CN112643043A - Preparation method of aptamer modified gold nano-star - Google Patents
Preparation method of aptamer modified gold nano-star Download PDFInfo
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- B22F9/00—Making metallic powder or suspensions thereof
- B22F9/16—Making metallic powder or suspensions thereof using chemical processes
- B22F9/18—Making metallic powder or suspensions thereof using chemical processes with reduction of metal compounds
- B22F9/24—Making metallic powder or suspensions thereof using chemical processes with reduction of metal compounds starting from liquid metal compounds, e.g. solutions
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- B22F1/00—Metallic powder; Treatment of metallic powder, e.g. to facilitate working or to improve properties
- B22F1/05—Metallic powder characterised by the size or surface area of the particles
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- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
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- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
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Abstract
The invention discloses a preparation method of a aptamer modified gold nano-star, which comprises the following steps: preparing anisotropic gold nanostar (GNSt) solution; adding aptamer aptamers to anisotropic gold nanostar (GNSt) solution and vortexing; adding citrate-HCl buffer solution; adding NaCl solution, whirling and standing for a certain time; centrifuging the solution, and dispersing the solution in ultrapure water again to obtain a required aptamer modified gold nano-star solution; and preserving the aptamer modified gold nano-star solution for later use.
Description
Technical Field
The invention belongs to the technical field of nano biological materials, and relates to a preparation method of a simple aptamer modified gold nano star.
Background
The raman spectroscopy is used as an analysis method to acquire fingerprints of analyte molecules, but due to the small raman scattering cross section and low sensitivity, the usage scenario of the raman spectroscopy is limited.
Surface Enhanced Raman Scattering (SERS) can significantly amplify raman signals by several orders of magnitude, which can improve the sensitivity of raman detection. In the prior art, noble metal Nanoparticles (NPs) are used as the active substrate material for Surface Enhanced Raman Spectroscopy (SERS), and when analyte molecules are located at the junctions between the nanoparticles, they are called "hot spots", in which case the raman signal of the detector is greatly enhanced. The process of forming "hot spots" between the nanoparticles is complex and difficult.
Among various biocompatible gold nanoparticles, anisotropic gold nanostars (GNSt) having a multi-branched structure on the surface exhibit unique physical and optical properties with a stronger raman enhancement factor than other gold nanoparticles. In addition, gold nanostars do not require particles to be close to creating "hot spots", which can be created more near their own protruding structures.
However, in the detection process, due to factors such as the electrical property, the size and the structure of the molecules of the detection object, the detection object is difficult to approach and gather in the 'hot spot' range of the gold nanostar, and a strong Raman signal is difficult to generate. Aptamers (aptamers), which are short single-stranded DNA or RNA oligonucleotides that fold into a unique secondary structure capable of specifically binding to a target with high affinity, have attracted considerable attention as targeting ligands for actively transporting drugs or nanoparticles. Thus, aptamers (aptamers) have created the possibility of capturing immobilized detector molecules to the gold nanostar "hot spot" regime.
The invention provides a preparation method of a nucleic acid aptamer modified gold nano-star, which aims to solve the problems that when the existing Raman spectroscopy is used as an analysis method, the Raman scattering cross section is small, the sensitivity is low, the process of forming a 'hot spot' by adopting noble metal nano-particles in surface enhanced Raman scattering is complex and has high difficulty, and a detected object is difficult to approach and enrich in the 'hot spot' range of the gold nano-star, so that a strong Raman scattering signal can be obtained.
Disclosure of Invention
The invention provides a preparation method of a nucleic acid aptamer modified gold nanostar, aiming at the problems that the existing Raman spectroscopy is small in Raman scattering cross section and low in sensitivity when used as an analysis method, the process of forming a 'hot spot' by adopting noble metal nanoparticles in surface enhanced Raman scattering is complex and difficult to achieve, a detected object is difficult to approach and enrich in the 'hot spot' range of the gold nanostar, and a strong Raman signal is difficult to generate. According to an embodiment of the invention, a method for preparing aptamer modified gold nano-star is provided, which comprises the following steps:
preparing anisotropic gold nanostar (GNSt) solution;
adding aptamer aptamers to anisotropic gold nanostar (GNSt) solution and vortexing;
adding citrate-HCl buffer solution;
adding NaCl solution, whirling and standing for a certain time;
centrifuging the solution, and dispersing the solution in ultrapure water again to obtain a required aptamer modified gold nano-star solution; and
and preserving the aptamer modified gold nano-star solution for later use.
In one embodiment of the present invention, preparing an anisotropic gold nanostar (GNSt) solution further comprises:
preparing a seed solution;
adding seed solution to HAuCl4Adding HCl into the solution;
AgNO is added while stirring3Solutions and ascorbic acid solutions; and
the solution was centrifuged and redispersed in ultrapure water to obtain an anisotropic gold nanostar (GNSt) solution.
In one embodiment of the invention, 1.5mL of a 1% by mass citrate solution is added to 10mL of boiling 0.001M HAuCl with stirring at 700rpm4After 15 minutes, cooling the solution to prepare a seed solution;
2mL of the seed solution was added to 200mL of 2.5X 10-4M HAuCl4To the solution, and 200. mu.L of 1M HCl was added;
while stirring for 30 seconds, 400. mu.L of 0.01M AgNO was added3Solution and 1mL ascorbic acid solution; and
the solution was centrifuged at 4000rpm for 1.5h at 4 ℃ and then re-dispersed in ultrapure water to obtain an anisotropic gold nanostar (GNSt) solution.
In one embodiment of the invention, the addition of aptamer aptamers to the anisotropic gold nanostar (GNSt) solution is at a ratio of 1:900 to 1:1100 molar ratio of anisotropic gold nanostar (GNSt) to aptamer.
In one embodiment of the invention, the addition of aptamer aptamers to the anisotropic gold nanostar (GNSt) solution is at a ratio of 1:1000 molar ratio of anisotropic gold nanostar (GNSt) to aptamer.
In one embodiment of the invention, the citrate-HCl buffer has a pH of 3.
In one embodiment of the invention, the NaCl solution is added at a concentration of 0.3M/L to 1M/L.
In one embodiment of the invention, the step of storing the aptamer-modified gold nanostar solution is to store the prepared aptamer-modified gold nanostar solution in an environment at 4 ℃.
According to another embodiment of the present invention, there is provided a method for preparing aptamer-modified gold nanostars, comprising:
aptamer was added at a GNSt to Aptamer molar ratio of 1:1000 was added to 0.6mL of GNSt solution at a concentration of 50nM/L and vortexed for 10 seconds;
0.6mL of 20nM citrate-HCl buffer was added;
adding 0.5mL of NaCl solution with the concentration of 0.3M/L-1M/L, and vortexing for 10 seconds;
the mixture was left to stand in a refrigerator at 4 ℃ for 24 hours;
the solution was re-dispersed in ultrapure water after centrifugation at 4000rpm for 10 minutes at 4 ℃; and
and storing the prepared aptamer modified gold nano star solution in a refrigerator at 4 ℃ for later use.
The invention provides a preparation method of aptamer modified gold nano-star, which comprises the steps of firstly preparing anisotropic gold nano-star (GNSt) solution, then adding aptamer into the GNSt solution, whirling, then adding citrate-HCl buffer solution, adding NaCl solution, whirling, standing, finally centrifuging the solution, and re-dispersing the solution into ultrapure water to obtain the aptamer modified gold nano-star solution. The preparation method of the aptamer modified gold nano-star based on the invention has the following advantages: 1) the prepared aptamer-modified gold nano star surface enhanced Raman scattering enhancement factor is high, and the detection is sensitive; 2) compared with the prior art, the reaction medicine is less in use, the incubation step is simple, surface protection is carried out without using surfactant sodium dodecyl sulfate or other molecular sodium phosphates, bovine serum albumin, tween and sucrose, and the preparation method is simple and rapid to operate, strong in repeatability and low in cost; 3) because the nucleic acid aptamer is combined on the surface of the gold nano-star to reach the optimal balance point, the synthesized nucleic acid aptamer modified gold nano-star is stable; 4) the method has wide applicability, can be suitable for preparing various aptamer modified gold nano-star of different types, can detect various drug molecules, and provides possibility for detecting excessive drug pollutants in an environmental water source, detecting the concentration of drugs in a human body and the like.
Drawings
To further clarify the above and other advantages and features of embodiments of the present invention, a more particular description of embodiments of the invention will be rendered by reference to the appended drawings. It is appreciated that these drawings depict only typical embodiments of the invention and are therefore not to be considered limiting of its scope. In the drawings, the same or corresponding parts will be denoted by the same or similar reference numerals for clarity.
Fig. 1 shows a flowchart 100 of a method for preparing aptamer-modified gold nanostars, according to an embodiment of the invention.
Fig. 2 shows a flow diagram 110 of a method of preparing an anisotropic gold nanostar (GNSt) solution, according to one embodiment of the invention.
Fig. 3 shows a transmission electron microscope TEM image of gold nanostars prepared by an example of the present invention.
Figure 4 shows UV-Vis spectra of different aptamer-modified gold nanostars formed by embodiments of the present invention.
Figure 5 illustrates a nucleic acid aptamer modified gold nanostar drug molecule SERS detection spectrum formed by one embodiment of the invention.
Figure 6 shows a SERS detection spectrum of aptamer-modified gold nanostar drug molecules formed by another embodiment of the invention.
Detailed Description
In the following description, the invention is described with reference to various embodiments. One skilled in the relevant art will recognize, however, that the embodiments may be practiced without one or more of the specific details, or with other alternative and/or additional methods, materials, or components. In other instances, well-known structures, materials, or operations are not shown or described in detail to avoid obscuring aspects of embodiments of the invention. Similarly, for purposes of explanation, specific numbers, materials and configurations are set forth in order to provide a thorough understanding of the embodiments of the invention. However, the invention may be practiced without specific details. Further, it should be understood that the embodiments shown in the figures are illustrative representations and are not necessarily drawn to scale.
Reference in the specification to "one embodiment" or "the embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment.
The invention provides a preparation method of aptamer modified gold nano-star, which comprises the steps of firstly preparing anisotropic gold nano-star (GNSt) solution, then adding aptamer into the GNSt solution, whirling, then adding citrate-HCl buffer solution, adding NaCl solution, whirling, standing, finally centrifuging the solution, and re-dispersing the solution into ultrapure water to obtain the aptamer modified gold nano-star solution. The preparation method of the aptamer modified gold nano-star based on the invention has the following advantages: 1) the prepared aptamer-modified gold nano star surface enhanced Raman scattering enhancement factor is high, and the detection is sensitive; 2) compared with the prior art, the reaction medicine is less in use, the incubation step is simple, surface protection is carried out without using surfactant sodium dodecyl sulfate or other molecular sodium phosphates, bovine serum albumin, tween and sucrose, and the preparation method is simple and rapid to operate, strong in repeatability and low in cost; 3) because the nucleic acid aptamer is combined on the surface of the gold nano-star to reach the optimal balance point, the synthesized nucleic acid aptamer modified gold nano-star is stable; 4) the method has wide applicability, can be suitable for preparing various aptamer modified gold nano-star of different types, can detect various drug molecules, and provides possibility for detecting excessive drug pollutants in an environmental water source, detecting the concentration of drugs in a human body and the like.
The method for preparing aptamer-modified gold nano-star according to the present invention is described in detail below with reference to fig. 1, and fig. 1 shows a flowchart 100 of a method for preparing aptamer-modified gold nano-star according to an embodiment of the present invention.
As shown in fig. 1, first, at step 110, an anisotropic gold nanostar (GNSt) solution is prepared.
In one embodiment of the present invention, as shown in fig. 2, an anisotropic gold nanostar (GNSt) solution is prepared comprising: first, at step 111, a seed solution is prepared; then, at step 112, the seed solution is added to HAuCl4Adding HCl into the solution; next, in step 113, AgNO is added while stirring3Solutions and ascorbic acid solutions; finally, at step 114, the solution is centrifuged and redispersed in ultrapure water to obtain an anisotropic gold nanostar (GNSt) solution.
In one embodiment of the invention, in a first step, a seed solution is prepared and 1.5mL of a 1% by mass citrate solution is added to 10mL of boiling 0.001M HAuCl solution with stirring at 700rpm4After 15 minutes, the solution is cooled; second, 2mL of seed solution was added to 200mL of 2.5X 10-4M HAuCl4To the solution, and 200. mu.L of 1M HCl was added; third, 400. mu.L of 0.01M AgNO was added while vigorously stirring for 30 seconds3Solution and 1mL ascorbic acid solution; finally, the solution was centrifuged at 4000rpm for 1.5h at 4 ℃ and then re-dispersed in ultrapure water to obtain an anisotropic gold nanostar (GNSt) solution.
Next, at step 120, the nucleic acid aptamer was added to the anisotropic gold nanostar (GNSt) solution and vortexed. In one embodiment of the invention, aptamer aptamers are added to an anisotropic gold nanostar (GNSt) solution at a molar ratio of 1:1000, followed by vortexing, which is the uniform mixing of the solution by vortexing, typically by vortexing with a vortexer. Wherein, the molar ratio of the anisotropic gold nano star (GNSt) to the aptamer is 1:1000 (namely the number ratio of the anisotropic gold nano star (GNSt) to the aptamer) is adopted, so that the aptamer is combined on the surface of the gold nano star to reach an optimal balance point, and the aptamer modified gold nano star synthesized in the way is most stable.
Then, at step 130, citrate-HCl buffer is added. In one embodiment of the invention, the citrate-HCl buffer is 20nM citrate-HCl buffer, pH 3.
Next, at step 140, NaCl solution is added and vortexed and allowed to stand for a period of time. In one embodiment of the invention, the NaCl solution is a high concentration NaCl solution of 0.3M/L to 1M/L.
Then, in step 150, the solution is centrifuged and re-dispersed in ultrapure water to obtain the desired aptamer-modified gold nanostar solution.
Finally, at step 160, the aptamer-modified gold nanostar solution is stored for use.
In one embodiment of the present invention, after preparing an anisotropic gold nanostar (GNSt) solution, first, a quantity of nucleic acid Aptamer is added according to a molar ratio of GNSt to Aptamer of 1:1000 was added to 0.6mL of GNSt solution at a concentration of 50nM/L and vortexed for 10 seconds; then, 0.6mL of 20nM citrate-HCl buffer, pH 3, was added; then, adding 0.5mL of NaCl solution with the concentration of 0.3M/L-1M/L, and vortexing for 10 seconds; then, the mixture was left in a refrigerator at 4 ℃ for 24 hours; next, the solution was re-dispersed in ultrapure water after centrifugation at 4000rpm for 10 minutes at 4 ℃; and finally, storing the prepared aptamer modified gold nano star solution in a refrigerator at 4 ℃ for later use.
FIG. 3 shows a transmission electron microscopy TEM image of anisotropic gold nanostars prepared by an embodiment of the present invention. As shown in FIG. 3, the diameter of the gold nanostar core formed by the method of the embodiment of the present invention is 38. + -.4 nm, the length of the branch structure is in the range of 7. + -.3 nm, and the number of branches is in the range of 8. + -.4. Anisotropic gold nanostars (GNSts) have a Zeta potential of about-27 mV and an equivalent hydrodynamic diameter of about 76 nm.
Figure 4 shows UV-Vis spectra of different aptamer-modified gold nanostars formed by embodiments of the present invention. As shown in FIG. 4, the UV-vis spectrum of anisotropic gold nanostars (GNSts) that were not modified with nucleic acid aptamers showed a SPRmax characteristic peak (surface plasmon resonance) at a wavelength of 737 nm. The SPRmax characteristic peak of the anisotropic gold nanostar is modified at the wavelength of 771nm through a theophylline aptamer. The SPRmax characteristic peak of the anisotropic gold nanostars modified by the ampicillin aptamer is at the wavelength of 799 nm. This is due to the change in dielectric constant experienced by GNSts after the citrate on the surface of the anisotropic gold nanostar has been replaced with a different aptamer.
The Zeta potential of anisotropic gold nanostars (GNSts) changes from negative to more negative upon binding to different aptamers. For example, the Zeta potential of the anisotropic gold nanostars (GNSts) prepared in one embodiment of the present invention is-27 mV, the Zeta potential of the anisotropic gold nanostars modified by theophylline aptamer is-40 mV, and the Zeta potential of the anisotropic gold nanostars modified by ampicillin aptamer is-37 mV. In addition, the equivalent hydrodynamic diameter of anisotropic gold nanostars (GNSts) bound to different aptamers may also vary. For example, the hydrodynamic diameter of the anisotropic gold nanostars (GNSts) prepared in yet another embodiment of the present invention was 76nm, the hydrodynamic diameter of the anisotropic gold nanostars modified by theophylline aptamer was 99nm, and the hydrodynamic diameter of the anisotropic gold nanostars modified by ampicillin aptamer was 104 nm. The above changes in Zeta potential and hydrodynamic diameter indicate successful modification of the aptamer.
Figure 5 illustrates a nucleic acid aptamer modified gold nanostar drug molecule SERS detection spectrum formed by one embodiment of the invention. As shown in FIG. 5, the theophylline aptamer modified anisotropic gold nanostar substrate is shown at 10-6After incubation in the M theophylline solution, a remarkable Raman peak enhancement is shown, which is consistent with the characteristic Raman peak of theophylline crystals. Wherein, 555cm-1The peak at position (E) is due to C ═ C-C distortion, 667cm-1The peak at (A) is associated with the O ═ C-N deformation, 928cm-1The peak at corresponds to N-CH3Symmetric stretching vibration, and 969cm-1Peak of (A) toDue to the N ═ C — H deformation. At 1248cm-1、1286cm-1And 1314cm-1The peak at (b) is due to C-N tensile vibration, 1665cm-1And 1706cm-1The peaks at (a) correspond to the C ═ O asymmetric stretch and the C ═ N stretch, respectively.
Figure 6 shows a SERS detection spectrum of aptamer-modified gold nanostar drug molecules formed by another embodiment of the invention. As shown in FIG. 6, the concentration of the ampicillin aptamer-modified anisotropic gold nanostar substrate was 10-6The M ampicillin solution also showed a significantly enhanced Raman peak after incubation, consistent with the characteristic Raman peak of ampicillin crystals. Wherein, 1004cm-1The peak at (A) is due to benzene ring C ═ C tensile vibration, 1070cm-1Peak at 1235cm, which is related to C-O-C stretching-1The peak at (a) corresponds to the C-N tensile vibration.
The preparation method of the aptamer modified gold nano-star provided by the invention has the following advantages: 1) the prepared aptamer-modified gold nano star surface enhanced Raman scattering enhancement factor is high, and the detection is sensitive; 2) compared with the prior art, the reaction medicine is less in use, the incubation step is simple, surface protection is carried out without using surfactant sodium dodecyl sulfate or other molecular sodium phosphates, bovine serum albumin, tween and sucrose, and the preparation method is simple and rapid to operate, strong in repeatability and low in cost; 3) because the nucleic acid aptamer is combined on the surface of the gold nano-star to reach the optimal balance point, the synthesized nucleic acid aptamer modified gold nano-star is stable; 4) the method has wide applicability, can be suitable for preparing various aptamer modified gold nano-star of different types, can detect various drug molecules, and provides possibility for detecting excessive drug pollutants in an environmental water source, detecting the concentration of drugs in a human body and the like.
While various embodiments of the present invention have been described above, it should be understood that they have been presented by way of example only, and not limitation. It will be apparent to persons skilled in the relevant art that various combinations, modifications, and changes can be made thereto without departing from the spirit and scope of the invention. Thus, the breadth and scope of the present invention disclosed herein should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.
Claims (9)
1. A preparation method of aptamer modified gold nano-star comprises the following steps:
preparing an anisotropic gold nano star GNSt solution;
adding aptamer aptamers to the anisotropic gold nanostar GNSt solution and vortexing;
adding citrate-HCl buffer solution;
adding NaCl solution, whirling and standing for a certain time;
centrifuging the solution, and dispersing the solution in ultrapure water again to obtain a required aptamer modified gold nano-star solution; and
and preserving the aptamer modified gold nano-star solution for later use.
2. The method of preparing aptamer-modified gold nanostars as claimed in claim 1, wherein preparing the anisotropic gold nanostars GNSt solution further comprises:
preparing a seed solution;
adding seed solution to HAuCl4Adding HCl into the solution;
AgNO is added while stirring3Solutions and ascorbic acid solutions; and
the solution was centrifuged and redispersed in ultrapure water to obtain an anisotropic gold nanostar GNSt solution.
3. The method of preparing anisotropic gold nanostar GNSt solution of claim 2, wherein:
1.5mL of a 1% by mass citrate solution was added to 10mL of boiling 0.001M HAuCl with stirring at 700rpm4After 15 minutes, cooling the solution to prepare a seed solution;
2mL of the seed solution was added to 200mL of 2.5X 10-4M HAuCl4To the solution, and 200. mu.L of 1M HCl was added;
while stirring for 30 seconds, 400. mu.L of 0.01M AgNO was added3Solution and 1mL ascorbic acid solution; and
the solution was centrifuged at 4000rpm for 1.5h at 4 ℃ and then redispersed in ultrapure water to obtain an anisotropic gold nanostar GNSt solution.
4. The method of claim 1, wherein the aptamer is added to the anisotropic gold nanostar GNSt solution at a molar ratio of 1:900 to 1:1100 of the aptamer to the aptamer.
5. The method of claim 1, wherein the adding of aptamer to the anisotropic gold nanostar GNSt solution is performed at a molar ratio of 1: 1000.
6. The method for preparing an aptamer-modified gold nanostar according to claim 1, wherein the pH of the citrate-HCl buffer is 3.
7. The method for preparing an aptamer-modified gold nanostar according to claim 1, wherein the concentration of the added NaCl solution is 0.3M/L to 1M/L.
8. The method for preparing a aptamer-modified gold nanostar according to claim 1, wherein the step of storing the aptamer-modified gold nanostar solution is to store the prepared aptamer-modified gold nanostar solution in an environment at 4 ℃.
9. A preparation method of aptamer modified gold nano-star comprises the following steps:
aptamer was added at a GNSt to Aptamer molar ratio of 1:1000 was added to 0.6mL of GNSt solution at a concentration of 50nM/L and vortexed for 10 seconds;
0.6mL of 20nM citrate-HCl buffer was added;
adding 0.5mL of NaCl solution with the concentration of 0.3M/L-1M/L, and vortexing for 10 seconds;
the mixture was left to stand in a refrigerator at 4 ℃ for 24 hours;
the solution was re-dispersed in ultrapure water after centrifugation at 4000rpm for 10 minutes at 4 ℃; and
and storing the prepared aptamer modified gold nano star solution in a refrigerator at 4 ℃ for later use.
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