A DNA-silver-platinum bimetallic nanozyme for the detection of β-amyloid oligomers, its preparation method and application

Abstract

This invention discloses a DNA-silver-platinum bimetallic nanozyme for the detection of β-amyloid oligomers, its preparation method, and its application. The DNA-silver-platinum bimetallic nanozyme of this invention is prepared through the following steps: first, a DNA sequence is synthesized, comprising a β-amyloid oligomer aptamer sequence and a DNA template sequence for synthesizing silver nanoclusters; then, silver ions are reduced onto the DNA template sequence using sodium borohydride to generate silver nanoclusters; subsequently, platinum ions undergo an electrocoupling reaction with silver, followed by further reduction with sodium borohydride to obtain the DNA-silver-platinum bimetallic nanozyme, which exhibits peroxidase-like catalytic activity. This invention also discloses the application of the above-mentioned DNA-silver-platinum bimetallic nanozyme in the detection of β-amyloid oligomers. The preparation method of this invention is simple and enables rapid, colorimetric detection of β-amyloid oligomers, showing broad application prospects in biochemical analysis.

Description

Technical Field

[0001] This invention belongs to the field of nanomaterials, and more specifically, relates to a DNA-silver-platinum bimetallic nanozyme for the detection of β-amyloid oligomers, its preparation method, and its application. Background Technology

[0002] Alzheimer's disease (AD) is the most common neurodegenerative disease. AD patients experience changes in brain morphology, including brain atrophy, thinning of the cerebral cortex, widening of sulci, and enlargement of the ventricles, leading to symptoms such as memory impairment, personality and behavioral changes, and motor dysfunction. It has become one of the leading causes of death among the elderly. Studies have shown that 10-15 years before the clinical onset of AD, β-amyloid protein (Aβ) self-assembles into toxic β-amyloid oligomers (AβO). AβO is considered one of the most effective early diagnostic biomarkers and therapeutic targets for AD. Since there is currently no specific drug to treat AD, early detection of AβO is of great significance for the prevention and diagnosis of AD.

[0003] Currently reported methods for detecting AβO mainly include neural imaging, optical detection, immunoassay, and electrochemical methods. However, these methods generally suffer from drawbacks such as expensive equipment, relatively complex operation, and high cost (Chinese Journal of Chem, 2018, 46(9):1339-1349). Nanozymes have advantages such as simple preparation, good stability, and high recycling efficiency. Based on their unique catalytic ability, they have been applied in fields such as biosensing, environmental protection, and disease treatment. In particular, the use of nanozymes can achieve highly sensitive and visual detection of the target based on the color change of the system solution, without the need for complex instruments and with simple operation.

[0004] Therefore, the present invention aims to develop a low-cost and easy-to-operate nanozyme for the colorimetric detection of AβO, providing a more sensitive and rapid new method for the detection of AβO. Summary of the Invention

[0005] One object of the present invention is to provide a simple one-step method for preparing DNA-silver-platinum bimetallic nanozymes, namely, synthesizing DNA-silver-platinum bimetallic nanozymes using DNA as a template, which does not require the addition of additional surfactants or high temperatures.

[0006] A second objective of this invention is to provide applications of the DNA-silver-platinum bimetallic nanozyme prepared by the above method.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a method for preparing a DNA-silver-platinum bimetallic nanozyme for the detection of β-amyloid oligomers, the method comprising the following steps:

[0009] (1) Synthesize a DNA sequence comprising a β-amyloid oligomer aptamer sequence and a DNA template sequence for synthesizing silver nanoclusters attached to its 5' end;

[0010] (2) Add silver nitrate solution, potassium tetrachloroplatinate solution and the DNA sequence from step (1) to the buffer solution, shake for 30s-2min, incubate in ice water for 20-30min, then add sodium borohydride, shake for 30s-2min, and incubate at room temperature for 3-6h to obtain DNA-silver-platinum bimetallic nanozyme.

[0011] The DNA-silver-platinum bimetallic nanozyme synthesized in this invention does not require the addition of additional surfactants and the reaction conditions are mild. Silver ions are reduced on the DNA template sequence by sodium borohydride to generate silver nanoclusters. Then, platinum ions undergo an electrocoupling reaction with silver, and the reduction is further carried out by sodium borohydride to obtain a DNA-silver-platinum bimetallic nanozyme with high peroxidase catalytic activity.

[0012] Furthermore, in step (1), the DNA template sequence used to synthesize the silver nanoclusters is 5'-CCCTTAATCCCC-3' (as shown in SEQ ID No. 1); the β-amyloid oligomer aptamer is 5'-GCCTGTGGTGTTGGGGCGGGTGCGTTTTTTTTTT-3' (as shown in SEQ ID No. 3). The adjustment of the number of T bases at the 3' end is within the scope of protection of this invention as long as it can achieve the present invention.

[0013] Furthermore, in step (2), the buffer solution is a buffer solution capable of generating DNA-silver-platinum bimetallic nanozymes and maintaining DNA stability;

[0014] Preferably, the buffer solution is a buffer solution with a pH of 7.2-7.4;

[0015] Preferably, the buffer solution is a chloride-free phosphate buffer solution with a pH of 7.2-7.4.

[0016] Furthermore, in step (2), the molar ratio of the DNA sequence to silver nitrate is 1:6; the molar ratio of potassium tetrachloroplatinate to silver nitrate is 0.2:1-10:1; and the molar ratio of silver nitrate to sodium borohydride is 1:1-1:40.

[0017] Secondly, the present invention also claims protection for the DNA-silver-platinum bimetallic nanozyme prepared by the above method.

[0018] The DNA-silver-platinum bimetallic nanozyme of the present invention has a spherical shape and a particle size of 0.8-5 nm.

[0019] Thirdly, the present invention also provides the application of the above-mentioned DNA-silver-platinum bimetallic nanozyme in the detection of β-amyloid oligomers.

[0020] According to a specific embodiment of the present invention, the DNA-silver-platinum bimetallic nanozyme was added to the sample to be tested for incubation, and then acetate buffer, hydrogen peroxide and 3,3',5,5'-tetramethylbenzidine were added to react. The color change of the reaction solution was observed and its absorbance was tested.

[0021] In the reaction system, the final concentration of the DNA-silver-platinum bimetallic nanozyme is 5 nM, the final concentration of hydrogen peroxide is 5 mM, and the final concentration of 3,3',5,5'-tetramethylbenzidine is 0.8 mM.

[0022] Preferably, the incubation conditions are: room temperature for 1-2 hours; the reaction conditions are: temperature 20-30℃, time 1-2 hours.

[0023] When the color of the reaction solution exhibits a visible hypochromic effect, changing from dark blue to light blue, and the absorption peak of the ultraviolet-visible spectrum at 652 nm continuously decreases, it indicates that the sample contains β-amyloid oligomers.

[0024] The beneficial effects of this invention are as follows:

[0025] 1. The nanozyme prepared by this invention directly utilizes the reduction effect of sodium borohydride and the electrocoupling reaction between platinum ions and silver to form a DNA-silver-platinum bimetallic nanozyme with high peroxidase catalytic activity. This method is simple, convenient to operate, and has mild reaction conditions.

[0026] 2. This invention uses a DNA template sequence to enrich silver on the template strand, followed by platinum formation on the silver. This preserves the aptamer's ability to complex β-amyloid oligomers, improving the sensitivity and selectivity of the sensing process.

[0027] 3. The DNA-silver-platinum bimetallic nanozyme prepared in this invention can rapidly catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) under H2O2 conditions, resulting in a visible color change from deep blue to light blue. Furthermore, thrombin, bovine serum albumin (BSA), trypsin, AβM, and AβF interfering agents have no significant effect on the colorimetric AβO catalysis of the DNA-Ag / Pt NCs-TMB-H2O2 system. This indicates that the DNA-Ag / Pt NCs mimic enzyme synthesized in this invention has good selectivity and anti-interference properties, exhibiting excellent specificity and selectivity for AβO. The detection concentration range for AβO can reach 0-3.5 μM, and the detection limit can be as low as 1.09 nM. It has advantages such as simple operation, good selectivity, high sensitivity, and strong visibility. Attached Figure Description

[0028] The specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

[0029] Figure 1 The structure of the DNA-silver-platinum bimetallic nanozyme and its working principle for detecting β-amyloid oligomers (AβO) are shown in the diagram.

[0030] Figure 2 The bar chart shows the absorption intensity at 652 nm of the absorption peak in the UV-Vis spectra of different DNA-silver-platinum bimetallic nanozymes synthesized by changing the DNA sequence in Example 1. The final concentration of the DNA-silver-platinum bimetallic nanozymes used was 5 nM (calculated based on the concentration of AβO aptamer), n = 3.

[0031] Figure 3 Aberration-corrected transmission electron microscopy (TEM) image of the DNA-silver-platinum bimetallic nanozyme prepared in Example 2 is shown.

[0032] Figure 4 The EDS spectrum of the DNA-silver-platinum bimetallic nanozyme prepared in Example 2 is shown.

[0033] Figure 5a The UV-Vis spectra of DNA-silver-platinum bimetallic nanozymes detecting different concentrations of AβO are shown in Application Example 1. The final concentration of the DNA-silver-platinum bimetallic nanozymes used was 5 nM (calculated based on the concentration of AβO aptamers).

[0034] Figure 5b The color changes of the DNA-Ag / PtNCs-TMB-H2O2 system at different concentrations of AβO are shown in Application Example 1.

[0035] Figure 5c The example shown in application example 1 illustrates Abs x The relationship curve between / Abs0 and cAβO;

[0036] Figure 6 The effects of thrombin, bovine serum albumin (BSA), trypsin, AβM, and AβF interfering agents on the catalytic colorimetric AβO of the DNA-Ag / Pt NCs-TMB-H2O2 system are shown in Application Example 2. Detailed Implementation

[0037] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments and accompanying drawings, further clarifies the invention. Those skilled in the art should understand that the specific description below is illustrative rather than restrictive and should not be construed as limiting the scope of protection of the present invention.

[0038] This invention provides a method for preparing DNA-silver-platinum bimetallic nanozymes, which can be applied to the detection of β-amyloid oligomers. Figure 1 As shown, the DNA-silver-platinum bimetallic nanozyme (DNA-Ag / Pt NCs) exhibits high peroxidase-like catalytic activity. After reacting with the colorimetric substrate, it forms a blue solution with a high absorbance. In the presence of the target analyte AβO, the aptamer binds to AβO, altering the microenvironment of the DNA-silver-platinum bimetallic nanozyme, changing its surface properties, and reducing the contact area with the substrate, thus significantly decreasing the nanozyme's catalytic activity. Higher AβO concentrations result in a more pronounced decrease in both the color and absorbance of the DNA-silver-platinum bimetallic nanozyme, thereby enabling the detection of AβO.

[0039] The following detailed description is based on specific embodiments.

[0040] Example 1

[0041] 1) Synthesizing different DNA sequences:

[0042] a)5'-CCCTTAATCCCC-3'; (SEQ ID No. 1)

[0043] b) 5'-GCCTGTGGTGTTGGGGCGGGTGCG-3'; (SEQ ID No. 2)

[0044] c) 5'-GCCTGTGGTGTTGGGGCGGGTGCGTTTTTTTTTT-3'; (SEQ ID No. 3)

[0045] d)5'-GCCTGTGGTGTTGGGGGCGGGTGCGAAAAAAAAAA-3'; (SEQ ID No. 4)

[0046] e) 5'-CCCTTAATCCCCGCCTGTGGTGTTGGGGCGGGTGCG-3'; (SEQ ID No. 5)

[0047] f) 5'-CCCTTAATCCCCGCCTGTGGTGTTGGGGCGGGTGCGTTTTTTTT TT-3'; (SEQ ID No. 6)

[0048] g) 5'-CCCTTAATCCCCTTTTTTTTTTGCCTGTGGTGTTGGGGCGGGTG CG-3'; (SEQ ID No. 7)

[0049] h) 5'-CCCTTAATCCCCAAAAAAAAAAGCCTGTGGTGTTGGGGCGGGT GCG-3'; (SEQ ID No. 8)

[0050] i) 5'-CCCTTAATCCCCGCCTGTGGTGTTGGGGGCGGGTGCGAAAAAAA AAA-3'; (SEQ ID No. 9)

[0051] 2) The different DNA sequences obtained in step 1) were added to 10mM PB buffer (pH 7.2-7.4) with silver nitrate and potassium tetrachloroplatinate in a molar ratio of 1:6:12. The mixture was shaken for 30s-2min and incubated in ice water for 20-30min. Then, freshly prepared sodium borohydride solution was added with a molar ratio of silver nitrate to sodium borohydride of 1:20. The mixture was shaken vigorously for 30s-2min and incubated at 25℃ for 5h to obtain DNA-Ag / Pt NCs nanozyme.

[0052] 3) Detection of oxidase catalytic activity

[0053] The DNA-Ag / Pt NCs nanozyme (160 μL, 100 nM, calculated based on the concentration of synthesized DNA) prepared using different DNA sequences in step 2) above was added to PB buffer solution (100 μL, pH = 7.4), followed by acetate buffer (1980 μL, pH = 4), hydrogen peroxide (320 μL, 50 mM), and 3,3',5,5'-tetramethylbenzidine (640 μL, 4 mM). The above mixed solution system was tested on a UV-Vis spectrophotometer with a selected wavelength range of 750-500 nm. The absorbance at the maximum absorption peak of 652 nm was collected and labeled Abs0. The results are as follows: Figure 2 As shown.

[0054] 4) AβO response performance testing

[0055] The DNA-Ag / Pt NCs nanozyme (160 μL, 100 nM, calculated based on the concentration of synthesized DNA) prepared using different DNA sequences in step 2) above was added to PB buffer solution (60 μL, pH = 7.4), and then incubated with AβO (40 μL, 1 mg / mL) at 25 °C for 2 h. Next, acetate buffer (1980 μL, pH = 4), hydrogen peroxide (320 μL, 50 mM), and 3,3',5,5'-tetramethylbenzidine (640 μL, 4 mM) were added to the incubated solution, and the reaction was carried out for 2 h. The above mixed solution system was tested on a UV-Vis spectrophotometer with a selected wavelength range of 750-500 nm. The absorbance at the maximum absorption peak of 652 nm was collected, labeled as Abs, and then normalized. The normalization process involved defining the maximum absorption peak intensity of the detection solution system without AβO as Abs0 and the maximum absorption peak intensity of the detection solution system containing AβO as Abs. The Abs / Abs0 ratio was used as the relative absorbance value, which is also the AβO response value of the nanozyme. The results are as follows: Figure 2 As shown.

[0056] from Figure 2 As can be seen, the larger the Abs0 value, the higher the catalytic activity of the nanozyme itself; the lower the Abs / Abs0 value, the more sensitive the nanozyme is to AβO. Therefore, the optimal DNA sequence was selected as SEQ ID No.6 (i.e., the 5' end of SEQ ID No.3 is linked to SEQ ID No.1). At this time, the nanozyme synthesized using this DNA sequence has both the best catalytic activity and the best AβO response sensitivity.

[0057] Example 2

[0058] 1) Synthetic DNA sequence:

[0059] 5'-CCCTTAATCCCCGCCTGTGGTGTTGGGGCGGGTGCGTTTTTTTTTT-3'; (SEQ ID No. 6)

[0060] 2) Add the different DNA sequences obtained in step 1), silver nitrate, and potassium tetrachloroplatinate to 10mM PB buffer (pH 7.2-7.4) in a molar ratio of 1:6:12, shake for 30s-2min, incubate in ice water for 20-30min, then add freshly prepared sodium borohydride solution (silver nitrate to sodium borohydride molar ratio of 1:30), shake vigorously for 30s-2min, and incubate at 25℃ for 5h to obtain DNA-Ag / Pt NCs nanozyme.

[0061] Aberration-corrected transmission electron microscopy (TEM) image of the DNA-Ag / Pt NCs nanozyme synthesized in the above steps is shown below. Figure 3 As shown, by Figure 3 The prepared DNA-silver-platinum bimetallic nanozymes are spherical in appearance, with a particle size of approximately 0.8-5 nm. The EDS spectrum of the DNA-Ag / Pt NCs nanozymes is shown below. Figure 4 As shown, the nanozyme is composed of Pt and Ag elements, and the atomic ratio of the elements is approximately 2.6:1.

[0062] Example 3

[0063] 1) Synthetic DNA sequence:

[0064] 5'-CCCTTAATCCCCGCCTGTGGTGTTGGGGCGGGTGCGTTTTTTTTTT-3'; (SEQ ID No. 6)

[0065] 2) The different DNA sequences obtained in step 1) were added to 10mM PB buffer (pH 7.2-7.4) with silver nitrate and potassium tetrachloroplatinate in a molar ratio of 1:6:1.2. The mixture was shaken for 30s-2min and incubated in ice water for 20-30min. Then, freshly prepared sodium borohydride solution was added with a molar ratio of silver nitrate to sodium borohydride of 1:20. The mixture was shaken vigorously for 30s-2min and incubated at 25℃ for 6h to obtain DNA-Ag / Pt NCs nanozyme.

[0066] Example 4

[0067] 1) Synthetic DNA sequence:

[0068] 5'-CCCTTAATCCCCGCCTGTGGTGTTGGGGCGGGTGCGTTTTTTTTTT-3'; (SEQ ID No. 6)

[0069] 2) The different DNA sequences obtained in step 1) were added to 10mM PB buffer (pH 7.2-7.4) with silver nitrate and potassium tetrachloroplatinate in a molar ratio of 1:6:120. The mixture was shaken for 30s-2min and incubated in ice water for 20-30min. Then, freshly prepared sodium borohydride solution was added with a molar ratio of silver nitrate to sodium borohydride of 1:40. The mixture was shaken vigorously for 30s-2min and incubated at 25℃ for 6h to obtain DNA-Ag / Pt NCs nanozyme.

[0070] Application Example 1: Detection of the response of β-amyloid oligomers (AβO).

[0071] The DNA-Ag / Pt NCs nanozyme (160 μL, 100 nM, calculated based on the concentration of synthesized DNA) prepared in Example 2 above was incubated with 100 μL of AβO at different concentrations (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.9, 1.1, 1.4, 2.1, 2.8, 3.5 μM) at 25 °C for 2 h, repeated 3 times. Then, acetate buffer (1980 μL, pH=4), hydrogen peroxide (320 μL, 50 mM), and 3,3',5,5'-tetramethylbenzidine (640 μL, 4 mM) were added to the incubated solution, and the reaction was allowed to proceed for 2 h. The above mixed solution system was tested on a UV-Vis spectrophotometer with a wavelength range of 750-500 nm, and the color change was observed. The results are as follows: Figure 5a and Figure 5b As shown. The results indicate that the DNA-Ag / Pt NCs nanozyme can catalyze the oxidation of TMB in the presence of H2O2, producing a significant UV-Vis characteristic absorption peak at 652 nm. Furthermore, the system color changes significantly from dark blue to light blue with increasing AβO concentration. The absorbance at the maximum absorption peak of 652 nm was collected and normalized. The normalization process was as follows: the maximum absorption peak intensity of the detection solution system without AβO (0 μM) was defined as Abs0, and the maximum absorption peak intensity of the detection solution systems containing different AβO concentrations (concentration labeled as x) was defined as Abs... x , with Abs x The / Abs0 value is used as a relative absorption value, such as Figure 5c As shown, the absorption intensity at 652 nm gradually decreases with increasing AβO concentration. The AβO concentration exhibits a good linear relationship in the range of 0 to 0.7 μM, and the linear equation is: Abs x / Abs0=-0.59497cAβO+0.96936,R 2 =0.995, detection limit is 1.09 nM.

[0072] Application Example 2: Interference-resistant detection of β-amyloid oligomers (AβO).

[0073] The DNA-Ag / Pt NCs nanozyme prepared in Example 2 was added to 60 μL of PB buffer solution. Accurately prepare standard solutions of 1 mg / ml thrombin, bovine serum albumin (BSA), trypsin, AβM, AβF, and AβO. Add 40 μL of each of the above-mentioned thrombin, BSA, trypsin, Aβ monomer (AβM), Aβ fibrils (AβF), AβO, and PB buffer to the system, and incubate at 25°C for 2 h. Then, add acetate buffer (1980 μL, pH=4), hydrogen peroxide (320 μL, 50 mM), and 3,3',5,5'-tetramethylbenzidine (640 μL, 4 mM) to the incubated solution and react for 2 h. Test the samples with different interfering substances on a UV-Vis spectrophotometer, selecting a wavelength range of 750-500 nm, repeating the test three times. The results are shown below. Figure 6 As shown. The maximum absorption peak intensity of the detection solution system with 40 μL Pb (blank) added is labeled as Abs0, and the maximum absorption peak intensity of the detection solution system containing different components (concentration labeled as x) is labeled as Abs. x Abs x The closer the / Abs0 value is to 1, the less interference exists. Figure 6 As can be seen, the absorption intensity at 652 nm decreased significantly after the addition of AβO, while the addition of other substances did not significantly interfere with the absorption of the nanozyme, indicating that the nanozyme has good selectivity.

[0074] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is impossible to exhaustively list all the implementation methods here. All obvious variations or modifications derived from the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. A method for preparing a DNA-silver-platinum bimetallic nanozyme for the detection of β-amyloid oligomers, characterized in that, The method includes the following steps: (1) A synthetic DNA sequence comprising a β-amyloid oligomer aptamer sequence and a DNA template sequence for synthesizing silver nanoclusters attached to its 5' end; the DNA template sequence for synthesizing silver nanoclusters is shown in SEQ ID No. 1; the β-amyloid oligomer aptamer is shown in SEQ ID No. 3; (2) Add silver nitrate solution, potassium tetrachloroplatinate solution and the DNA sequence from step (1) to a buffer solution, shake for 30s-2min, incubate in ice water for 20-30min, then add sodium borohydride, shake for 30s-2min, and incubate at room temperature for 3-6h to obtain DNA-silver-platinum bimetallic nanozyme; the buffer solution is a chloride-free phosphate buffer with pH 7.2-7.4; the molar ratio of the DNA sequence to silver nitrate is 1:6; the molar ratio of potassium tetrachloroplatinate to silver nitrate is 0.2:1-10:1; the molar ratio of silver nitrate to sodium borohydride is 1:1-1:

40.

2. A DNA-silver-platinum bimetallic nanozyme prepared by the preparation method described in claim 1.

3. The DNA-silver-platinum bimetallic nanozyme according to claim 2, characterized in that, The DNA-silver-platinum bimetallic nanozyme is spherical in shape with a particle size of 0.8-5 nm.

4. The application of the DNA-silver-platinum bimetallic nanozyme as described in claim 2 or 3 in the preparation of products for detecting β-amyloid oligomers.

5. The application according to claim 4, characterized in that, The DNA-silver-platinum bimetallic nanozyme was added to the sample to be tested and incubated. Then, acetate buffer, hydrogen peroxide and 3,3',5,5'-tetramethylbenzidine were added to react. The color change of the reaction solution was observed and its absorbance was tested.

6. The application according to claim 5, characterized in that, In the reaction system, the final concentration of the DNA-silver-platinum bimetallic nanozyme is 5 nM, the final concentration of hydrogen peroxide is 5 mM, and the final concentration of 3,3',5,5'-tetramethylbenzidine is 0.8 mM.

7. The application according to claim 5, characterized in that, The incubation conditions are: room temperature incubation for 1-2 hours; the reaction conditions are: temperature 20-30℃, time 1-2 hours.

8. The application according to claim 5, characterized in that, When the color of the reaction solution exhibits a visible hypochromic effect, changing from dark blue to light blue, and the absorption peak of the ultraviolet-visible spectrum at 652 nm continuously decreases, it indicates that the sample contains β-amyloid oligomers.

Images