjects enrolled in this study underwent imaging examinations. As Figure 31 and 32 showed, there were bleeding points and proliferative membranes in the fundus of DR patients. As Figure 33 and 34 showed, extensive retinal non-perfusion areas and obvious fluorescence leakage of neovascularization were shown in both eyes of DR patients. As Figure 35 showed, macular edema and detachment of the retinal neurosensory epithelium were shown. To further evaluate the clinical application value of this nanozyme SERS sensor, SERS detection was performed on the expression levels of VEGF in the sera of healthy people and DR patients. As Figure 36 and 37 showed, the intensity of the characteristic peak at 1062 cm -1 in DR patients was significantly higher than that in healthy people. Substituting the signal intensity into the linear regression equation (as Figure 30 showed), the expression levels of VEGF in the sera of healthy people and DR patients were calculated, and then the SERS results were compared with ELISA. As shown in Table 2, compared with the healthy control group, VEGF was highly expressed in the sera of DR patients. The SERS detection results were highly consistent with ELISA, fully confirming the reliability of this method.

[0133]

[0134] Table 2 Detection of VEGF expression levels in the sera of healthy people and DR patients by SERS and ELISA

[0135] Although the embodiments of the present invention have been shown and described, for those of ordinary skill in the art, it can be understood that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. Preparation method of a nanozyme SERS sensor for detecting diabetic retinopathy, characterized in that: It includes the following steps: Step 1: Synthesize bimetallic gold-palladium nanorods (Au@PdNRs) using the seed growth method; Step 2: Synthesize gold octahedra (AuTOHs) using the seed growth method, and form a neatly arranged and compact AuTOHs array through self-assembly at the oil-water interface; Step 3: Modify single-stranded DNA1 (ssDNA1) on the surface of the Au@PdNRs synthesized in Step 1 to prepare a nanozyme probe. Hybridize the aptamer of vascular endothelial growth factor (VEGF) with single-stranded DNA2 (ssDNA2) and then modify it on the surface of the AuTOHs array assembled in Step 2 to prepare a capture substrate. The nanozyme probe and the capture substrate jointly construct a nanozyme SERS sensor.

2. The preparation method of the nanozyme SERS sensor for detecting diabetic retinopathy according to claim 1, characterized in that: The specific implementation method of Step 1 is as follows: (1) First, mix 0.196 mL of chloroauric acid (HAuCl4) solution (25.4 mM) with 9.78 mL of deionized water, then add 9.9 mL of cetyltrimethylammonium bromide (CTAB) solution (0.2 M), and mix well. Then, take 2 mL of freshly prepared sodium borohydride (NaBH4) solution (0.0055 M) and inject it into the above mixed solution under stirring at 1100 rpm. Stir continuously for 120 seconds, and the color of the solution changes from yellow to light brown. Let it stand for 1 hour to complete the preparation of gold seeds. Subsequently, take 50 mL of deionized water preheated to 50 °C, dissolve CTAB (0.14 M) and sodium oleate (0.026 M). After the solution cools to 30 °C, add 500 mL of silver nitrate (AgNO3) solution (3.95 mM), 500 mL of HAuCl4 solution (1.0 mM), 4.2 mL of 37% hydrochloric acid solution, and 2.5 mL of ascorbic acid (AA) solution (12 mM) in sequence. Under stirring at 800 rpm, react for 30 minutes. Finally, centrifuge the product at 9000 rpm for 15 minutes, and disperse the precipitate fully in a 7 mM CTAB solution to obtain gold nanorods (AuNRs). (2) Synthesize Au@PdNRs using the seed growth method. Dissolve 0.0354 g of PdCl2 powder in 2.4 mL of HCl (0.2 M), continuously dissolve it in a 60 °C water bath for 60 minutes, and further dilute it with deionized water to 20 mL to prepare a chloropalladic acid (H2PdCl4) (10 mM) solution. Add 4 mL of H2PdCl4 (10 mM) solution and 100 mM AA to 70 mL of CTAB (7 mM), then add 80 mL of deionized water, and mix under vigorous stirring for 5 minutes to prepare the growth solution. Then, add the AuNRs solution (seeds) prepared in step (1) to the above growth solution, and continuously stir at 25 °C for 3 hours to complete the growth.

3. The method for preparing a nanozyme SERS sensor for detecting diabetic retinopathy according to claim 2, characterized in that: In step (2), the volume ratio of the AuNRs solution to the growth solution is 1:

10.

4. The preparation method of the nanozyme SERS sensor for detecting diabetic retinopathy according to claim 1, characterized in that: The specific implementation method of Step 2 is as follows: (a) AuTOHs were synthesized by the seed growth method. First, 19.5 mL of CTAB (0.1 M) solution, 0.3 mL of HAuCl4 (0.01 M), and 0.2 mL of H2PtCl6 (0.1 M) were uniformly mixed. Then, 1.8 mL of freshly prepared ice-cold NaBH4 (0.01 M) solution was added, and the mixture was stirred at a low speed of 300 rpm until the solution turned light brown and a small amount of foam was generated. 40 μL of the reaction solution was mixed with 20 mL of deionized water to obtain a seed dilution solution for the next step. The seed dilution solution must be used within 30 minutes to avoid crystallization. Subsequently, 1 mL of HAuCl4 (0.01 M), 10 μL of H2PtCl6 (0.01 M), and 0.5 mL of AA (0.1 M) solution were successively added to 10 mL of cetyltrimethylammonium chloride (CTAC) (25 mM) solution and mixed well. Then, a certain volume of the seed dilution solution was added. After gently shaking and mixing, the mixture was placed in an oven at 45 °C and allowed to react statically for 7 minutes. The solution finally turned dark red, and AuTOHs were obtained. (b) The AuTOHs solution prepared in step (a) was mixed with n-hexane to generate an oil-water liquid-liquid interface. An equal volume of absolute ethanol was quickly added to the reaction system. The absolute ethanol drove the self-assembly of nanoparticles at the oil-water interface to form a closely arranged monolayer AuTOHs array. After about 2 minutes, a clean silicon wafer hydrophilized with piranha solution was contacted with the array parallelly and slowly. The monolayer AuTOHs array was transferred to the surface of the silicon wafer and dried in a desiccator for 15 minutes to obtain the AuTOHs array.

5. The preparation method of the nanozyme SERS sensor for detecting diabetic retinopathy according to claim 4, wherein: In step (a), the volume of the seed dilution solution is 1 mL, and in step (b), the volume ratio of the AuTOHs solution to n-hexane is 1:

1.

6. The preparation method of the nanozyme SERS sensor for detecting diabetic retinopathy according to claim 1, wherein: The specific implementation method of step three is as follows: (A) First, ssDNA1 (0.1 mM) activated by 40 mL of TCEP was added to 1 mL of the Au@PdNRs solution prepared in step one, and the mixture was incubated at 37 °C. ssDNA1 was modified onto the surface of Au@PdNRs through the thiol group at the 5ˊ end. Then, 15 μL of 1 wt% bovine serum albumin (BSA) solution was added to the mixture to block the non-specific binding sites on the particle surface, and the reaction continued for 1.5 hours. Finally, the product was centrifuged at a speed of 12,000 rpm for 7 minutes to remove the excess reagents and nucleic acid chains, thereby obtaining a nanozyme probe (ssDNA1-functionalized Au@PdNRs). The nucleotide sequence of ssDNA1 is 5ˊ-SH-AATCTACCCGGCCCGT-3ˊ. (B) Pre-hybridize ssDNA2 and aptamer with phosphate buffer (8 mM) containing magnesium chloride (MgCl2) (0.8 mM) and sodium chloride (NaCl) (20 mM). After incubation for 1.5 hours, mix 800 μL of ssDNA2 (0.1 mM) with 80 μL of TCEP (1 M) solution and react at room temperature for 60 minutes to activate the terminal thiol groups of the nucleic acid duplex. Drop the activated nucleic acid duplex onto the surface of the Au TOHs array and incubate at 37 °C. The nucleic acid duplex is modified onto the surface of the Au TOHs array through the 5ˊ-terminal thiol group of ssDNA2. Finally, wash repeatedly with phosphate PBS buffer to obtain the capture substrate (Au TOHs array functionalized with ssDNA2 and aptamer). The nucleotide sequences of the ssDNA2 and aptamer are 5ˊ-SH-AAACGGGCCGGGTAGA-3ˊ and 5ˊ-TTTTTTTTTTGTGGGGGTGGACGGG CCGGGTAGA-3ˊ. (C) Drop 200 mL of the nanozyme probe prepared in step (A) onto the capture substrate assembled in step (B) to construct a nanozyme SERS sensor for detecting DR biomarkers.

7. The preparation method of the nanozyme SERS sensor for detecting diabetic retinopathy according to claim 6, characterized in that: In step (A), the optimal incubation time for Au@PdNRs and TCEP-activated ssDNA1 at 37 °C is 2 hours to allow ssDNA1 to bind fully to Au@Pd NRs. In step (B), when the activated nucleic acid duplex is dropped onto the surface of the Au TOHs array, the optimal incubation time at 37 °C is 2 hours to allow the duplex to bind completely to the Au TOHs array.

8. A method for preparing a nanozyme SERS sensor for detecting diabetic retinopathy according to claim 7, to prepare a nanozyme SERS sensor for detecting diabetic retinopathy.

9. Application of a nanozyme SERS sensor for detecting diabetic retinopathy, comprising the method for preparing a nanozyme SERS sensor for detecting diabetic retinopathy according to any one of claims 1 to 8, characterized in that: Comprising the following steps: First step: Add VEGF into the serum to prepare VEGF solutions with different concentrations. Drop 200 mL of VEGF solutions with different concentrations onto the nanozyme SERS sensor for detecting DR biomarkers prepared as claimed in claim 8, carry out a hybridization reaction at a specific incubation temperature, and wash away the excess nanozyme probes with PBS buffer. Then, adjust a mixture of 500 mL of TMB and H2O2 with sodium acetate buffer and drop it onto the nanozyme SERS sensor, and perform SERS detection at room temperature to obtain the signal of oxTMB; According to the characteristic peak of oxTMB at 1062 cm -1 Make a fitting curve between the logarithm of the VEGF concentration and the SERS signal intensity at the characteristic peak; Second step: Collect 30 samples each of healthy human serum and DR patient serum. Drop 200 mL of the clinical sample onto the nanozyme SERS sensor for detecting DR biomarkers prepared as claimed in claim 8, and perform a hybridization reaction at a specific incubation temperature. Wash away the excess nanozyme probe with PBS buffer. Then, adjust a mixture of 500 mL of TMB and H2O2 with sodium acetate buffer and drop it onto the platform, and perform SERS testing at room temperature. Step 3: Substitute the characteristic peak intensity at 1062 cm -1 of the clinical sample detected in Step 2 into the working curve determined in Step 1 to measure the concentration of VEGF in the clinical sample.

10. Use of the nanozyme SERS sensor for detecting diabetic retinopathy according to claim 9, characterized in that: In the first step, the optimal incubation temperature is 30 °C, the optimal pH is 4.5, the optimal incubation time is 14 minutes, and the optimal concentrations of TMB and H2O2 dropped onto the surface of the nanozyme SERS sensor are 1.0 mM and 0.6 mM, respectively.

Images