CN116237535A - Platinum-coated gold nano bipyramid synthesized by DNA (deoxyribonucleic acid) mediation and preparation method and application thereof - Google Patents

Platinum-coated gold nano bipyramid synthesized by DNA (deoxyribonucleic acid) mediation and preparation method and application thereof Download PDF

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CN116237535A
CN116237535A CN202310130103.9A CN202310130103A CN116237535A CN 116237535 A CN116237535 A CN 116237535A CN 202310130103 A CN202310130103 A CN 202310130103A CN 116237535 A CN116237535 A CN 116237535A
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鲁娜
蔡梦超
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Shanghai University of Engineering Science
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Abstract

The invention relates to the technical field of nano biosensors, and discloses a platinum-coated gold nano bipyramid colorimetric sensor synthesized by DNA (deoxyribonucleic acid) mediation, a preparation method and application thereof. The preparation method comprises the following steps: adding DNA sequence into gold nanometer bipyramidIncubating in solution, and adding H 2 PtCl 6 And ascorbic acid, and incubating again to obtain the platinum-coated gold nano bipyramid. The obtained platinum-coated gold nano bipyramid has peroxidase activity, can be used for preparing a peroxidase catalyst, can be used for preparing a colorimetric sensor, and is used for detecting alkaline phosphatase. The detection process has mild conditions, does not need other reagents, realizes convenient and rapid detection of the activity of alkaline phosphatase, has low detection cost, simple operation and sensitive response to alkaline phosphatase, has the detection limit as low as 0.35mU/mL and has the detection range as wide as 0-12.5 mU/mL; the measurement result of the actual sample is accurate and reliable, and the detection of the alkaline phosphatase activity in human serum can be realized.

Description

Platinum-coated gold nano bipyramid synthesized by DNA (deoxyribonucleic acid) mediation and preparation method and application thereof
Technical Field
The invention belongs to the field of nano biological sensors, and particularly relates to a preparation method and application of a platinum-coated gold nano bipyramid colorimetric sensor synthesized by DNA (deoxyribonucleic acid) mediation.
Background
Alkaline phosphatase (ALP) is a biological enzyme that is widely distributed in the liver, bones, intestines, kidneys and placenta of humans and is capable of catalyzing the removal of phosphate groups of one molecule from polyphosphorylated substances such as nucleic acids, proteins and small molecules. Numerous studies have shown that abnormalities in ALP activity are closely associated with a variety of diseases including bone injury, liver dysfunction and prostate cancer. Therefore, it is commonly used as a clinical biomarker for diagnosis and treatment of these diseases. In addition, ALP is often used in various enzyme-linked immunosorbent assays (ELISA). Based on these broad needs, it is important to develop an effective, low cost and easy to use assay for ALP activity. The existing alkaline phosphatase detection methods mainly comprise a fluorescence method, an electrochemical method, a colorimetric method and the like.
The appearance of nano enzyme is a great progress in the field of artificial simulated enzyme research, breaks through the limitation of low catalytic efficiency of the conventional artificial simulated enzyme, and is widely applied to a plurality of fields such as disease diagnosis and treatment, environmental monitoring, sewage treatment and the like. In recent years, various nanomaterials have been found to have natural enzymatic activity, and nanomaterials having peroxide-like activity have been applied to the preparation of colorimetric biosensors. By means of the catalytic capability of the nano material, the method simulates the activity of the peroxidase-like enzyme, exerts the advantages of low cost, high stability, easiness in storage and the like, directly realizes the catalysis of biochemical reaction, and further carries out quick and sensitive qualitative and quantitative analysis on the target object by naked eye observation or detection of the change of the absorbance of the solution by a spectrophotometer.
Disclosure of Invention
The invention aims to provide a platinum-coated gold nano bipyramid synthesized by DNA mediation and a preparation method thereof; the invention also aims to provide application of the platinum-coated gold nano bipyramid and a method for detecting alkaline phosphatase.
The technical scheme of the invention is that the preparation method of the platinum-coated gold nano bipyramid colorimetric sensor synthesized by DNA mediation comprises the following steps:
adding the DNA sequence into a gold nano bipyramid solution for incubation for 15-60min; then add H 2 PtCl 6 And Ascorbic Acid (AA), and incubating for 60-300min again to obtain the platinum-coated gold nano bipyramid synthesized by the DNA mediation. The gold nanometer bipyramid has a bipyramid structure, the length of the bipyramid is 60-100nm, and the width of the bipyramid is 25-50nm.
Preferably, the incubation time of the DNA sequence and the gold nano bipyramid solution is 25-40min, and in a preferred mode of the invention, the incubation time is 30min; adding H 2 PtCl 6 And an incubation time of 75-120min after ascorbic acid, in a preferred mode of the invention, the incubation time is 90min.
In the reaction system, gold nanometer bipyramid, DNA sequence and H 2 PtCl 6 The molar ratio of (2) is 1:15000-40000:1600-2400, preferably 1:28000-32000:1700-2000; h 2 PtCl 6 The molar ratio of the sodium ascorbate to the ascorbic acid is 1:2-4, preferably 1:2.6-3.2, in a preferred embodiment of the invention, H 2 PtCl 6 The molar ratio of the sodium ascorbate to the ascorbic acid is 1:3.
the concentration of the gold nano bipyramid in the reaction system is 0.06-0.15nmol/L, preferably 0.08-0.12nmol/L, and in a preferred mode of the invention, the concentration of the gold nano bipyramid is 0.09-0.1nmol/L.
The DNA sequence is Poly T, poly C or Poly A, preferably Poly T; it is 12-20 nucleotides in length, preferably 15 nucleotides in length. In a preferred embodiment of the invention, the DNA sequence is Poly T 15 。
The preparation method of the gold nanometer bipyramid comprises the following steps:
(1) To contain HAuCl 4 Adding NaBH to a solution of surfactant and citric acid 4 Heating the solution at 60-90 deg.c for 1.5-4 hr to form gold seed;
said HAuCl 4 With citric acid, naBH 4 And the molar ratio of the surfactant is 1:15-30:2-4:150-300, atIn a preferred embodiment of the present invention, the molar ratio is 1:20:2.5:200; HAuCl in the reaction System 4 The content of (C) is 0.1-0.4mol/L, preferably 0.2-0.25mol/L; preferably, the surfactant is cetyltrimethylammonium chloride, CTAC;
(2) To a surfactant-containing HAuCl 4 、AgNO 3 Adding the gold seed solution into the mixed solution of HCl and ascorbic acid AA, reacting for 1.5-4h at 20-40 ℃, centrifuging at 6000-15000rpm, taking precipitate, and washing to obtain gold nano bipyramids; in a preferred mode of the invention, the reaction is carried out for 2 hours at 30 ℃;
jin Chong and HAuCl in terms of Au 4 、AgNO 3 The molar ratio of ascorbic acid to surfactant is 1:30-50:6-15:60-90;
the HCl content in the reaction system is 0.01-0.025mol/L, preferably 0.015-0.02mol/L; the content of the surfactant is 0.06-0.12mol/L, preferably; the gold species content is 0.01-0.025mmol/L, preferably 0.01-0.015mmol/L; the surfactant content is 0.06-0.15mol/L, preferably 0.08-0.1mol/L; the surfactant is cetyltrimethylammonium chloride CTAC.
The platinum-coated gold nano bipyramids prepared by the method are provided with platinum nano particles distributed on the surfaces of the gold nano bipyramids; the particle size of the platinum nano particles is 0.5-5nm, and the gold nano bipyramids have a bipyramid structure, the length of the platinum nano particles is 60-100nm, and the width of the platinum nano particles is 25-50nm. .
The platinum-coated gold nano bipyramid has peroxidase activity, can be used for preparing a peroxidase catalyst, can be used for preparing a colorimetric sensor, and is used for detecting alkaline phosphatase.
A method of detecting alkaline phosphatase comprising the steps of:
(1) Mixing and incubating a sample to be tested with L-ascorbic acid-2-phosphate and a buffer solution with pH=7-7.4;
(2) Adding platinum-coated gold nano bipyramid, TMB and H 2 O 2 Buffer at ph=10-11, color change was observed after incubation or absorbance at 652nm was detected.
And comparing the detection result with a blank sample or a standard curve to obtain a qualitative or quantitative result.
In the step (1), the concentration of L-ascorbic acid-2-phosphoric acid is 2.5-3mol/L, and the incubation is carried out for 20-60min at the temperature of 35-40 ℃; preferably at 37℃for 30min; the buffer was Tris-HCl buffer at pH 7.4.
In the step (2), the concentration of the platinum-coated gold nano bipyramid in the reaction system is 4-10 mug/mL, the concentration of TMB is 0.4-1mmol/L, and H 2 O 2 The concentration of (2) is 0.15-0.25mmol/L, the buffer is acetic acid-acetate buffer, the preferable pH is 4, and the concentration is 10mmol/L. In a preferred mode of the invention, the concentration of the platinum-coated gold nano bipyramid is 5 mug/mL, the concentration of TMB is 0.5mmol/L, H 2 O 2 The concentration of (2) was 0.2mmol/L, and the buffer was acetic acid-acetate buffer having a pH=4 and a concentration of 10mmol/L.
The platinum-coated gold nano bipyramid synthesized by the DNA mediation method is used for detecting alkaline phosphatase, the detectable activity range of an alkaline phosphatase sample to be detected is 0-12.5 mU/mL, and the detection limit is as low as 0.35mU/mL.
The beneficial effects of the invention are as follows: the method has mild detection process conditions, does not need other reagents, realizes convenient and rapid detection of the alkaline phosphatase activity, and has low detection cost and simple operation; the activity of alkaline phosphatase is detected by a platinum-coated gold nano bipyramid colorimetric sensor synthesized by DNA mediation, the detection limit is as low as 0.35mU/mL, and the detection range is as wide as 0-12.5 mU/mL; the platinum-coated gold nano bipyramid colorimetric sensor synthesized by DNA mediation is used for detecting the activity of alkaline phosphatase, the response to alkaline phosphatase is sensitive, the measurement result of an actual sample is accurate and reliable, and the detection of the activity of alkaline phosphatase in human serum is realized.
Drawings
FIG. 1 is a flow chart of a solution for synthesizing Au NBPs@Pt by mediation of DNA of different sequences;
fig. 2 is a TEM image of AuNBPs prepared in example 1;
FIG. 3 is a TEM image of Au NBPs@Pt prepared in example 1;
FIG. 4 is a UV-Vis spectrum of Au NBPs@Pt prepared in example 1;
FIG. 5 is a diagram of example 2A 15 And C 15 Mediating prepared Au NBPs@PtTEM image;
FIG. 6 shows peroxidase activity comparisons of Au NBPs and Au NBPs@Pt prepared by different methods;
FIG. 7 is a schematic diagram of the detection of alkaline phosphatase by a platinum-coated gold nano bipyramid colorimetric sensor for DNA-mediated synthesis;
FIG. 8 is a graph of UV-Vis variation spectrum of a platinum-coated gold nano bipyramid colorimetric sensor synthesized by adding DNA to alkaline phosphatase with different activities;
FIG. 9 is a graph showing the linear relationship between activity of A652 and alkaline phosphatase in the present invention; the inset is a digital photograph of the solution when different concentrations of ALP were added;
FIG. 10 shows the selectivity of DNA mediated synthesis of platinum-coated gold nano bipyramid colorimetric sensors for detecting alkaline phosphatase activity.
Detailed Description
The invention will be further described with reference to the following embodiments in order to make the technical means, the creation features, the achievement of the objects and the effects of the invention easy to understand.
Example 1
(1) Preparation of gold seeds
To a 100mL beaker (washed with ultrapure water, dried for later use) was added 50mM cetyltrimethylammonium chloride CTAC 10mL,10mM chloroauric acid 0.25mL and 0.2M citric acid solution 0.25mL and mixed well, and then 0.25mL of freshly prepared 25mM NaBH was injected 4 Rapidly stirring for 1min. The solution turned from colorless to brown. The solution was then heated at 80 ℃ for 2 hours, changing its color from brown to red, representing the formation of gold species.
(2) Preparation of gold nanometer bipyramid
To a solution containing 100mL of 0.1M cetyltrimethylammonium bromide CTAB, 2mL of 1M HCl, 5mL of 10mM HAuCl 4 1mL of 10mM AgNO 3 And 0.8mL of 100mM ascorbic acid AA solution was added with 5mL of gold seed solution, and reacted at 30℃for 2 hours. The gold nano bipyramid Au NBPs solution obtained was centrifuged at 10000rpm for 10min, the supernatant was discarded and the precipitate was redispersed in deionized water.
FIG. 2 is a transmission electron microscope image of gold nano bipyramid Au NBPs showing that the gold nano bipyramid has a length of about 60-100nm and a width of about 20-35nm.
(3) DNA-mediated synthesis of platinum-coated gold nano bipyramid
The DNA-mediated synthesis process of platinum-coated gold nano bipyramids is shown in figure 1. To 270. Mu.L of the 0.1nM gold nanoparticle bipyramid solution which was redispersed by centrifugation, 150. Mu.M of DNA sequence T was added 15 5.4. Mu.L, for 30min. A further 10mM H was added 2 PtCl 6 5mu L and 0.1M ascorbic acid AA1.5 mu L, incubating for 90min, centrifugally washing, and dispersing the precipitate in deionized water to obtain the platinum-coated gold nano bipyramid Au NBPs@Pt solution synthesized by DNA mediation.
The transmission electron microscope image of the platinum-coated gold nano bipyramid Au NBPs@Pt is shown in FIG. 3, and the Pt nano particles are attached to the gold nano bipyramid, and the particle size is about 0.5-5nm.
The UV-Vis spectrum of Au NBPs@Pt is shown in FIG. 4, which shows absorption peaks around 530nm and 760 nm.
Example 2
With A 15 Or C 15 Substitution sequence T 15 With the rest of example 1, the obtained product has a radio-electron microscope image as shown in fig. 5A and B, and no obvious Pt nanoparticles are found on the gold nanoparticle bipyramid surface.
The peroxidase activity of the products prepared in examples 1 and 2 was measured on gold nanobipyramids by the following method:
to the wells were added 100. Mu.g/mL of the Au NBPs@Pt solution obtained by mediation of the different DNA sequences, or 10. Mu.L of the 100. Mu.g/mL gold nanoparticle bipyramid solution, followed by 10mM TMB 10. Mu.L, 4M H 2 O 2 10. Mu.L of the solution was made up to 200. Mu.L in 10mM acetic acid-sodium acetate buffer having a pH of 4, and reacted for 10 minutes after mixing the solution uniformly, and the ultraviolet absorption spectrum of the mixed solution was measured by an ultraviolet-visible absorption spectrophotometer, and the absorbance at 652nm was recorded.
Gold nano bipyramid and DNA sequence T 15 、A 15 And C 15 The comparison of mediated platinum-coated gold nano bipyramid Au NBPs@Pt peroxidase activity is shown in FIG. 6, and the result shows that the gold nano bipyramid has no peroxidase activity and uses a DNA sequence T 15 The mediated platinum-coated gold nano bipyramid has the highest peroxidase activity.
Example 3
Use of example 1polyT 15 The platinum-coated gold nano bipyramid nano enzyme Au NBPs@Pt which is mediated and synthesized is used as a colorimetric sensor for detecting the activity of alkaline phosphatase, and the process and the principle are shown in figure 7. The hydrogen peroxide and TMB produce water and oxidize TMB under the action of nano enzyme and display blue color. The alkaline phosphatase decomposes the ascorbic acid diphosphate, the generated ascorbic acid reduces oxidized TMB into TMB, the color of a reaction system becomes light, and visual detection or quantitative detection of absorbance is realized.
The method comprises the following steps:
(1) 50. Mu.L of alkaline phosphatase with different activities and 150. Mu.L of Tris-HCl buffer with pH 7.4 containing 3.75mM L-ascorbic acid-2-phosphate were added to a1.5 mL centrifuge tube, and incubated at 37℃for 30min;
(2) To the wells were added sequentially 100. Mu.g/mL of Au NBPs@Pt solution 10. Mu.L, 10mM TMB 10. Mu.L, 4M H 2 O 2 10mu L of 10mM acetic acid-sodium acetate buffer solution with pH of 4, and reacting for 10min after uniformly mixing the systems; adding 5mu L of the mixed solution in the step (1) into the system, and uniformly mixing; the total volume of the system was 200 μl; the final activities of alkaline phosphatase in the system were 0.625mU/mL, 1.25mU/mL, 2.5mU/mL, 3.75mU/mL, 5mU/mL, 5.625mU/mL, 7.5mU/mL, 10mU/mL, 12.5mU/mL and 0mU/mL, respectively; measuring ultraviolet absorption spectrum of the mixed solution by an ultraviolet-visible absorption spectrophotometer, and recording absorbance at 652 nm;
(3) The absorbance of oxTMB at 652nm was recorded and the results are shown in fig. 8, which illustrate that the absorbance of the solution gradually decreased as the ALP activity increased;
and drawing an absorbance fitting straight line at 652nm of an ultraviolet spectrum with TMB as a color developing agent, namely an alkaline phosphatase activity-absorbance standard working curve, as shown in fig. 9, and showing that the method has an excellent detection effect on ALP activity; FIG. 9 is a photograph showing the digital representation of the solution when ALP is added at various concentrations, and it can be seen that the color of the reaction system becomes lighter as the activity of alkaline phosphatase increases, so that visual detection can be achieved;
(4) And (3) operating the alkaline phosphatase sample to be detected according to the steps (1) and (2), respectively measuring the absorbance of the alkaline phosphatase sample at 652nm with TMB as a substrate by using an ultraviolet-visible absorption spectrophotometer, and comparing the absorbance with a standard working curve through calculation to obtain the activity of the alkaline phosphatase.
Example 4
The selective verification of the platinum-coated gold nano bipyramid as a colorimetric sensor for detecting the activity of alkaline phosphatase is as follows:
(1) 50. Mu.L of different kinds of amino acids or proteases and 150. Mu.L of Tris-HCl buffer of pH 7.4 containing 3.75mM L-ascorbic acid-2-phosphate were added to a1.5 mL centrifuge tube, respectively, and incubated at 37℃for 30min; meanwhile, an alkaline phosphatase experimental group is added for comparison;
(2) To the wells were added sequentially 100. Mu.g/mL of Au NBPs@Pt solution 10. Mu.L, 10mM TMB 10. Mu.L, 4M H 2 O 2 10mu L of 10mM sodium acetate buffer solution with pH of 4, and reacting for 10min after uniformly mixing the systems; adding 5mu L of the mixed solution in the step (1) into the system, and uniformly mixing to ensure that the total volume of the system is 200 mu L; the absorbance of the above mixed solution at 652nm was measured with an ultraviolet-visible absorption spectrophotometer. The results are shown in FIG. 10.
FIG. 10 is a bar graph of selectivity of alkaline phosphatase activity detection using a DNA-mediated platinum-coated nano-bipyramid colorimetric sensor, with Acetylcholinesterase (ACHE), pepsin (Pep), papain (Pap), lysine (Lys), tryptophan (Tyr), glucose oxidase (GOx), glutathione Reductase (GR), blank (Blank) in order from left to right.
As can be seen from FIG. 10, A is present only in the presence of alkaline phosphatase 652 The absorbance of (A) decreases without adding alkaline phosphatase 652 The absorbance of the ALP is not changed, that is, other amino acids or proteases cannot influence the detection of alkaline phosphatase, so that the sensor can be used for stably and high-selectively performing colorimetric quantitative detection of ALP activity.
The foregoing has shown and described the basic principles and main features of the present invention and the advantages of the present invention. It will be appreciated by persons skilled in the art that the present invention is not limited to the embodiments described above, but is capable of numerous variations and modifications without departing from the spirit and scope of the invention as hereinafter claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims (10)

1. A method for synthesizing platinum-coated gold nano bipyramids by using DNA mediation, which is characterized by comprising the following steps:
adding the DNA sequence into a gold nano bipyramid solution for incubation, and then adding H 2 PtCl 6 And ascorbic acid, and incubating again to obtain the platinum-coated gold nano bipyramid.
2. The method according to claim 1, wherein the gold nanobipyramid has a bipyramid structure with a length of 60-100nm and a width of 25-50nm.
3. The method of claim 1, wherein the DNA sequence is Poly T, poly C or Poly a of 12-20 nucleotides in length.
4. The method according to claim 1, wherein gold nano bipyramid and DNA sequence, H in the reaction system 2 PtCl 6 The molar ratio of (2) is 1:15000-40000:1600-2400, H 2 PtCl 6 The molar ratio of the sodium ascorbate to the ascorbic acid is 1:2-4; the gold nano bipyramid concentration in the reaction system is 0.06-0.15nmol/L.
5. The method according to claim 1, wherein the gold nano bipyramid is prepared by the following steps:
(1) To contain HAuCl 4 Adding NaBH to a solution of surfactant and citric acid 4 Heating the solution at 60-90 deg.c for 1.5-4 hr to form gold seed;
said HAuCl 4 With citric acid, naBH 4 And the molar ratio of the surfactant is 1:15-30:2-4:150-300; HAuCl in the reaction System 4 The content of (2) is 0.1-0.4mol/L;
(2) To a surfactant-containing HAuCl 4 、AgNO 3 Adding the gold seed solution into the mixed solution of HCl and ascorbic acid AA, reacting for 1.5-4h at 20-40 ℃, centrifuging at 6000-15000rpm, taking precipitate, and washing to obtain gold nano bipyramids;
jin Chong and HAuCl in terms of Au 4 、AgNO 3 The molar ratio of ascorbic acid to surfactant is 1:30-50:6-15:60-90;
the HCl content in the reaction system is 0.01-0.025mol/L, the surfactant content is 0.06-0.12mol/L, and the surfactant content is 0.06-0.15mol/L.
6. A platinum-coated gold nano bipyramid, which is characterized in that platinum nano particles are distributed on the surface of the gold nano bipyramid by the method of any one of claims 1-5, the particle size of the platinum nano particles is 0.5-5nm, and the gold nano bipyramid has a bipyramid structure, the length of which is 60-100nm, and the width of which is 25-50nm.
7. The use of platinum-coated gold nano bipyramid according to claim 6 for preparing a peroxidase or a colorimetric sensor.
8. The use of platinum coated gold nano bipyramids according to claim 6 for detecting alkaline phosphatase or preparing alkaline phosphatase detection reagent.
9. A reagent or kit for detecting alkaline phosphatase, comprising the platinum-coated gold nano bipyramid according to claim 6.
10. A method for detecting alkaline phosphatase, comprising the steps of:
(1) Mixing and incubating a sample to be tested with L-ascorbic acid-2-phosphate and a buffer solution with pH=7-7.4;
(2) Adding the platinum-coated gold nano bipyramid, TMB and H of claim 6 2 O 2 Buffer at ph=10-11, color change was observed after incubation or absorbance at 652nm was detected.
CN202310130103.9A 2023-02-17 2023-02-17 Platinum-coated gold nano bipyramid synthesized by DNA (deoxyribonucleic acid) mediation and preparation method and application thereof Pending CN116237535A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117554362A (en) * 2024-01-10 2024-02-13 中国科学院烟台海岸带研究所 Method for detecting cyanide at normal temperature with high sensitivity and nano colorimetric analysis kit

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117554362A (en) * 2024-01-10 2024-02-13 中国科学院烟台海岸带研究所 Method for detecting cyanide at normal temperature with high sensitivity and nano colorimetric analysis kit
CN117554362B (en) * 2024-01-10 2024-03-12 中国科学院烟台海岸带研究所 Method for detecting cyanide at normal temperature with high sensitivity and nano colorimetric analysis kit

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