CN113310966A - Single nuclear-satellite assembly surface enhanced Raman molecular ruler and application thereof - Google Patents
Single nuclear-satellite assembly surface enhanced Raman molecular ruler and application thereof Download PDFInfo
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- CN113310966A CN113310966A CN202110630094.0A CN202110630094A CN113310966A CN 113310966 A CN113310966 A CN 113310966A CN 202110630094 A CN202110630094 A CN 202110630094A CN 113310966 A CN113310966 A CN 113310966A
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Abstract
The invention discloses a single nuclear-satellite assembly Surface Enhanced Raman Spectroscopy (SERS) molecular ruler and application thereof in detecting single tetrahedral DNA configuration change. The molecular ruler comprises nuclear gold nanoparticles, tetrahedral DNA and satellite gold nanoparticles, wherein the satellite gold nanoparticles are connected with the nuclear gold nanoparticles through the tetrahedral DNA. In Hg2+Under the drive, the configuration of the tetrahedral DNA is changed, the distance between the nuclear nanogold and the satellite nanogold is reduced, and the plasmon coupling effect is enhanced, so that the SERS strength of the nuclear-satellite nano assembly is greatly increased; the process can be observed in real time through a DFM and Raman spectrum combined device, the intensity of the SERS spectrum of a single nuclear-satellite assembly body is increased in a step shape, and the tetrahedral DNA configuration change can be monitored in real time at a single molecular level.
Description
Technical Field
The invention belongs to the technical field of single molecule detection, and particularly relates to a single nuclear-satellite assembly Surface Enhanced Raman Spectroscopy (SERS) molecular ruler and application thereof in detection of single tetrahedral DNA configuration change.
Background
The DNA aptamer is DNA based on the driving structure change of a specific target molecule and has a powerful biological application function. The conformation change of a single DNA aptamer is tracked in real time at a single molecule level, so that the method is favorable for better understanding of a reaction mechanism and a kinetic mechanism of the DNA aptamer and is greatly helpful for researching molecular biology, biochemistry and medicine. Currently common methods of single molecule technology include Surface Enhanced Raman (SERS) spectroscopy and fluorescence spectroscopy. Especially, SERS spectrum has high sensitivity and rich fingerprint information, so that it is widely used in single molecule detection. However, SERS spectroscopy is rarely able to monitor changes in single molecule conformation in real time, and its random time fluctuations and brownian dynamics of raman probe molecules in hot spot regions lead to fluctuations in SERS intensity, making detection more complex. Although these drawbacks can be avoided using the Tip Enhanced Raman Spectroscopy (TERS) approach, the TERS technology also has its drawbacks, e.g., the TERS metal tips are easily broken during scan testing. In addition, TERS equipment is very expensive. In order to solve these problems, it is urgently required to design a low-cost and reliable method.
SERS spectral information of single plasma nanoparticles can be tracked in real time by combining a Dark Field Microscope (DFM) and a Raman spectrum technology. By utilizing the SERS spectrum changing along with time, the DFM and the Raman spectrum can be combined to monitor the change of the distance between two adjacent plasmas in real time, so that the continuously changed distance between the plasmas is converted into the response of a series of SERS signals, and a new possibility is provided for designing an excellent SERS molecular ruler. Within a certain distance range, the electromagnetic coupling effect and the distance accord with a simple exponential relationship, and a molecular ruler probe can be constructed by utilizing the relationship, so that an effective means is provided for researching the conformation change of biomolecules at a single molecule level.
Disclosure of Invention
Aiming at the defects of the existing SERS single molecule technology, the invention provides a single nuclear-satellite assembly Surface Enhanced Raman Spectroscopy (SERS) molecular ruler and application thereof in detecting single tetrahedral DNA configuration change.
In order to achieve the purpose, the invention adopts the following technical scheme:
a single nuclear-satellite assembly surface enhanced Raman molecular ruler comprises nuclear gold nanoparticles, tetrahedral DNA and satellite gold nanoparticles, wherein the satellite gold nanoparticles are connected with the nuclear gold nanoparticles through the tetrahedral DNA;
three vertexes of the bottom surface of the tetrahedral DNA are connected with the nuclear gold nanoparticles, a fourth vertex of the tetrahedral DNA is connected with the satellite gold nanoparticles, any one of three edges between the fourth vertex and the three vertexes of the bottom surface is a single chain, and the other five edges of the tetrahedral DNA are double chains;
the single strand of the tetrahedral DNA is Hg-containing2+Single-stranded DNA of the aptamer.
Further, the molecular ruler comprises at least 8 tetrahedral DNAs and at least 8 satellite gold nanoparticles.
Further, the particle size of the nuclear gold nanoparticle is 80-150nm, and the particle size of the satellite gold nanoparticle is 20-30 nm.
Further, the particle size of the nuclear gold nanoparticle is 100nm, and the particle size of the satellite gold nanoparticle is 20 nm.
Further, three vertexes of the bottom surface of the tetrahedral DNA are connected with the nuclear gold nanoparticle through covalent bonds, and the fourth vertex of the tetrahedral DNA is connected with the satellite gold nanoparticle through covalent bonds.
The preparation method of the single nuclear-satellite assembly surface enhanced Raman molecular ruler comprises the following steps:
step 2, modifying tetrahedral DNA on the surface of the nuclear gold nano-particles;
step 3, modifying 4-mercaptobenzoic acid on the surface of the satellite gold nanoparticles;
and 4, modifying the satellite gold nanoparticles obtained in the step 3 on the surfaces of the nuclear gold nanoparticles obtained in the step 2 to obtain the nuclear-satellite assembly SERS molecular ruler.
The application of the single nuclear-satellite assembly surface enhanced Raman molecular ruler in detecting the configuration change of the single tetrahedral DNA.
Further, the application is to Hg2+The solution is dripped on the surface of the ITO glass sheet adsorbed with the nuclear-satellite structure assembly, and the change of the SERS spectrum of a single nuclear-satellite nano assembly is continuously observed in real time by utilizing a dark field microscope and Raman spectrum combined device.
Further, the Hg2+The concentration of the solution is 10-100 nM. Preferably, Hg2+The concentration of the solution was 50 nM.
As shown in FIG. 1, the single nuclear-satellite assembly surface enhanced Raman molecular ruler of the present invention is a nuclear-satellite assembly assembled by using tetrahedral DNA as a linker molecule, wherein one side of the tetrahedral DNA is composed of Hg-containing nanoparticles and satellite gold nanoparticles2+Single-stranded DNA of the aptamer. In Hg2+In the presence of Hg2+And Hg2+Strong affinity between aptamers, Hg2+The aptamer becomes a hairpin structure, resulting in the tetrahedral DNA transitioning from a relaxed state to a strained state. As a result, the spacing between the nuclear gold nanoparticles and the satellite gold nanoparticles is reduced. Due to the surface plasmon coupling effect, the SERS intensity of the nuclear-satellite nano assembly is greatly increased, and the nuclear-satellite nano assembly becomes an ideal optical molecular ruler for monitoring the configuration change of single tetrahedral DNA. And continuously observing the change of SERS intensity by using a dark field optical microscope (DFM) and Raman spectrum combined instrument, and monitoring the process of the change of the single tetrahedral DNA configuration in real time.
Has the advantages that:
1. the SERS molecular ruler of the single nuclear-satellite assembly has the capability of detecting the change of the tetrahedral DNA detection configuration in single-level real time, and has the advantages of simple structure, convenience in manufacturing and the like. In one embodiment of the invention, 50nM Hg is added2+Then, 1073 cm-1The step height of the SERS intensity is about 2.16-2.7 a.u, and each step of the SERS intensity corresponds to a single tetrahedral DNA structure change event.
2. Compared with the traditional plasma molecular ruler, the SERS molecular ruler is more sensitive to the change of the distance between particles.
3. The defect that the traditional SERS single-molecule technology can hardly monitor the change of single-molecule conformation in real time is overcome.
Drawings
Fig. 1 is a schematic structural diagram and a detection principle of the SERS molecular ruler of the single nuclear-satellite assembly of the present invention.
FIG. 2 shows the addition of 50nM Hg to example 12+And then, a waterfall graph of the SERS spectrum of the nuclear-satellite nano assembly changing along with time.
FIG. 3 shows the addition of 50nM Hg to example 12+Then, 1073 cm-1Change of SERS intensity with time (data point of black dot), 1073 cm-1The fit of SERS intensity at (horizontal line) versus time (vertical line) represents the step height of the SERS intensity.
FIG. 4 shows the addition of 10 nM (a) and 100nM (c) Hg, respectively, to example 12+Then, a waterfall graph of SERS spectrum of the nuclear-satellite nano assembly changing along with time; b is the addition of 10 nM Hg2+Then, 1073 cm-1SERS intensity over time; d is the addition of 100nM Hg2+Then, 1073 cm-1The SERS intensity of (a) varies with time. Wherein: 1073 cm-1The fit of SERS intensity at (horizontal line) versus time (vertical line) represents the SERS intensity step height.
Detailed Description
The invention is described in further detail below with reference to the figures and the specific examples, which should not be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention. The experimental methods and reagents of the formulations not specified in the examples are in accordance with the conventional conditions in the art.
Example 1
The DNA sequences used in this example are shown below:
Tetra1，5’-SH-AGCATTACAGCTTGCTACACGATTCAGA CTTAGGAATGTTCGACATGCGAGGGTCCAATACCG-3’（SEQ ID NO.1）
Tetra2, 5’-SH-ACGAACATTCCTAAGTCTGAAATTTATC ACCCGCCATAGTAGACGTATCACCAGGCAGTTGAG-3’ （SEQ ID NO.2）
Tetra 3, 5’-SH-ACGTGTAGCAAGCTGTAATCGACGTTCTTTC TTCCCCTTGTTTGTTGTACTATGGCGGGTGATAAA-3’ （SEQ ID NO.3）
Tetra 4, 5’-ACGGTATTGGACCCTCGCATGACTCAACTGCCTGGTGATACGA(A)15-3’ （SEQ ID NO.4）
the above DNA sequences were purchased from Takara Biotechnology co.ltd (chinese grand union).
The tetrahedral DNA is formed by mixing four single-stranded DNA strands (Tetra 1, Tetra 2, Tetra 3 and Tetra 4) at equal ratio and annealing.
The lower division part in the Tetra 3 sequence can correspond to Hg2+Binding forms a hairpin structure that converts tetrahedral DNA from a relaxed state to a strained state.
In this embodiment, 100nm AuNPs are used as the nuclear gold nanoparticles, and 20nm AuNPs are used as the satellite gold nanoparticles.
The 20nm AuNPs and the 100nm AuNPs used in the experiment are synthesized and prepared by adopting a seed growth method.
The nuclear-satellite assembly SERS molecular ruler is constructed by connecting 20nm AuNP on the surface of 100nm AuNP by using tetrahedral DNA as a connecting molecule. The specific method comprises the following steps:
(1) the ITO glass sheet is soaked in an ethanol solution (10: 2 v/v) of 3-Mercaptopropyltriethoxysilane (MPTES) for 3 hours, taken out, repeatedly washed by ethanol, and dried by nitrogen. Then, the ITO glass sheet was immersed in 5 ml of an ethanol solution (0.02 nM) of 100nM AuNPs for 3 hours, taken out, repeatedly rinsed with ultrapure water, and blow-dried with nitrogen to obtain an ITO glass sheet having 100nM AuNPs fixed thereto.
(2) 4 pieces of single-stranded DNA (Tetra 1, Tetra 2, Tetra 3 and Tetra 4) in TM buffer solution (20 mM Tris, 50 mM MgCl)2pH 8.0) in equal proportions. The resulting mixed solution was heated to 95 ℃ and stored at 95 ℃ for 5 minutes, then cooled to 4 ℃ over 5 minutes and held at 4 ℃ for 5 minutes,obtaining DNA with a tetrahedral structure.
(3) 200 μ L of tetrahedral DNA solution (1 μ M) was first dropped onto the surface of 100nm AuNPs modified ITO glass plate and incubated at room temperature in a shaker at 30 rpm/min for 3 hours. The ITO glass slide was then rinsed with ultra pure water and dried in a weak stream of nitrogen.
(4) mu.L of 4-mercaptobenzoic acid (1 mM) is added into 20nM AuNPs aqueous solution (0.02 nM) and incubated for 30 min, so as to obtain the 4-mercaptobenzoic acid functionalized and modified 20nM AuNPs.
(5) And (2) dripping 100 mu L of 20nm AuNPs on an AuNPs @ tetrahedral DNA modified ITO glass slide, incubating for 3 hours at room temperature in an oscillator at 30 rpm/min, then washing the surface of the ITO glass slide with ultrapure water, and drying in nitrogen to obtain the SERS molecular ruler of the nuclear-satellite assembly.
The method for detecting the tetrahedral DNA configuration change of the single nuclear-satellite assembly SERS molecular ruler at the single molecular level comprises the following specific steps: the ITO glass plate having the core-satellite structure assembly adsorbed thereon was placed on a motorized translation stage, and 200. mu.L of 50nM Hg was then placed thereon2+The solution is dripped on the surface of an ITO glass sheet, and the change of the SERS spectrum of a single nuclear-satellite nano assembly is continuously observed in real time. The wavelength of excitation light used in Raman spectrum measurement is 633 nm, the laser power is 29.2 muW, the exposure time is 5 seconds, and the configuration of tetrahedral DNA is observed to change after mercury ions are added. 50nM Hg was added2+The time-varying SERS spectra of the mononuclear-satellite nano-assemblies are shown in FIG. 2, where 1075 cm is measured-1The SERS intensity at (a) increases stepwise (as shown in fig. 3). The step heights for SERS intensity are about 2.16-2.7 a.u. Each step in SERS intensity corresponds to a single tetrahedral DNA structure altering event.
In order to fully understand the potential application prospect of the SERS molecular ruler, SERS molecular ruler experiments are respectively carried out under low concentration (10 nM) and high concentration (100 nM). When Hg is contained2+At lower concentrations, only one SERS intensity step signal was observed (fig. 4a and b). The reason for this is Hg2+In small quantities, only with a small amount of unfolded Hg2+Aptamer-bound population, Hg2+Aptamer binding to Hg2+The probability of (2) is low. In contrast, high Hg concentrations2+The addition of (a) will, at a given time, simultaneously give rise to multiple independent tetrahedral DNA structure change events (fig. 4c and d) because it produces a SERS intensity step height 2-4 times the SERS intensity step height produced by a single tetrahedral DNA structure change event. To better record single tetrahedral DNA structural change events, the optimal Hg was chosen2+Concentration is very important because a trade-off needs to be made between the probability of a single tetrahedral DNA structure change event occurring and the number of tetrahedral DNA structure change events occurring at a particular time. Thus, 50nM Hg was selected2+A single tetrahedral DNA structure-changing event is driven as an optimal concentration.
Sequence listing
<110> Nanjing post and telecommunications university
<120> single nuclear-satellite assembly surface enhanced Raman molecular ruler and application thereof
<130> 20210604
<160> 4
<170> SIPOSequenceListing 1.0
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ccg 63
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acgaacattc ctaagtctga aatttatcac ccgccatagt agacgtatca ccaggcagtt 60
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acgtgtagca agctgtaatc gacgttcttt cttccccttg tttgttgtac tatggcgggt 60
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acggtattgg accctcgcat gactcaactg cctggtgata cgaaaaaaaa aaaaaaaa 58
Claims (10)
1. A single nuclear-satellite assembly surface enhanced Raman molecular ruler is characterized in that: the nano-gold nanoparticle comprises a nano-gold nanoparticle, tetrahedral DNA and a satellite nano-gold nanoparticle, wherein the satellite nano-gold nanoparticle is connected with the nano-gold nanoparticle through the tetrahedral DNA;
three vertexes of the bottom surface of the tetrahedral DNA are connected with the nuclear gold nanoparticles, a fourth vertex of the tetrahedral DNA is connected with the satellite gold nanoparticles, any one of three edges between the fourth vertex and the three vertexes of the bottom surface is a single chain, and the other five edges of the tetrahedral DNA are double chains;
the single strand of the tetrahedral DNA is Hg-containing2+Single-stranded DNA of the aptamer.
2. The single nuclear-satellite assembly surface enhanced raman molecular ruler of claim 1, wherein: the molecular ruler comprises at least 8 tetrahedral DNAs and at least 8 satellite gold nanoparticles.
3. The single nuclear-satellite assembly surface enhanced raman molecular ruler of claim 1, wherein: the particle size of the nuclear gold nanoparticle is 80-150nm, and the particle size of the satellite gold nanoparticle is 20-30 nm.
4. The single nuclear-satellite assembly surface enhanced raman molecular ruler of claim 3, wherein: the particle size of the nuclear gold nanoparticles is 100nm, and the particle size of the satellite gold nanoparticles is 20 nm.
5. The single nuclear-satellite assembly surface enhanced raman molecular ruler of claim 1, wherein: three vertexes of the bottom surface of the tetrahedral DNA are connected with the nuclear gold nanoparticles through covalent bonds, and the fourth vertex of the tetrahedral DNA is connected with the satellite gold nanoparticles through covalent bonds.
6. The method for preparing the surface-enhanced Raman molecular ruler of the single nuclear-satellite assembly according to claim 1, wherein the method comprises the following steps: the method comprises the following steps:
step 1, preparing nuclear gold nanoparticles, tetrahedral DNA and satellite gold nanoparticles respectively;
step 2, modifying tetrahedral DNA on the surface of the nuclear gold nano-particles;
step 3, modifying 4-mercaptobenzoic acid on the surface of the satellite gold nanoparticles;
and 4, modifying the satellite gold nanoparticles obtained in the step 3 on the surfaces of the nuclear gold nanoparticles obtained in the step 2 to obtain the nuclear-satellite assembly SERS molecular ruler.
7. Use of the single nuclear-satellite assembly surface enhanced raman molecular ruler of claim 1 for detecting changes in the configuration of a single tetrahedral DNA.
8. Use according to claim 7, characterized in that: the application is to add Hg2+The solution is dripped on the surface of the ITO glass sheet adsorbed with the nuclear-satellite structure assembly, and the change of the SERS spectrum of a single nuclear-satellite nano assembly is continuously observed in real time by utilizing a dark field microscope and Raman spectrum combined device.
9. Use according to claim 7, characterized in that: the Hg is2+The concentration of the solution is 10-100 nM.
10. Use according to claim 9, characterized in that: what is needed isHg is described2+The concentration of the solution was 50 nM.
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| CN104458704A (en) * | 2014-12-24 | 2015-03-25 | 中国科学院合肥物质科学研究院 | Method for detecting low-concentration mercury ions based on DNA modified SERS substrate |
| CN110066851A (en) * | 2019-04-26 | 2019-07-30 | 南京邮电大学 | Tetrahedron DNA mediates assembling fluorescence nano bioprobe, preparation method and application |
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| CN111781186A (en) * | 2020-06-12 | 2020-10-16 | 南京邮电大学 | SERS sensor for integrally detecting tumor protein and nucleic acid marker and preparation method thereof |
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| CN104458704A (en) * | 2014-12-24 | 2015-03-25 | 中国科学院合肥物质科学研究院 | Method for detecting low-concentration mercury ions based on DNA modified SERS substrate |
| CN110066851A (en) * | 2019-04-26 | 2019-07-30 | 南京邮电大学 | Tetrahedron DNA mediates assembling fluorescence nano bioprobe, preparation method and application |
| CN110243891A (en) * | 2019-07-23 | 2019-09-17 | 青岛农业大学 | A kind of label-free homogeneous electrochemical biosensor method detecting cancer cell |
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