A DNA-mediated synthesized platinum-coated gold nanobicone and its preparation method and application

Abstract

The present invention relates to the technical field of nano-biosensors, and discloses a DNA-mediated synthesized platinum-coated gold nano-bicone colorimetric sensor, a preparation method thereof and an application thereof. The preparation method comprises the following steps: adding a DNA sequence into a gold nano-bicone solution for incubation, then adding H2PtCl6 and ascorbic acid, and incubating again to obtain platinum-coated gold nano-bicones. The obtained platinum-coated gold nano-bicones have peroxidase activity, can be used for preparing peroxidase catalysts, and can be used for preparing colorimetric sensors for detecting alkaline phosphatase. The detection process has mild conditions, does not require other reagents, realizes the convenient and rapid detection of alkaline phosphatase activity, has low detection cost, simple operation, sensitive response to alkaline phosphatase, a detection limit as low as 0.35 mU / mL, and a detectable range as wide as 0-12.5 mU / mL; the measurement results of actual samples are accurate and reliable, and the detection of alkaline phosphatase activity in human serum can be realized.

Description

Technical Field

[0001] The present invention belongs to the field of nanobiosensors, and specifically relates to a preparation method and application of a DNA-mediated synthesized platinum-coated gold nanobiconical colorimetric sensor. Background Art

[0002] Alkaline phosphatase (ALP) is a biological enzyme widely distributed in human liver, bone, intestine, kidney and placenta. This enzyme can catalyze the removal of one phosphate group from polyphosphorylated substances (such as nucleic acids, proteins and small molecules). A large number of studies have shown that abnormal ALP activity is closely related to various diseases, including bone injury, liver dysfunction and prostate cancer. Therefore, it is commonly used as a clinical biomarker for the diagnosis and treatment of these diseases. In addition, ALP is often used in various enzyme-linked immunosorbent assays (ELISAs). Based on these extensive demands, it is of great significance to develop effective, low-cost and easy-to-use ALP activity analysis methods. Currently, the detection methods of alkaline phosphatase mainly include fluorescence method, electrochemical method, colorimetric method, etc.

[0003] The emergence of nanozymes is a major progress in the field of artificial enzyme mimics, breaking through the limitation of low catalytic efficiency of previous artificial enzyme mimics. Currently, it has been widely applied to many fields such as disease diagnosis and treatment, environmental monitoring and sewage treatment. In recent years, people have found that a variety of nanomaterials have natural enzyme activity, and the nanomaterials with peroxidase-like activity have been applied to the preparation of colorimetric biosensors. By virtue of the catalytic ability of the nanomaterials themselves, simulating peroxidase-like activity, and giving play to their advantages such as low cost, high stability and easy storage, the catalytic of biochemical reactions can be directly realized, and then the target can be quickly and sensitively qualitatively and quantitatively analyzed by naked-eye observation or by detecting the change of the absorbance of the solution with a spectrophotometer. Summary of the Invention

[0004] The present invention aims to provide a DNA-mediated synthesized platinum-coated gold nanobicone and its preparation method; another object of the present invention is to provide the application of the above platinum-coated gold nanobicone and a method for detecting alkaline phosphatase.

[0005] The technical solution of the present invention is a preparation method of a DNA-mediated synthesized platinum-coated gold nanobiconical colorimetric sensor, including the following steps:

[0006] Add a DNA sequence into a gold nanobicone solution and incubate for 15 - 60 min; then add H2PtCl6 and ascorbic acid (AA), and incubate again for 60 - 300 min to obtain a DNA-mediated synthesized platinum-coated gold nanobicone. The gold nanobicone has a biconical structure, with a length of 60 - 100 nm and a width of 25 - 50 nm.

[0007] Preferably, the incubation time of the DNA sequence with the gold nanobipyramid solution is 25 - 40 min. In a preferred embodiment of the present invention, the incubation time is 30 min; after adding H2PtCl6 and ascorbic acid, the incubation time is 75 - 120 min. In a preferred embodiment of the present invention, the incubation time is 90 min.

[0008] In the reaction system, the molar ratio of gold nanobipyramids to the DNA sequence and H2PtCl6 is 1:15000 - 40000:1600 - 2400, preferably 1:28000 - 32000:1700 - 2000; the molar ratio of H2PtCl6 to ascorbic acid is 1:2 - 4, preferably 1:2.6 - 3.2. In a preferred embodiment of the present invention, the molar ratio of H2PtCl6 to ascorbic acid is 1:3.

[0009] The concentration of gold nanobipyramids in the reaction system is 0.06 - 0.15 nmol / L, preferably 0.08 - 0.12 nmol / L. In a preferred embodiment of the present invention, the concentration of gold nanobipyramids is 0.09 - 0.1 nmol / L.

[0010] The DNA sequence is Poly T, Poly C or Poly A, preferably Poly T; its length is 12 - 20 nucleotides, preferably 15 nucleotides. In a preferred embodiment of the present invention, the DNA sequence is Poly T. 15 。

[0011] The preparation method of the gold nanobipyramids is as follows:

[0012] (1) Add a NaBH4 solution to a solution containing HAuCl4, a surfactant, and citric acid, stir, and then heat at 60 - 90 °C for 1.5 - 4 h to form gold seeds;

[0013] The molar ratio of HAuCl4 to citric acid, NaBH4, and the surfactant is 1:15 - 30:2 - 4:150 - 300. In a preferred embodiment of the present invention, the molar ratio is 1:20:2.5:200; the content of HAuCl4 in the reaction system is 0.1 - 0.4 mol / L, preferably 0.2 - 0.25 mol / L; preferably, the surfactant is cetyltrimethylammonium chloride CTAC;

[0014] (2) Add the above gold seed solution to a mixed solution containing a surfactant, HAuCl4, AgNO3, HCl, and ascorbic acid AA, react at 20 - 40 °C for 1.5 - 4 h, and centrifuge at 6000 - 15000 rpm to collect the precipitate and wash to obtain gold nanobipyramids; in a preferred embodiment of the present invention, react at 30 °C for 2 h;

[0015] In terms of Au, the molar ratio of the gold seeds to HAuCl4, AgNO3, ascorbic acid, and surfactant is 1:30 - 50:6 - 15:60 - 90;

[0016] The HCl content in the reaction system is 0.01 - 0.025 mol / L, preferably 0.015 - 0.02 mol / L; the surfactant content is 0.06 - 0.12 mol / L, preferably; the gold seed content is 0.01 - 0.025 mmol / L, preferably 0.01 - 0.015 mmol / L; the surfactant content is 0.06 - 0.15 mol / L, preferably 0.08 - 0.1 mol / L; the surfactant is cetyltrimethylammonium chloride CTAC.

[0017] A kind of platinum-coated gold nanobicone prepared by the above method, with platinum nanoparticles distributed on the surface of the gold nanobicone; the particle size of the platinum nanoparticles is 0.5 - 5 nm, the gold nanobicone has a biconical structure, its length is 60 - 100 nm, and its width is 25 - 50 nm.

[0018] The above platinum-coated gold nanobicone has peroxidase activity, can be used to prepare peroxidase catalysts, and can be used to prepare colorimetric sensors for detecting alkaline phosphatase.

[0019] A method for detecting alkaline phosphatase, comprising the following steps:

[0020] (1) Mix the sample to be tested with L-ascorbic acid-2-phosphate and a buffer solution with pH = 7 - 7.4 and incubate;

[0021] (2) Add platinum-coated gold nanobicone, TMB, H2O2, and a buffer solution with pH = 10 - 11, and observe the color change or detect the absorbance at 652 nm after incubation.

[0022] Compare the detection result with a blank sample or a standard curve to obtain qualitative or quantitative results.

[0023] In step (1), the concentration of L-ascorbic acid-2-phosphate is 2.5 - 3 mol / L, and incubate at 35 - 40 °C for 20 - 60 min; preferably incubate at 37 °C for 30 min; the buffer solution is Tris-HCl buffer solution with pH 7.4.

[0024] In step (2), the concentration of the gold-coated platinum nanobipyramids in the reaction system is 4-10 μg / mL, the concentration of TMB is 0.4-1 mmol / L, the concentration of H2O2 is 0.15-0.25 mmol / L, the buffer solution is acetic acid-acetate buffer solution, and the preferred pH is 4 and the concentration is 10 mmol / L. In a preferred embodiment of the present invention, the concentration of the gold-coated platinum nanobipyramids is 5 μg / mL, the concentration of TMB is 0.5 mmol / L, the concentration of H2O2 is 0.2 mmol / L, and the buffer solution is acetic acid-acetate buffer solution with pH = 4 and concentration 10 mmol / L.

[0025] Using the DNA-mediated synthesized gold-coated platinum nanobipyramids of the present invention to detect alkaline phosphatase, the detectable activity range of the alkaline phosphatase sample to be detected is 0-12.5 mU / mL, and the detection limit is as low as 0.35 mU / mL.

[0026] The beneficial effects of the present invention are as follows: The detection process of the present invention has mild conditions, does not require other reagents, realizes the convenient and rapid detection of alkaline phosphatase activity, has low detection cost and simple operation; the colorimetric sensor for detecting alkaline phosphatase activity by DNA-mediated synthesized gold-coated platinum nanobipyramids has a detection limit as low as 0.35 mU / mL and a detectable range as wide as 0-12.5 mU / mL; using the colorimetric sensor for detecting alkaline phosphatase activity by DNA-mediated synthesized gold-coated platinum nanobipyramids, it is sensitive to alkaline phosphatase, the measurement results of actual samples are accurate and reliable, and the detection of alkaline phosphatase activity in human serum is realized. Description of the Drawings

[0027] Figure 1 Flow chart of synthesizing Au NBPs@Pt solution mediated by different sequences of DNA;

[0028] Figure 2 TEM image of AuNBPs prepared in Example 1;

[0029] Figure 3 TEM image of Au NBPs@Pt prepared in Example 1;

[0030] Figure 4 UV-Vis spectrogram of Au NBPs@Pt prepared in Example 1;

[0031] Figure 5 For Example 2 using A 15 and C 15 TEM image of Au NBPs@Pt mediated for preparation;

[0032] Figure 6 Comparison of peroxidase activities of Au NBPs and Au NBPs@Pt prepared by different methods;

[0033] Figure 7 Schematic diagram of a DNA-mediated synthesized platinum-coated gold nanobicone colorimetric sensor for detecting alkaline phosphatase;

[0034] Figure 8 UV-Vis change spectra of adding a DNA-mediated synthesized platinum-coated gold nanobicone colorimetric sensor to alkaline phosphatase with different activities;

[0035] Figure 9 Linear relationship diagram of A652 and alkaline phosphatase activity in the present invention; The inset is the digital photo of the solution when adding different concentrations of ALP;

[0036] Figure 10 Selectivity of a DNA-mediated synthesized platinum-coated gold nanobicone colorimetric sensor for detecting alkaline phosphatase activity. Detailed implementation mode

[0037] In order to make the technical means, creative features, achieved purposes and functions of the present invention easy to understand, the present invention will be further described below in conjunction with specific embodiments.

[0038] Example 1

[0039] (1) Preparation of gold seeds

[0040] Add 10 mL of 50 mM cetyltrimethylammonium chloride CTAC, 0.25 mL of 10 mM chloroauric acid and 0.25 mL of 0.2 M citric acid solution into a 100 mL beaker (washed and dried with ultrapure water for standby), mix them evenly, and then inject 0.25 mL of freshly prepared 25 mM NaBH4 and stir rapidly for 1 min. The solution changes from colorless to brown. Then, the solution is heated at 80 °C for 2 hours, and its color changes from brown to red, indicating the formation of gold seeds.

[0041] (2) Preparation of gold nanobicones

[0042] Inject 5 mL of gold seed solution into a solution containing 100 mL of 0.1 M cetyltrimethylammonium bromide CTAB, 2 mL of 1 M HCl, 5 mL of 10 mM HAuCl4, 1 mL of 10 mM AgNO3 and 0.8 mL of 100 mM ascorbic acid AA, and react at 30 °C for 2 hours. The obtained gold nanobicone Au NBPs solution is centrifuged at 10000 rpm for 10 min, the supernatant is discarded, and the precipitate is redispersed in deionized water.

[0043] Figure 2 Transmission electron microscope image of gold nanobicone Au NBPs, showing that the length of the gold nanobicone is about 60 - 100 nm and the width is about 20 - 35 nm.

[0044] (3) Synthesis of DNA-Mediated Platinum-Coated Gold Nanobipyramids

[0045] The synthesis process of DNA-mediated platinum-coated gold nanobipyramids is as Figure 1 follows. To 270 μL of 0.1 nM re-dispersed gold nanobipyramid solution, 5.4 μL of 150 μM DNA sequence T 15 was added and incubated for 30 min. Then, 5 μL of 10 mM H2PtCl6 and 1.5 μL of 0.1 M ascorbic acid AA were added and incubated for 90 min. After centrifugation and washing, the precipitate was dispersed in deionized water to obtain the DNA-mediated synthesized platinum-coated gold nanobipyramid Au NBPs@Pt solution.

[0046] The transmission electron microscope image of platinum-coated gold nanobipyramid Au NBPs@Pt is as Figure 3 shown, showing Pt nanoparticles attached to the gold nanobipyramids with a particle size of about 0.5 - 5 nm.

[0047] The UV-Vis spectrum of Au NBPs@Pt is as Figure 4 shown, showing absorption peaks near 530 nm and 760 nm.

[0048] Example 2

[0049] Using A 15 or C 15 to replace sequence T 15 , and the rest the same as in Example 1, the radio microscope images of the obtained products are as Figure 5 shown in A and B, and no obvious Pt nanoparticles are seen on the surface of the gold nanobipyramids.

[0050] The peroxidase activities of the gold nanobipyramids and the products prepared in Examples 1 and 2 were detected by the following method:

[0051] Add 10 μL of 100 μg / mL Au NBPs@Pt solution obtained by different DNA sequences mediation, or 10 μL of 100 μg / mL gold nanobipyramid solution to the wells, then add 10 μL of 10 mM TMB and 10 μL of 4 M H2O2, and make up the volume to 200 μL with 10 mM acetic acid-sodium acetate buffer at pH 4. After mixing the system, react for 10 min, and measure the ultraviolet absorption spectrum of the mixed solution with an ultraviolet-visible absorption spectrophotometer, and record the absorbance at a wavelength of 652 nm.

[0052] The comparison of peroxidase-like activities of gold nanobipyramids and DNA sequences T 15 , A 15 and C 15 mediated platinum-coated gold nanobipyramid Au NBPs@Pt is as Figure 6, The results showed that the gold nanobipyramids had no peroxidase activity, and the obtained platinum-coated gold nanobipyramids mediated by the DNA sequence T 15 had the highest peroxidase activity.

[0053] Example 3

[0054] Using the polyT in Example 1 15 mediated platinum-coated gold nanobipyramid nanozyme Au NBPs@Pt as a colorimetric sensor to detect the activity of alkaline phosphatase, and the process and principle are as Figure 7 shown. Hydrogen peroxide reacts with TMB under the action of the nanozyme to generate water and oxidized TMB, which shows blue. Alkaline phosphatase decomposes ascorbic acid diphosphate, and the generated ascorbic acid reduces oxidized TMB to TMB, and the color of the reaction system becomes lighter, realizing visual detection or quantitative detection by detecting its absorbance.

[0055] The steps are as follows:

[0056] (1) Add 50 μL of alkaline phosphatase with different activities and 150 μL of pH 7.4 Tris-HCl buffer containing 3.75 mM L-ascorbic acid-2-phosphate to a 1.5 mL centrifuge tube, and incubate at 37 °C for 30 min;

[0057] (2) Sequentially add 10 μL of 100 μg / mL Au NBPs@Pt solution, 10 μL of 10 mM TMB, 10 μL of 4 M H2O2, and 10 mM acetic acid-sodium acetate buffer with pH 4 to the well, mix the system and react for 10 min; add 5 μL of the mixed solution in (1) to the system and mix well; the total volume of the system is 200 μL; the final activities of alkaline phosphatase in the system are 0.625 mU / mL, 1.25 mU / mL, 2.5 mU / mL, 3.75 mU / mL, 5 mU / mL, 5.625 mU / mL, 7.5 mU / mL, 10 mU / mL, 12.5 mU / mL, and 0 mU / mL, respectively; measure the ultraviolet absorption spectrum of the mixed solution with a UV-visible absorption spectrophotometer, and record the absorbance at a wavelength of 652 nm;

[0058] (3) Record the absorbance of oxTMB at a wavelength of 652 nm, and the results are as Figure 8 shown, indicating that as the activity of ALP increases, the absorbance of the solution gradually decreases;

[0059] And draw the absorbance fitting line at 652 nm of the ultraviolet spectrum with TMB as the chromogenic agent, that is, the alkaline phosphatase activity-absorbance standard working curve, as Figure 9 shown, indicating that this method has excellent detection effect on the activity of ALP; Figure 9The illustration is a digital photo of the solution with different concentrations of ALP added. It can be seen that as the activity of alkaline phosphatase increases, the color of the reaction system gradually fades, so visual detection can be achieved;

[0060] (4) The alkaline phosphatase sample to be tested is operated according to steps (1) and (2), and the absorbance at a wavelength of 652 nm with TMB as the substrate is measured respectively using a UV-visible absorption spectrophotometer. Through calculation and comparison with the standard working curve, the activity of alkaline phosphatase can be obtained.

[0061] Example 4

[0062] The selectivity verification of the platinum-coated gold nanobipyramid as a colorimetric sensor for detecting the activity of alkaline phosphatase is as follows:

[0063] (1) Add 50 μL of different kinds of amino acids or proteases and 150 μL of Tris-HCl buffer solution with pH 7.4 containing 3.75 mM L-ascorbic acid-2-phosphate to a 1.5 mL centrifuge tube, and incubate at 37 °C for 30 min; at the same time, set up an experimental group with alkaline phosphatase added for comparison;

[0064] (2) Add 10 μL of 100 μg / mL Au NBPs@Pt solution, 10 μL of 10 mM TMB, 10 μL of 4 M H2O2, and 10 mM sodium acetate buffer solution with pH 4 to the well in sequence. After mixing the system, react for 10 min; add 5 μL of the mixed solution in (1) to the system and mix evenly to make the total volume of the system 200 μL; measure the absorbance of the above mixed solution at 652 nm using a UV-visible absorption spectrophotometer. The results are as Figure 10 .

[0065] Figure 10 is a bar chart of the selectivity of using a DNA-mediated platinum-coated gold nanobipyramid colorimetric sensor to detect the activity of alkaline phosphatase. From left to right in the bar chart are acetylcholinesterase (ACHE), pepsin (Pep), papain (Pap), lysine (Lys), tryptophan (Tyr), glucose oxidase (GOx), glutathione reductase (GR), and blank (Blank).

[0066] From Figure 10 it can be seen that only in the presence of alkaline phosphatase does the absorbance of A 652 decrease. Without adding alkaline phosphatase, the absorbance of A 652 does not change. That is to say, other amino acids or proteases will not affect the detection of alkaline phosphatase, indicating that this sensor can complete the colorimetric quantitative detection of ALP activity with high selectivity and stability.

[0067] The foregoing has shown and described the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments, and there will be various changes and improvements without departing from the spirit and scope of the present invention, and these changes and improvements fall within the scope of the present invention claimed. The scope of the present invention claimed is defined by the appended claims and their equivalents.

Claims

1. A method for DNA-mediated synthesis of platinum-coated gold nanobipyramids, characterized in that, It includes the following steps: Add the DNA sequence into the gold nanobipyramid solution for incubation, then add H2PtCl6 and ascorbic acid, and incubate again to obtain platinum-coated gold nanobipyramids; The DNA sequence is Poly T with a length of 12 - 20 nucleotides; The preparation method of the gold nanobipyramids is as follows: (1) Add the NaBH4 solution into the solution containing HAuCl4, surfactant and citric acid, stir and heat at 60 - 90 °C for 1.5 - 4 h to form gold seeds; The molar ratio of HAuCl4 to citric acid, NaBH4 and surfactant is 1:15 - 30:2 - 4:150 - 300; the content of HAuCl4 in the reaction system is 0.1 - 0.4 mol / L; (2) Add the above gold seed solution into the mixed solution containing surfactant, HAuCl4, AgNO3, HCl and ascorbic acid AA, react at 20 - 40 °C for 1.5 - 4 h, centrifuge at 6000 - 15000 rpm, take the precipitate and wash to obtain gold nanobipyramids; Calculated by Au, the molar ratio of gold seeds to HAuCl4, AgNO3, ascorbic acid and surfactant is 1:30 - 50:6 - 15:60 - 90.

2. The method according to claim 1, wherein The gold nanobipyramids have a bipyramidal structure, with a length of 60 - 100 nm and a width of 25 - 50 nm.

3. The method according to claim 1, wherein In the reaction system, the molar ratio of gold nanobipyramids to DNA sequence, H2PtCl6 is 1:15000 - 40000:1600 - 2400, and the molar ratio of H2PtCl6 to ascorbic acid is 1:2 - 4; the concentration of gold nanobipyramids in the reaction system is 0.06 - 0.15 nmol / L.

4. The method according to claim 1, wherein In the reaction system of step (2), the content of HCl is 0.01 - 0.025 mol / L, and the content of surfactant is 0.06 - 0.15 mol / L.

5. A platinum-coated gold nanobicone, characterized in that, Prepared by the method according to any one of claims 1 - 4, platinum nanoparticles are distributed on the surface of the gold nanobipyramids. The particle size of the platinum nanoparticles is 0.5 - 5 nm, and the gold nanobipyramids have a bipyramidal structure, with a length of 60 - 100 nm and a width of 25 - 50 nm.

6. Application of the platinum-coated gold nanobipyramids according to claim 5 in the preparation of peroxidase nanozymes or colorimetric sensors.

7. Application of the platinum-coated gold nanobipyramids according to claim 5 in the detection of alkaline phosphatase or the preparation of alkaline phosphatase detection reagents.

8. A reagent or kit for detecting alkaline phosphatase, characterized in that, It contains the platinum-coated gold nanobipyramids according to claim 5.

9. A method for detecting alkaline phosphatase, characterized in that, It includes the following steps: (1) Mix the sample to be tested with L-ascorbic acid-2-phosphate and a buffer solution with pH = 7 - 7.4 and incubate; (2) Add the platinum-coated gold nanobipyramids according to claim 5, TMB, H2O2, and a buffer solution with pH = 10 - 11, and observe the color change or detect the absorbance at 652 nm after incubation.

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