CN108444969B - Method for detecting nucleic acid structure based on surface enhanced Raman spectroscopy - Google Patents
Method for detecting nucleic acid structure based on surface enhanced Raman spectroscopy Download PDFInfo
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- CN108444969B CN108444969B CN201711251899.4A CN201711251899A CN108444969B CN 108444969 B CN108444969 B CN 108444969B CN 201711251899 A CN201711251899 A CN 201711251899A CN 108444969 B CN108444969 B CN 108444969B
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- 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
- G01N21/65—Raman scattering
- G01N21/658—Raman scattering enhancement Raman, e.g. surface plasmons
Abstract
The invention relates to a method for detecting a nucleic acid structure based on surface enhanced Raman spectroscopy, which comprises the following steps: (1) preparing iodine ion modified and cleaned silver nanoparticle sol; (2) adding a DNA sample to be detected into a silver nanoparticle centrifugal tube modified by iodide ions, adding a buffer solution with the pH value of 3.0-5.0, and adding an aluminum ion or titanium ion aggregating agent for surface enhanced Raman spectroscopy detection. The method effectively detects the secondary structure of the nucleic acid, has the advantages of simplicity, rapidness and the like of SERS, and has extremely high reproducibility and credibility. The method overcomes the difficulty that the secondary structure of the nucleic acid is difficult to detect in the traditional SERS detection, has the advantages of simple and rapid operation steps, high sensitivity and good reproducibility, obtains signals with extremely high signal-to-noise ratio, and realizes the high-sensitivity detection of various secondary structures of the nucleic acid.
Description
Technical Field
The invention belongs to the field of nucleic acid structure detection, and particularly relates to a method for detecting a nucleic acid structure based on surface enhanced Raman spectroscopy.
Background
Deoxyribonucleic acid, also known as deoxyribonucleic acid, is a biological macromolecule that can constitute genetic instructions to guide biological development and functioning of vital functions. The main function is information storage, which can be compared to a "blueprint" or "recipe". The instructions contained therein are required to construct other compounds within the cell, such as proteins and ribonucleic acids. A DNA fragment carrying a protein code is called a gene. The SERS method is adopted, so that many details of the secondary structure of the nucleic acid at the molecular level are shown, and a novel method is provided for better research on the structure of the nucleic acid.
Surface Enhanced Raman Spectroscopy (SERS) is an important method for identifying and analyzing substances as fingerprint spectroscopy. The surface enhanced Raman greatly improves the detection capability of the Raman technology, and the qualitative analysis of the substance has the advantages of simple operation, high sensitivity and the like. Many researchers choose to use raman spectroscopy to study nucleic acid bases and nucleic acid base homologues, and provide a great deal of experimental data and direct evidence for further studying the structural changes of nucleic acid molecules and analyzing the role of nucleic acid molecules in organisms.
However, it is relatively difficult to apply the surface-enhanced raman technique to the field of quantitative analysis because the condition of the surface-enhanced raman is not easy to control, and the substrate types are many, and different factors such as the surface roughness of the metal nanoparticles can affect the intensity of the surface-enhanced raman spectrum. The prior art relies on strong interactions between the sequence containing the label and the substrate for quantitative analysis of the secondary structure of the nucleic acid, but the label itself may have an effect on the secondary structure of the nucleic acid formed. Therefore, the actual structure of the nucleic acid cannot be detected truly, and the cost is high. Surface enhanced raman spectroscopy, while successfully applied to label-free analysis of oligonucleotides and single-stranded DNA, is still a challenge to detect complex DNA secondary structures.
Disclosure of Invention
The invention aims to provide a method for detecting a nucleic acid structure based on surface enhanced Raman spectroscopy, which overcomes the difficulty that the secondary structure of nucleic acid is difficult to detect in the traditional SERS detection, has the advantages of simple and rapid operation steps, high sensitivity and good reproducibility, obtains a signal with extremely high signal-to-noise ratio, and realizes the high-sensitivity detection of various secondary structures of nucleic acid.
The purpose of the invention is realized by the following technical scheme:
a method for detecting nucleic acid structures based on surface enhanced Raman spectroscopy, comprising the steps of:
(1) preparing iodine ion modified and cleaned silver nanoparticle sol;
(2) adding a DNA sample to be detected into a silver nanoparticle centrifugal tube modified by iodide ions, adding a buffer solution with the pH value of 3.0-5.0, and adding an aluminum ion or titanium ion aggregating agent for surface enhanced Raman spectroscopy detection.
As a more excellent technical scheme of the invention: the aggregating agent is aluminum ions.
As a more excellent technical scheme of the invention: the pH value of the acidic buffer solution is 4.5.
As a more excellent technical scheme of the invention: the volume ratio of the aluminum ion or titanium ion aggregating agent in the step (2) to the sample to be detected is 2: 5.
as a more excellent technical scheme of the invention: the synthesis method of the iodine ion-modified silver nanoparticles in the step (1) is as follows: under the heating condition of a water phase, 0.036g of silver nitrate dissolved in 200mL of water is added with 4mL of sodium citrate with the mass concentration of 1%, the maximum ultraviolet absorption value of the obtained silver nano-ions is 415nm, 10mL of silver sol is centrifuged for 15 minutes (5000r/h) at the low temperature of 4 ℃, supernatant liquid is removed, 50ul of the centrifuged silver sol is taken, 50ul of a cleaning agent (KI) is added, the mixture is kept at the room temperature for 1 hour, and the mixture is placed in a refrigerator at the temperature of 4 ℃ and cooled for 1 hour.
The invention has the following beneficial effects:
1. the iodine ion cleaning method can effectively remove impurities on the surface of the silver nanoparticles, and improves the accuracy of a spectrogram;
2. the aluminum ion or titanium ion aggregating agent is introduced into the silver nanoparticles to form a high-quality 'hot spot', and an enhanced SERS signal with strong repeatability can be obtained, so that structural information with rich secondary structures of nucleic acid, such as glucoside folding, Hoogsteen hydrogen bond and InterBase hydrogen bond, is obtained; the method effectively detects the secondary structure of the nucleic acid, has the advantages of simplicity, rapidness and the like of SERS, and has extremely high reproducibility and credibility.
3. The invention is a SERS detection method for qualitatively and quantitatively analyzing DNA sequence structure, which is carried out in solution, does not cause any label on a DNA chain, can be widely applied to the detection of nucleic acid secondary structure, is expected to be called as a new method for researching DNA structure in the whole genome range, and provides technical means for the function research of biomacromolecule, the interaction between micromolecule ligand and DNA, and the like.
Drawings
FIG. 1 is a diagram of the structure of the G-quad to be tested according to the present invention;
FIG. 2 is a projection electron microscope image of the sol to be detected which is aggregated under the guidance of aluminum ions according to the present invention;
FIG. 3 is a SERS spectrum of example 1 of the present invention;
FIG. 4 is a SERS spectrum of example 1, comparative example 1 and comparative example 2 of the present invention;
FIG. 5 is a SERS spectrum of examples 1, 2, 3, 4 and 5 of the present invention;
fig. 6 is a SERS spectrum of example 6 of the present invention.
Detailed Description
The present invention will be further described with reference to the following examples, but the present invention is not limited to the following examples.
The synthesis method of the iodine modified silver nanoparticles in the following examples specifically comprises the following steps:
example 1:
DNA generally exists in a double-stranded form, and forms a triple-stranded structure and a quadruple-stranded structure. As shown in FIG. 1, G-quadruplex is one of four-stranded DNA structures formed by a G (guanine) -rich sequence in a Na + or K + solution, and each layer of the structure is formed by G and G through Hoogsteen hydrogen bond to form G-quartz, and each base is a hydrogen bond donor and acceptor. The structure may be composed of 4, 2 or 1 nucleotide chains. G-quatruplex is typically formed by planar base stacking of two or more G-quatets. G-quadruplex is a ubiquitous DNA secondary structure in humans. The method for detecting the structure of the probe comprises the following steps:
(1) referring to a classical lee method, 0.036g of silver nitrate dissolved in 200mL of water is added with 4mL of sodium citrate with the mass concentration of 1%, the maximum ultraviolet absorption value of the obtained silver nano-ions is 415nm, 10mL of silver sol is centrifuged for 15 minutes (5000r/h) at the low temperature of 4 ℃, the supernatant is removed, 50ul of the centrifuged silver sol is taken, 50ul (KI) of a cleaning agent is added, the mixture is kept at room temperature for 1 hour, and the mixture is placed in a refrigerator at the temperature of 4 ℃ for cooling for 1 hour;
(2) adding 50ul of fitted DNA (TG5T) G-quadruplex sample into the silver nanoparticles modified by the iodide ions obtained in the step (1), adding 16ul of acidic buffer solution with the pH value of 4.5, finally adding 20ul of aluminum ions (0.01M), and then performing Raman detection with the laser wavelength of 633 nm.
Comparative example 1 differs from example 1 in that no aggregating agent is added.
Comparative example 2 is different from example 1 in that a magnesium ion aggregating agent is added.
The sol to be detected, which is synthesized in this embodiment 1 and guided by the aluminum ions, is subjected to scanning by a projection electron microscope, and the result is shown in fig. 2, where the addition of the aluminum ions can be seen, so that the silver nanoparticles are aggregated, and a high-quality hot spot is generated. The sol to be detected which is synthesized in the embodiment 1 and is guided and gathered by the aluminum ions is taken for SERS detection, and a spectrogram shown in fig. 3 is obtained, and a DNA signal has a very high signal-to-noise ratio. As shown in FIG. 4, when the spectrum of the aggregating agent in comparative example 1 without adding the aggregating agent and the spectrum of the magnesium ion in comparative example 2 are compared with those of example 1, no signal can be clearly observed when no aggregating agent a is added, a weak signal is observed when a magnesium ion b line is added, and an O6 internbase H-bond (marked by g-quadruplex formation) peak is observed, the signal-to-noise ratio of the c-line spectrum of example 1 is obviously improved, all the spectrum peaks can be clearly observed, and the resolution is extremely high.
Example 2:
this example is different from example 1 in that the buffer solution added in step (2) has a pH of 3.0.
Example 3
This example is different from example 1 in that the buffer solution added in step (2) has a pH of 5.0.
Example 4
This example is different from example 1 in that the pH of the buffer solution added in step (2) was 4.0.
Example 5
This example differs from example 1 in that the buffer solution added in step (2) has a pH of 3.5.
The sol to be detected which is collected under the guidance of the aluminum ions synthesized in the embodiments 2, 3, 4 and 5 is taken for SERS detection, and a spectrogram shown in fig. 5 is obtained, and all spectral peaks can be clearly observed, so that the method has extremely high resolution.
Example 6
This example is different from example 1 in that the aluminum ion aggregating agent was replaced with a titanium ion aggregating agent.
In conclusion, the detection platform is prepared, the nucleic acid secondary structure without the mark can be conveniently and directly detected, the iodide ions are used as a cleaning agent to remove impurities on the surface of the silver sol, and the aluminum ions are added to well gather the silver nanoparticles cleaned by the iodide ions to generate high-quality hot spots, so that the high-sensitivity detection of various nucleic acid secondary structures is realized.
The above is a preferred example of the present invention and is not intended to limit the technical scope of the present invention in any way. Therefore, any minor modifications, equivalent changes and modifications made to the above embodiments according to the technical spirit of the present invention are within the scope of the present invention.
Claims (4)
1. A method for detecting a nucleic acid structure based on surface enhanced Raman spectroscopy, comprising the steps of:
(1) preparing iodine ion modified and cleaned silver nanoparticle sol;
(2) adding a DNA sample to be detected into a silver nanoparticle centrifugal tube modified by iodide ions, adding a buffer solution with the pH value of 3.0-5.0, and adding an aluminum ion or titanium ion aggregating agent for surface enhanced Raman spectroscopy detection.
2. The method for detecting nucleic acid structures based on surface enhanced raman spectroscopy of claim 1, wherein: the pH value of the acidic buffer solution is 4.5.
3. The method for detecting nucleic acid structures based on surface enhanced raman spectroscopy of claim 1, wherein: the volume ratio of the cationic aggregating agent to the sample to be detected in the step (2) is 2: 5.
4. the method for detecting nucleic acid structures based on surface enhanced raman spectroscopy of claim 1, wherein: the synthesis method of the iodine ion-modified silver nanoparticles in the step (1) is as follows: under the heating condition of a water phase, 0.036g of silver nitrate dissolved in 200mL of water is added with 4mL of sodium citrate with the mass concentration of 1%, the maximum ultraviolet absorption value of the obtained silver nano-ions is 415nm, 10mL of silver sol is centrifuged at the low temperature of 4 ℃ for 15 minutes at a speed of 5000r/h, supernatant liquid is removed, 50ul of the centrifuged silver sol is taken, 50ul of cleaning agent KI is added, the mixture is kept at the room temperature for 1 hour, and the mixture is placed in a refrigerator at the temperature of 4 ℃ and cooled for 1 hour.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102175664A (en) * | 2011-02-17 | 2011-09-07 | 福建师范大学 | Method for detecting surface enhanced Raman spectra of blood RNA |
| CN103529011A (en) * | 2012-07-06 | 2014-01-22 | 南京大学 | Method for detecting DNA (deoxyribonucleic acid) through high sensitivity Raman spectrum |
| CN104406952A (en) * | 2014-11-19 | 2015-03-11 | 上海纳米技术及应用国家工程研究中心有限公司 | Method for preparing SERS (Surface Enhanced Raman Scattering) substrate based on rolling circle amplification technology and core-shell gold nano structures |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102175664A (en) * | 2011-02-17 | 2011-09-07 | 福建师范大学 | Method for detecting surface enhanced Raman spectra of blood RNA |
| CN103529011A (en) * | 2012-07-06 | 2014-01-22 | 南京大学 | Method for detecting DNA (deoxyribonucleic acid) through high sensitivity Raman spectrum |
| CN104406952A (en) * | 2014-11-19 | 2015-03-11 | 上海纳米技术及应用国家工程研究中心有限公司 | Method for preparing SERS (Surface Enhanced Raman Scattering) substrate based on rolling circle amplification technology and core-shell gold nano structures |
Non-Patent Citations (2)
| Title |
|---|
| Label-Free Surface-Enhanced Raman Spectroscopy Detection of DNA with Single-Base Sensitivity;Li-Jia Xu et al.;《JOURNAL OF THE AMERICAN CHEMICAL SOCIETY》;20150402;第137卷;第5149-5154页 * |
| 纳米结构金属表面吸附功能分子的拉曼光谱研究;季媛;《中国优秀硕士学位论文全文数据库 工程科技Ⅰ辑》;20040315(第01期);第B014-501页 * |
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