Method for detecting comprehensive structure information of nucleic acid based on surface enhanced Raman spectroscopy
By cleaning the impurities of silver and gold nanoparticles and using barium ions to promote their aggregation, the problem of impurity signal interference and low aggregation efficiency of SERS technology when detecting nucleic acid structure is solved, and high sensitivity detection of comprehensive nucleic acid structural information is achieved.
Patent Information
- Application Number
- CN202411961740.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-30
- Publication Date
- 2025-04-11
AI Technical Summary
The existing SERS technology has problems such as impurity signal interference and low nanoparticle aggregation efficiency when detecting nucleic acid structures, which leads to difficulty in detecting internal structure information of nucleic acids.
By cleaning the impurities of silver and gold nanoparticles with iodine and bromide ions, the combined barium ions promote the aggregation of nanoparticles, thereby eliminating impurity signal interference and significantly enhancing the Raman signal.
It effectively eliminates the interference of impurity signals on nucleic acid signals, significantly enhances the Raman signal, and realizes high sensitivity detection of comprehensive structural information of nucleic acids.
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Figure CN119799848A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of nucleic acid structure detection, and specifically relates to a method for detecting comprehensive nucleic acid structure information based on surface enhanced Raman spectroscopy. Background Art
[0002] DNA (Deoxyribonucleic Acid) is an important genetic material that is widely present in the cell nucleus and mitochondria. A single strand of DNA is composed of multiple deoxyribonucleotide monomers polymerized by phosphodiester bonds. As the basic building block of DNA, nucleotides are composed of a phosphate group, a deoxyribose molecule, and a base molecule. The bases of DNA include thymine (T) and cytosine (C) of the pyrimidine class, and guanine (G) and adenine (A) of the purine class. The different arrangements and combinations of these four bases create genetic diversity. In addition to the typical double-stranded (DS), DNA sequences can also adopt various non-canonical secondary structures, including G-quadruplex (G4) and i-motif (iM). These structures have been shown to exist in cells, and their formation can significantly affect DNA replication, transcription, and stability, and is associated with many diseases. For example, the G-rich sequence in the telomere forms a G4 structure, which can inhibit telomerase activity, thereby inhibiting tumor cell growth, making G4 a target for tumor drug design.
[0003] Recently, surface enhanced Raman spectroscopy (SERS) has emerged as a valuable, fast, and cost-effective technique for identifying secondary DNA structural features. Its high sensitivity in detecting stretching and vibrational modes of molecular chemical bonds, coupled with compatibility with aqueous conditions, enables it to provide unique structural information even at micromolar or nanomolar concentrations of the analyte. The technique is mainly achieved by exciting nanostructures such as Ag, Au, and Cu at appropriate laser wavelengths to produce an electromagnetic field enhancement effect. SERS technology inherits the ability of Raman spectroscopy to provide molecular fingerprint information with higher sensitivity. There are currently two SERS detection methods: indirect and direct. Indirect methods detect the target through external labels, and the signal obtained is not the signal of the target itself. In contrast, label-free direct SERS detection methods can observe the internal structure of the target and are therefore more suitable for detailed studies of the structural properties of nucleic acids.
[0004] Metal nanoparticles (Ag, Au, Cu, etc.) are one of the commonly used SERS substrates. Aggregated metal nanoparticles can generate a large number of "hot spots", which can significantly enhance the signal of nucleic acids. One method to synthesize silver and gold nanoparticles is to use sodium citrate for reduction. However, this will cause the surface of the two nanoparticles to be covered by reducing agents such as citrate plasma, forming SERS signals generated by impurities, which interfere with the nucleic acid signal, thereby bringing difficulties to the detection of internal structural information of nucleic acids. In addition, there are also differences between gold and silver nanoparticle substrates in detecting nucleic acid structures, and both have advantages and disadvantages. Therefore, SERS detection of nucleic acid structure still faces great challenges. Summary of the invention
[0005] Based on this, the present invention provides a method for detecting comprehensive structural information of nucleic acids based on surface enhanced Raman spectroscopy. The method can clean impurities on the surface of silver and gold nanoparticles through iodine ions and bromide ions, respectively, and remove the adsorption of impurity ions such as citrate on the surface of silver and gold nanoparticles, thereby eliminating the interference of SERS signals generated by impurities on nucleic acid signals. At the same time, barium ions can promote the aggregation of silver and gold nanoparticles, thereby generating a large number of "hot spots", thereby achieving a significant enhancement effect of Raman signals.
[0006] The objective of the present invention is achieved through the following technical solutions:
[0007] The present invention provides a method for detecting comprehensive structural information of nucleic acid based on surface enhanced Raman spectroscopy, comprising the following steps:
[0008] Step 1, preparing silver and gold nanoparticle colloids;
[0009] Step 2: centrifuge the two nanoparticle colloids synthesized above at 4° C. and 8000 rpm for 15 minutes, remove the supernatant, and obtain silver and gold sols;
[0010] Step 3: respectively mixing the silver and gold sols obtained in step 2 with potassium salt solution and incubating overnight;
[0011] Step 4: Take 30 μL of the two sol mixtures obtained in step 3 and mix them with 30 μL of nucleic acid sample respectively as the sample to be tested;
[0012] Step 5, adding aggregating agent BaCl2 solution to the two samples to be tested obtained in step 4 respectively;
[0013] Step six: Use a Raman spectrometer to detect the two substrate sample mixtures obtained in step five.
[0014] Furthermore, the synthesis method of the silver and gold nanoparticle colloids in step 1 is as follows: 0.036 g of silver nitrate is dissolved in 200 mL of water, heated to boiling, 0.04 g of trisodium citrate dihydrate solid is added, and stirred for 40 minutes to obtain dark green silver nanoparticle colloid; 1 mL of a 1% trisodium citrate aqueous solution is added to 100 mL of a 0.01% HAuCl4 boiling solution to obtain red gold nanoparticle colloid.
[0015] Furthermore, the specific steps of step three are: adding 30 μL of 1 mM KI solution to 30 μL of silver sol, and adding 30 μL of 1 mM KBr solution to 30 μL of gold sol.
[0016] Furthermore, in step five, the volume ratio of the aggregating agent BaCl2 solution to the sample to be tested is preferably 1:1.
[0017] Furthermore, the concentration of the BaCl2 solution in step five is 50 mM.
[0018] Compared with the prior art, the present invention has the following beneficial effects:
[0019] 1. Iodide ions and bromide ions can clean impurities on the surface of silver and gold nanoparticles, respectively, and remove the adsorption of impurity ions such as citrate on the surface of silver and gold nanoparticles, thereby eliminating the interference of SERS signals generated by impurities on nucleic acid signals;
[0020] 2. Barium ions can promote the aggregation of silver and gold nanoparticles, thereby generating a large number of "hot spots" and achieving a significant enhancement of the Raman signal;
[0021] 3. Through the simultaneous detection of two nanoparticles, the details of the nucleic acid structure can be fully detected and different nucleic acid structures can be distinguished. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art; obviously, the drawings described below are some embodiments of the present invention, and for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.
[0023] Figure 1 are the G4, iM and DS nucleic acid structures to be tested in the present invention;
[0024] Figure 2 The transmission electron microscope images of two kinds of sols to be tested in which barium ions are aggregated according to the present invention;
[0025] Figure 3 is the SERS spectrum of Example 1 of the present invention;
[0026] Figure 4 The SERS spectra of Example 1 of the present invention and Comparative Example 1;
[0027] Figure 5 is the SERS spectrum of Example 2 of the present invention;
[0028] Figure 6 The SERS spectra of Example 2 of the present invention and Comparative Example 2;
[0029] Figure 7 This is the SERS spectrum of Example 3 of the present invention;
[0030] Figure 8 The SERS spectra of Example 3 of the present invention and Comparative Example 3;
[0031] Fig. 9 is the SERS spectrum of Example 4 of the present invention;
[0032] Fig.10 The SERS spectra of Example 4 of the present invention and Comparative Example 4;
[0033] Fig.11 is the SERS spectrum of Example 5 of the present invention;
[0034] Fig.12 The SERS spectra of Example 5 of the present invention and Comparative Example 5;
[0035] Fig.13 is the SERS spectrum of Example 6 of the present invention;
[0036] Fig.14 The SERS spectra of Example 6 of the present invention and Comparative Example 6;
[0037] Fig.15 This is the principal component analysis PCA diagram of Example 7 of the present invention. DETAILED DESCRIPTION
[0038] In order to make the purpose, technical effect and technical solution of the embodiment of the present invention clearer, the technical solution in the embodiment of the present invention is clearly and completely described below in conjunction with the drawings in the embodiment of the present invention; it is obvious that the described embodiment is a part of the embodiment of the present invention. Based on the embodiment disclosed in the present invention, other embodiments obtained by ordinary technicians in this field without making creative work should all fall within the scope of protection of the present invention.
[0039] Several specific examples are provided below to verify the effect of the method. It should be noted that the containers and reagents used in the following examples, unless otherwise specified, can be obtained from commercial sources. The model of the Raman spectrometer used is LabRAM Soleil (Horiba, France). Raman test parameters: laser detection wavelength is 638nm, scanning time is 10s, accumulation time is 1 time, and laser energy is 15mw.
[0040] Example 1: This example provides a method for detecting G4 based on silver nanomaterial as a SERS substrate, comprising the following steps:
[0041] Step 1, dissolving 0.036 g of silver nitrate in 200 mL of water, heating to boiling, adding 0.04 g of trisodium citrate dihydrate solid, and stirring for 40 minutes to obtain dark green silver nanoparticle colloid;
[0042] Step 2, centrifuging the synthesized silver nanoparticle colloid at 4° C. and 8000 rpm for 15 minutes, removing the supernatant to obtain a silver sol;
[0043] Step 3: Take 30 μL of the silver sol obtained in step 2, mix it with 30 μL of 1 mM KI solution, and incubate overnight;
[0044] Step 4: Take 30 μL of the sol mixture obtained in step 3 and mix it with 30 μL of the nucleic acid sample to be tested;
[0045] Step 5: Add 30 μL of 50 mM BaCl2 solution to the mixed solution obtained in step 4;
[0046] Step 6: Use a Raman spectrometer to detect the substrate sample mixture in step 5.
[0047] Comparative Example 1: The difference from Example 1 is that an aluminum ion aggregating agent is added.
[0048] Figure 1 The G4, iM and DS structures to be tested are shown. G-quadruplex is a DNA secondary structure formed by guanine (G)-rich oligonucleotides through Hoogsteen hydrogen bonding interactions. iM is formed by cytosine (C)-rich sequences under weak acid conditions, in which two parallel chains are connected by half-protonated C:CH+ base pairs and intercalated with the other two antiparallel chains. DS is generally a structure formed by AT, CG complementary pairing.
[0049] Figure 2 Transmission electron micrographs of gold and silver nanoparticle substrates are shown, showing the synthesis of both nanoparticles.
[0050] Figure 3The addition of Ba into the silver nanoparticle matrix 2+ The SERS spectrum after mixing with G4 shows good spectrum signal.
[0051] like Figure 4 As shown, Example 1 adds Ba 2+ Aggregating agent and Al in Comparative Example 1 3+ It can be clearly observed that the 653cm -1 and 1582cm -1 At the corresponding peak position, the effect of Example 1 is better than that of Comparative Example 1. This shows that in the silver substrate, Ba 2+ Good results for structural testing.
[0052] Example 2: This example provides a method for detecting G4 based on a gold nanomaterial as a SERS substrate, comprising the following steps:
[0053] Step 1, adding 1 mL of 1% sodium citrate trisodium aqueous solution into 100 mL of 0.01% HAuCl4 boiling solution to obtain red gold nanoparticle colloid;
[0054] Step 2, centrifuging the synthesized gold nanoparticle colloid at 4° C. and 8000 rpm for 15 minutes, removing the supernatant to obtain a gold sol;
[0055] Step 3: Take 30 μL of the gold sol obtained in step 2, mix it with 30 μL of 1 mM KBr solution, and incubate overnight;
[0056] Step 4: Take 30 μL of the sol mixture obtained in step 3 and mix it with 30 μL of the nucleic acid sample to be tested;
[0057] Step 5: Add 30 μL of 50 mM BaCl2 solution to the mixed solution obtained in step 4;
[0058] Step 6: Use a Raman spectrometer to detect the substrate sample mixture in step 5.
[0059] Comparative Example 2: The difference from Example 2 is that a magnesium ion aggregating agent is added.
[0060] Figure 5 Shows the addition of Ba to the gold nanoparticle substrate 2+ The SERS spectrum after mixing with G4 shows good spectrum signal.
[0061] like Figure 6 As shown, Example 2 adds Ba 2+ Aggregating agent and Mg in Comparative Example 2 2+It can be clearly observed that the 1324cm -1 and 1340cm -1 At the corresponding peak position, the effect of Example 2 is better than that of Comparative Example 2. This shows that in the gold substrate, Ba 2+ Good effect for structural detection. In addition, compared with Example 1 and Example 2, the peak positions of the G4 structure obtained by the two substrates are different. The silver nanoparticles obtain the signal of the G plane of G4, while the gold nanoparticles obtain the signal of the glycosidic bond of G4. The two can be combined to obtain more comprehensive structural information of G4.
[0062] Example 3: The difference between this example and Example 1 is that the nucleic acid structure to be tested is iM.
[0063] Comparative Example 3: The difference from Example 3 is that an aluminum ion aggregating agent is added.
[0064] Figure 7 The addition of Ba into the silver nanoparticle matrix 2+ The SERS spectrum after mixing with iM shows good spectrum signal.
[0065] like Figure 8 As shown, Example 3 adds Ba 2+ Aggregating agent and Al in Comparative Example 1 3+ It can be clearly observed that the 787cm corresponding to the iM structure -1 and 1403cm -1 At the corresponding peak position, the effect of Example 3 is better than that of Comparative Example 3. This shows that in the silver substrate, Ba 2+ Good results for structural testing.
[0066] Embodiment 4: The difference between this embodiment and embodiment 2 is that the structure to be tested is iM.
[0067] Comparative Example 4: The difference from Example 3 is that a magnesium ion aggregating agent is added.
[0068] Fig. 9 Shows the addition of Ba to the gold nanoparticle substrate 2+ The SERS spectrum after mixing with iM shows good spectrum signal.
[0069] like Fig.10 As shown, Example 4 adds Ba 2+ Aggregating agent and Mg in Comparative Example 4 2+ It can be clearly observed that the 787cm corresponding to the iM structure -1 and 1403cm -1At the corresponding peak position, the effect of Example 4 is better than that of Comparative Example 4. This shows that in the gold substrate, Ba 2+ Good results for structural testing.
[0070] Embodiment 5: The difference between this embodiment and embodiment 1 is that the structure to be tested is DS.
[0071] Comparative Example 5: The difference from Example 5 is that an aluminum ion aggregating agent is added.
[0072] Fig.11 The addition of Ba into the silver nanoparticle matrix 2+ The SERS spectrum after mixing with DS shows good spectrum signal.
[0073] like Fig.12 As shown, Example 5 adds Ba 2+ Aggregating agent and Al in Comparative Example 4 3+ It can be clearly observed that the 1577cm -1 At the corresponding peak position, the effect of Example 5 is better than that of Comparative Example 5. This shows that in the silver substrate, Ba 2+ Good results for structural testing.
[0074] Embodiment 6: The difference between this embodiment and embodiment 2 is that the structure to be tested is DS.
[0075] Comparative Example 6: The difference from Example 6 is that a magnesium ion aggregating agent is added.
[0076] Fig.13 Shows the addition of Ba to the gold nanoparticle substrate 2+ The SERS spectrum after mixing with DS shows good spectrum signal.
[0077] like Fig.14 As shown, Example 6 adds Ba 2+ Aggregating agent and Mg in Comparative Example 6 2+ It can be clearly observed that the 804cm corresponding to the DS structure -1 At the corresponding peak position, the effect of Example 6 is better than that of Comparative Example 6. This shows that in the gold substrate, Ba 2+ Good effect for structural detection. In addition, compared with Example 6, the DS structure peaks obtained by the two substrates are different. The silver nanoparticles obtain the signal of DS hydrogen bond, while the gold nanoparticles obtain the signal of DS phosphate skeleton. The two can be combined to obtain more comprehensive structural information of DS.
[0078] Example 7: Silver nanoparticles modified with iodide ions and barium ions (AgIBaNPs) and gold nanoparticles modified with bromide ions and barium ions (AuBrBaNPs) were used as substrates to detect more PCA classification diagrams of different G4, iM and DS sequences.
[0079] Step 1, preparing silver and gold nanoparticle colloids;
[0080] The specific steps of step 1 are as follows: dissolving 0.036 g of silver nitrate in 200 mL of water, heating to boiling, adding 0.04 g of trisodium citrate dihydrate solid, and stirring for 40 minutes to obtain dark green silver nanoparticle colloid; adding 1 mL of 1% trisodium citrate aqueous solution to 100 mL of 0.01% HAuCl4 boiling solution to obtain red gold nanoparticle colloid;
[0081] Step 2, centrifuging the two synthesized nanoparticle colloids at 4° C. and 8000 rpm for 15 minutes, and removing the supernatant;
[0082] Step 3: Take 30 μL of the silver and gold sols obtained in step 2, add 30 μL of 1 mM KI solution to the silver sol, and add 30 μL of 1 mM KBr solution to the gold sol, and incubate overnight;
[0083] Step 4: Take 30 μL of the two sol mixtures obtained in step 3 and mix them with 30 μL of the nucleic acid sample to be tested;
[0084] Step 5: Add 30 μL of 50 mM BaCl2 solution to each of the two mixed solutions obtained in step 4;
[0085] Step 6: Use a Raman spectrometer to detect the two substrate sample mixtures in step 5.
[0086] Fig.15 The PCA graph of the principal component analysis of the SERS spectra of AgIBaNPs and AuBrBaNPs obtained in Example 7 for detecting G4, iM and DS as substrates is shown. It can be seen that AgIBaNPs can distinguish G4 and iM with different numbers of layers, but cannot distinguish G4 and DS with different conformations. AuBrBaNPs can distinguish G4 and DS with different conformations, but cannot distinguish G4 and iM with different numbers of layers. Therefore, the combination of the two substrates can obtain more comprehensive nucleic acid structure information.
[0087] The above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the same. Although the present invention has been described in detail with reference to the above embodiments, a person skilled in the art can still modify or make equivalent substitutions to the specific implementations of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention are within the scope of protection of the claims of the present invention to be approved.
Claims
1. A method for detecting comprehensive structural information of nucleic acids based on surface enhanced Raman spectroscopy, characterized in that: The steps include: Step 1, preparing silver and gold nanoparticle colloids; Step 2: centrifuge the two nanoparticle colloids synthesized above at 4° C. and 8000 rpm for 15 minutes, remove the supernatant, and obtain silver and gold sols; Step 3: respectively mixing the silver and gold sols obtained in step 2 with potassium salt solution and incubating overnight; Step 4: Take 30 μL of the two sol mixtures obtained in step 3 and mix them with 30 μL of nucleic acid sample respectively as the sample to be tested; Step 5, adding aggregating agent BaCl2 solution to the two samples to be tested obtained in step 4 respectively; Step six: Use a Raman spectrometer to detect the mixture of the two substrate samples obtained in step five.
2. A method for detecting comprehensive structural information of nucleic acid based on surface enhanced Raman spectroscopy according to claim 1, characterized in that: The synthesis method of the silver and gold nanoparticle colloid in the step 1 is as follows: 0.036g of silver nitrate is dissolved in 200mL of water, and after heating to boiling, 0.04g of trisodium citrate dihydrate solid is added, and after stirring for 40 minutes, dark green silver nanoparticle colloid is obtained; 1mL of a 1% trisodium citrate aqueous solution is added to 100mL of a 0.01% HAuCl4 boiling solution to obtain red gold nanoparticle colloid.
3. The method for detecting comprehensive structural information of nucleic acid based on surface enhanced Raman spectroscopy according to claim 1, characterized in that: The specific steps of step three are: adding 30 μL of 1 mM KI solution to 30 μL of silver sol, and adding 30 μL of 1 mM KBr solution to 30 μL of gold sol.
4. The method for detecting comprehensive structural information of nucleic acid based on surface enhanced Raman spectroscopy according to claim 1, characterized in that: In the step 5, the volume ratio of the aggregating agent BaCl2 solution to the sample to be tested is 1:
1.
5. The method for detecting comprehensive structural information of nucleic acid based on surface enhanced Raman spectroscopy according to claim 1, characterized in that: The concentration of the BaCl2 solution in step 5 is 50 mM.