A quantitative detection method for mercury ions based on surface enhanced Raman scattering technology

Abstract

The invention discloses a method for quantitatively detecting mercury ions based on surface enhanced Raman scattering technology, wherein a gold nano-finger array structure is first obtained by nano-imprinting, and then a specific single-stranded DNA is modified on the Au nano-finger array structure by sulfur-hydrogen bonds, and the finger array structure collapses during the volatilization of the DNA solution, and finally, the single-stranded DNA lying on the Au surface is converted into a double-stranded DNA spiral space structure perpendicular to the Au surface by titrating the mercury ion solution, so that the terminal base molecules in the DNA are away from the coupling enhanced electromagnetic field formed in the gap, and thus the SERS signal thereof is reduced. The invention can simultaneously realize the interference-free high selectivity and high sensitivity detection of Hg ions in a mixed solution.

Description

Technical Field

[0001] The invention belongs to the technical field of analysis and detection, and in particular relates to a method for quantitatively detecting mercury ions based on surface enhanced Raman scattering technology. Background Art

[0002] Mercury(II) (Hg 2+ ) is a pollutant widely found in water environments and may cause serious harm to human health. Mercury ions, especially methylmercury, can accumulate in the human body through the food chain and induce permanent damage to the kidneys, central nervous system, and digestive system, leading to a variety of chronic diseases (New England Journal of Medicine, 2003, 349(18):1731-1737). 2+ The high selectivity and high sensitivity of Hg quantitative detection has been a long-term concern. 2+ Detection methods include organic dye probes, conjugated polymers, and electrochemical anodic stripping voltammetry. These methods usually rely on coordination chemistry and the oxidation-reduction properties of mercury ions, so the detection systems are mostly limited by chemical reaction conditions and the use environment (laboratory). Recently, some biorecognition materials can be used to improve Hg 2+ The detection specificity of the aptamer includes aptamer DNA, nuclease and protein. For DNA aptamers, the base molecule thymine-Hg 2+ -thymine (T-Hg 2+ The specific interaction of Hg-T) has attracted special attention in biological research. Using this interaction, researchers have developed a series of new Hg-T detection methods through electrochemical, fluorescence spectroscopy and colorimetric detection. 2+ Detection probes and sensors. For example, Yang et al. used single-stranded DNA modified with fluorescein and gold nanoparticles to detect Hg by fluorescence and colorimetry. 2+ Ions (Analytical Chemistry, 2008, 80(23):9021-9028). Chen et al. used a colorimetric paper analysis device based on gold nanoparticles to detect mercury ions (Analytical Chemistry, 2014, 86(14):6843-6849).

[0003] At present, surface enhanced Raman scattering (SERS) based on the optical effect of plasmons on the surface of metal micro-nanostructures has been widely used in physics, chemistry, materials science, biomedicine and other fields. Its enhancement principle is based on the resonant excitation of the surface plasmon effect of metal nanostructures, thereby forming an enhanced electromagnetic field on the surface of the metal nanostructure, so that the Raman signal of the molecules near the enhanced electromagnetic field on the metal surface is greatly enhanced. Generally, the enhancement factor of the Raman signal is proportional to the fourth power of the enhanced electromagnetic field intensity. Compared with other measurement methods, Raman detection has molecular recognition and fingerprint properties, and is insensitive to aqueous solutions, which is particularly useful for the detection of biological molecules. However, the disadvantage of Raman spectroscopy is that the signal is very weak, and the use of surface enhanced Raman technology has solved this problem. Therefore, surface enhanced Raman spectroscopy can be truly applied to the field of trace detection of biological molecules.

[0004] How to control the excitation of the local light field and obtain a strong local light field state density is crucial to its efficiency in enhancing SERS. Compared with the resonant excitation of a single nanostructure, the metal coupling structure will produce a stronger local light field due to the gap plasmon coupling effect between the structures, and therefore has attracted widespread attention. In 1999, Academician Xu Hongxing of Wuhan University discovered for the first time that the experimental phenomenon of SERS single-molecule detection (Phys. Rev. Lett., 1999, 83(21):4357-4360) was caused by the coupled electromagnetic field in the ultra-small gap between Ag nanoparticles, and the field enhancement can reach 10 3 times, making the SERS enhancement factor reach 10 12 , thus explaining the single-molecule detection phenomenon of SERS. In recent years, Professor Shuang Zhang of the University of Birmingham in the UK and others have used the curled nanobowl combined with the medium and gold nanoparticle deposition to obtain the gold coupling structure and achieved wide-band single-molecule SERS detection (Nat. Commun., 2018, 9(1): 5248-5255).

[0005] For metal coupling structures, it has been recognized that for ultra-small gap plasmon systems that can achieve ultra-strong coupled electromagnetic field enhancement, both classical electromagnetic theory and quantum effects need to be considered. Classical electromagnetic theory shows that the smaller the gap, the stronger the coupled electromagnetic field strength exponentially increases; while the quantum effect shows that the smaller the gap (for example, below 1nm), the gradually attenuated coupled electromagnetic field strength. This is due to the non-localization of electrons and the tunneling effect. The electron density at the interface on both sides of the gap will decrease, resulting in a decrease in the coupled electric field strength. Therefore, under the joint influence of classical electromagnetic theory and quantum effects, the gap in the metal coupling structure reaches a sub-nanometer size, which can make the coupled electromagnetic field reach the level of single-molecule Raman detection. At present, based on nano-metal particles in solution, the gap between particles can be more accurately controlled by DNA methods, and the size can be reduced to 1-2 nanometers. However, the use of colloidal particle solutions as an enhancement technology has great limitations in actual use and cannot meet actual needs. In addition, the controllability of the enhanced hot spot area is poor, and the molecule to be tested cannot be accurately placed in the coupled electromagnetic field area, and Raman detection at the single-molecule level cannot be accurately achieved. However, due to the limitation of precision, the metal nanoarray structure prepared by physical method is unable to provide surface-enhanced Raman substrate with high enhancement factor and good reproducibility, and it is also unable to realize Raman detection at single-molecule level.

[0006] In the early stage, we combined EBL, lithography and nanoimprinting techniques to realize an ordered structure of flexible metal nano-finger arrays with precisely adjustable structural units (ACS Nano, 2017, 11(6): 5836-5843; Small, 2018, 14: 1801146-1801155; ZL201710825180.0; ZL201910666854.6). This metal nano-finger array can be used to collapse the flexible nano-fingers that are close to each other by titrating the solvent and utilizing the surface tension during its evaporation process. Through this technology, DNA modification technology can be combined to connect to the Au surface in advance, collapse, and then come together to form an Au nanofinger / DNA / Au nanofinger coupling structure. Since the spatial longitudinal dimension of DNA is at the sub-nanometer level, the coupling structure formed can achieve an enhanced electromagnetic field under the regulation of quantum plasmons. With the help of this structure, SERS detection at the single-molecule level can be achieved; in addition, more importantly, if this DNA is used to capture the substance to be tested in the solution, high selectivity and high sensitivity measurement of the substance to be tested can be achieved.

[0007] For SERS in Hg 2+In terms of detection applications, studies have reported that after combining different base molecules in single-stranded DNA with Au nanoparticles, under the action of Hg ions, the single-stranded DNA structure will attach to the Au surface and become a double-stranded DNA spiral space structure perpendicular to the Au surface, so that different base sequences in the DNA are at different distances from the Au surface. Since the enhanced electromagnetic field is the strongest on the surface and then decays exponentially in free space, the different distances cause different base sequences in the DNA to feel different enhanced electromagnetic fields, and ultimately make the intensities of their corresponding SERS characteristic peaks different, thereby achieving quantitative detection of Hg ions (Sensors Actuators B (2018) 365-372). However, due to the need for monitoring the field environment, handheld Raman is often used to monitor the environment, which places high requirements on the design of SERS enhanced substrates. The above-mentioned SERS enhancement factor based on nanoparticles is low. In addition, since nanoparticles are mainly used in liquid phase environments, the particle distribution is highly volatile, and the application is subject to certain limitations. However, due to the limitation of resolution, the metal nanoarray structure based on nanometer and micron microfabrication technology cannot provide a surface enhanced Raman substrate with high enhancement factor and good reproducibility to solve the quantitative SERS detection of Hg ions. We propose to use our flexible Au nanofinger array ordered structure to combine with DNA to first construct the Au nanofinger / DNA / Au nanofinger coupling structure. Since the spatial longitudinal dimension of DNA is at the sub-nanometer level, the coupling structure formed can realize the enhanced electromagnetic field under the control of quantum plasmons; secondly, the DNA in this coupling structure interacts specifically with Hg ions to change the single-stranded DNA structure from being attached to the Au surface to a double-stranded DNA helical spatial structure perpendicular to the Au surface. The different intensities of the SERS characteristic peaks corresponding to the different enhanced electromagnetic fields felt by different base sequences are used to achieve highly selective and highly sensitive quantitative detection of Hg ions. Summary of the invention

[0008] The purpose of the present invention is to solve the deficiencies of the prior art and provide a method for preparing a surface enhanced Raman substrate with high selectivity and high sensitivity for detecting Hg ions, and to detect mercury ions by using this method. The method is simple and reasonable to prepare, can be prepared in large quantities and at low cost, can achieve high selectivity and high sensitivity monitoring of Hg ions in a mixed solution for Hg ion monitoring, and can be applied to portable devices such as outdoor handheld Raman. By utilizing the ordered structure of a flexible Au nano-finger array and combining with DNA, firstly, an Au nano-finger / DNA / Au nano-finger coupling structure is constructed. Since the spatial longitudinal size of DNA is at the sub-nanometer level, the coupling structure formed can realize the enhanced electromagnetic field under the control of quantum plasmons; secondly, by utilizing the specific interaction between DNA and Hg ions in this coupling structure, the single-stranded DNA structure is changed from being attached to the Au surface to a double-stranded DNA helical spatial structure perpendicular to the Au surface, and the different intensities of the SERS characteristic peaks corresponding to the different enhanced electromagnetic fields felt by different base sequences are utilized to achieve high selectivity and high sensitivity quantitative detection of Hg ions.

[0009] To achieve the above object, the present invention adopts the following technical solutions:

[0010] A method for quantitatively detecting mercury ions based on surface enhanced Raman scattering technology comprises the following steps:

[0011] 1) Prepare the ordered array structure of Au nano-finger by nanoimprinting, Au thin film deposition and reactive ion beam etching;

[0012] 2) The thiolated single-stranded DNA solution was subjected to thiol activation and desalting treatment, and then all DNA samples were heated in hot water at 90°C for 20 minutes and then rapidly cooled in an ice water bath;

[0013] 3) dropping the single-stranded DNA solution treated in step 2 onto the Au nano-finger ordered array structure prepared in step 1 for incubation. Due to the good affinity between the terminal base molecules in the DNA and Au, the single-stranded DNA is mainly attached to the surface of the Au nano-finger in a lying form;

[0014] 4) Take out the Au nanofinger ordered array structure modified with single-stranded DNA treated in step 3, rinse with ultrapure water to remove the unconnected and excess single-stranded DNA solution, soak it in ultrapure water for two hours, take it out and dry it under natural conditions;

[0015] 5) During the evaporation of the solution, the micro-surface tension makes the four adjacent nano-fingers close to each other, forming a collapsed Au finger array structure with tetramers as structural units. A sub-nanometer gap twice the size of DNA is formed between the finger structures, which can generate an enhanced coupled electromagnetic field regulated by quantum plasmons, and the enhanced electromagnetic field in the tetramer coupled structure does not depend on the polarization of the incident light;

[0016] 6) titrating a solution containing mercury ions on the SERS enhanced substrate based on the Au nano-finger closed array structure obtained in step 5, since the mercury ions and the middle base molecules in the two adjacent single-stranded DNAs form a base molecule-Hg-base molecule, the single-stranded DNA lying on the Au surface is transformed into a double-stranded DNA spiral space structure perpendicular to the Au surface, so that the terminal base molecules in the DNA are dissociated from the Au surface, thereby being away from the coupled enhanced electromagnetic field formed in the gap, while the middle base molecules are closer to the Au surface, so that their SERS signals are reduced relative to the middle base molecules;

[0017] 7) The quantitative and highly selective detection of Hg ions of different concentrations can be achieved by enhancing the ratio of the Raman characteristic peaks of the terminal base molecules and the intermediate base molecules in DNA.

[0018] In the above steps, steps 1-5 have actually become independent technical solutions for mercury ion detection, and steps 6-7 are specific case detection methods.

[0019] The height of a single nano-finger in the Au nano-finger ordered array structure in the above step 1 is 300 nanometers, including a 250-nanometer polymer column and 50 nanometers of Au on top, with a diameter of 80 nanometers. The distance between adjacent fingers is 120 nanometers, in order to form a tetramer coupling structure in the subsequent collapse process. The column material is a UV-curable imprinted polymer glue with strong thermal stability.

[0020] The thiolated single-stranded DNA sequence in the above step 2 is 5'-HS-(CH2) 6-TTTTTTTTTTGGGGGGGGGAAAAAAAA, wherein T, G, and A are base molecules with different structures, and is reduced with tri-(2-formylethyl)phosphine hydrochloride (TCEP) reducing agent for use.

[0021] The DNA incubation in step 3 above is carried out in a refrigerator at 4°C and high humidity for 12 hours overnight, so that one end of the single-stranded DNA is tightly connected to the Au surface by a sulfhydryl bond. Due to the good affinity between the other end base molecule in the DNA and Au, the single-stranded DNA is mainly attached to the surface of the Au nano-finger in a lying form;

[0022] The intermediate base molecule in the above step 6 is the T sequence in DNA, forming a T-Hg-T structure, triggering the formation of a double-stranded DNA helical spatial structure.

[0023] The terminal base molecule and the middle base molecule in the DNA in the above step 7 are the G sequence and the A sequence, and the ratio of the two is used to quantitatively measure the concentration of Hg ions.

[0024] Based on the flexible Au nano-finger array structure combined with DNA modification technology, the present invention constructs a coupling structure with an ultra-small gap of sub-nanometer DNA size, and utilizes the relative change of the enhancement factor of the Raman characteristic peaks of different DNA sequences caused by the interaction between DNA and Hg ions to develop a quantitative detection method for mercury ions based on surface enhanced Raman scattering technology. In our design, since the longitudinal size of DNA is at the sub-nanometer level, when the nano-fingers are close together, the localized surface plasmon dipole modes of a single nano-fingers will couple with each other to form two coupling modes of bonding and anti-bonding, among which the bonding coupling mode will induce an ultra-strong local enhanced electromagnetic field. In our design, the enhancement factor of this local enhanced electromagnetic field is 10 8 The sensitivity of SERS at the single-molecule level can be achieved above the order of magnitude. In addition, in the preparation of the flexible Au nano-finger array structure, by designing the appropriate size and period, we can realize a coupling structure with tetramers as the structural unit. This tetramer structure has an unpolarized response to the incident laser. Therefore, in SERS detection, there is no need to adjust the position of the SERS substrate to excite the plasmon coupling mode. If it is a non-tetramer structure and a dimer structure, then only when the direction of the incident photoelectric magnetic field is along the direction of the dimer gap can the local electromagnetic field coupling mode be excited. This will place high demands on operators in practical applications, thereby reducing practical applicability. Then, through the interaction between DNA and Hg, a T-Hg-T structure is formed, which triggers the formation of a double-stranded DNA helical spatial structure. The ratio of the Raman characteristic peaks of the terminal base molecules and the intermediate base molecules in the DNA is enhanced to achieve the detection of Hg ions of different concentrations, and the minimum detection concentration is 10 -9 mol / L. This detection scheme uses DNA to form a sub-nanometer gap layer in the Au finger coupling structure, and uses the changes in the spatial structure caused by the specific binding of base molecules in DNA and Hg ions to simultaneously achieve non-interference, highly selective and highly sensitive detection of Hg ions in mixed solutions.

[0025] Beneficial effects of the present invention:

[0026] 1. By combining Au flexible nano-fingers with DNA incubation technology, under the action of micro-capillary force, a sub-nanometer DNA gap with uniform gap size is obtained. The gap size is determined by twice the longitudinal dimension of DNA, rather than the resolution of nanoimprinting, and has the characteristic of gap size protection.

[0027] 2. Due to the sub-nanometer longitudinal size characteristics of DNA, the coupled electromagnetic field can achieve electromagnetic field enhancement with the participation of quantum plasmons, and the enhancement multiple can realize SERS detection at the single-molecule level.

[0028] 3. In addition to being a gap layer, DNA also plays a role in specific binding with Hg ions. This allows different sequences in DNA with changed spatial structure to feel the influence of enhanced electromagnetic fields at close range. This is different from what has been reported in the literature, and can achieve ultra-low concentration Hg ion detection. At the same time, this characteristic interaction can achieve highly selective detection of Hg ions in mixed solutions, eliminating interference from other possible substances in the solution. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1Schematic diagram of the quantitative detection method of mercury ion surface enhanced Raman scattering based on Au flexible nano-finger array structure: (a) For a specific single-stranded DNA sequence, thiolated DNA has two functional segments: one area is composed of continuous T bases near the end of the thiol group as a recognition segment, and the other is a signal output end composed of continuous G and A. The inherent Raman signal intensity ratio of G and A bases can be used to detect the direction of DNA; (b) Specific single-stranded DNA molecules are conformed on the Au surface through sulfur-gold bonds. Due to the good affinity between the terminal base molecule A sequence in DNA and Au, the single-stranded DNA is mainly attached to the Au surface in a lying form; (c) Hg2+ ions specifically bind to base T to form a T-Hg2+-T pair, triggering the formation of a three-dimensional structure, so that the single-stranded DNA lying on the Au surface becomes a double-stranded DNA spiral spatial structure perpendicular to the Au surface; (d) On the Au After DNA incubation on the nanofinger substrate, single-stranded DNA randomly conforms on the Au surface and then collapses to form an Au nanofinger / DNA / Au nanofinger coupling structure. Finally, under the action of Hg ions, the spatial configuration of DNA changes, and the ratio of the Raman characteristic peaks of the A sequence and the G sequence is used to achieve quantitative detection of Hg ion concentration. (e) The nanofinger is placed in a DNA solution for incubation. One end of the DNA is connected to the Au finger surface through a thiol group, and the other end is also connected to the Au surface through the A sequence, so that the single-stranded DNA is mainly attached to the Au surface in a lying form. (f) After incubation, the Au nanofinger couples to form a tetramer, and gaps are formed between the fingers due to the presence of DNA. (g) Mercury ion solution is added to form T-Hg2+-T pairs, triggering the formation of a DNA three-dimensional structure perpendicular to the Au finger surface.

[0030] Figure 2 :(a) Scanning electron microscope image (SEM) of the ordered array of Au flexible nano-finger prepared by nanoimprinting described in the present invention; (b) Scanning electron microscope image (SEM) of the Au nano-finger / DNA / Au nano-finger tetramer coupling structure formed after the DNA incubation process and the collapse process; (c) Transmission electron microscope result (TEM) along the cross-sectional structure of two adjacent fingers. Since DNA is a molecular structure, it cannot be seen during FIB processing, and only the fingers can be seen close together; (d) Optical transmission spectrum measurement of Au nano-finger before closure and after DNA modification closure. It can be seen that after closure, the resonance position red-shifts from 600 nanometers to 650 nanometers. This is because the bonding mode formed after coupling has lower energy.

[0031] Figure 3 :(a) Using the DNA-modified Au nano-finger coupling structure as the SERS enhanced substrate, the Raman spectra of Hg ion concentration changes from 10 -3 mol / L to 10-9 mol / L; (b) Select the Raman peak 736cm corresponding to sequence A and sequence G -1 and 650cm -1 The ratio I(736cm -1 ) / I(650cm -1 ) and Hg 2+ Ion concentration fitting curve.

[0032] Figure 4 :(a) Taking the tetramer nanofinger in the realization as a model, the extinction spectrum of Au nanofinger before and after coupling is simulated by Comsol, and the resonance position is red-shifted from 530 nm to 650 nm; (b) The three-dimensional electric field distribution of the coupling structure, the maximum electromagnetic field enhancement is more than 100, so the SERS enhancement factor is 10 8 Magnitude. DETAILED DESCRIPTION

[0033] Example 1

[0034] The method for preparing the Au nano-finger / DNA / Au nano-finger coupling structure comprises the following steps:

[0035] Step 1: Prepare an ordered array structure of Au nano-finger by nano-imprinting, Au thin film deposition and reactive ion beam etching. The height of a single nano-finger is 300 nanometers, including a 250-nanometer polymer pillar and 50 nanometers of Au on top, with a diameter of 80 nanometers. The distance between adjacent fingers is 120 nanometers, as shown in the scanning electron microscope photo of Figure 2 (a).

[0036] Step 2: The thiolated single-stranded DNA sequence was selected as 5'-HS-(CH2) 6-TTTTTTTTTTGGGGGGGGGAAAAAAAA, where T, G, and A are base molecules with different structures, and was reduced with tri-(2-formylethyl)phosphine hydrochloride (TCEP) reducing agent for use. The thiolated single-stranded DNA solution was subjected to thiol activation and desalting treatment, and then all DNA samples were heated in 90°C hot water for 20 minutes and then rapidly cooled in an ice water bath;

[0037] Step 3: drip the single-stranded DNA solution treated in step 2 onto the Au nano-finger ordered array structure prepared in step 1 for incubation. The DNA incubation is carried out in a refrigerator at 4°C and high humidity for 12 hours overnight, so that one end of the single-stranded DNA is tightly connected to the Au surface by a sulfhydryl bond. Due to the good affinity between the other end base molecule in the DNA and Au, the single-stranded DNA is mainly attached to the Au nano-finger surface in a lying form.

[0038] Step 4: Take out the Au nano-finger ordered array structure modified with single-stranded DNA processed in step 3, rinse with ultrapure water to remove the unconnected and excess single-stranded DNA solution, soak it in ultrapure water for two hours, take it out and dry it under natural conditions;

[0039] Step 5: During the evaporation of the solution, the micro-surface tension makes the four adjacent nano-fingers close to each other, forming a collapsed Au finger array structure with a tetramer as the structural unit. A sub-nanometer gap twice the size of DNA is formed between the finger structures, which can generate an enhanced coupled electromagnetic field regulated by quantum plasmons, and the enhanced electromagnetic field in the tetramer coupled structure does not depend on the polarization of the incident light. For details, see Figure 2 (b) SEM image, Figure 2 The cross-sectional electron microscopy image in (c) further confirms that the nano-fingers are close to each other. In addition, through the optical transmission spectrum measurement of the Au nano-fingers before closure and after DNA modification closure, it can be seen that after closure, the resonance position red-shifts from 600 nanometers to 650 nanometers. This is because the bonding mode formed after coupling has lower energy. For details, see Figure 2 (d) As shown. Furthermore, electromagnetic field simulation of the above structure was carried out using Comsol electromagnetic field simulation software, which confirmed that the coupled electromagnetic field enhancement was more than 100 times, so the SERS enhancement factor reached 10 8 Magnitude, see Figure 4 shown.

[0040] Example 2

[0041] The highly selective and sensitive quantitative detection of Hg ions based on the Au nanofinger / DNA / Au nanofinger coupling structure includes the following steps:

[0042] Step 1: a solution containing mercury ions is titrated on a SERS enhanced substrate of a DNA-modified Au nano-finger closed array structure. Since the mercury ions and the middle base molecules in two adjacent single-stranded DNAs form a base molecule-Hg-base molecule, the single-stranded DNA lying on the Au surface is transformed into a double-stranded DNA spiral space structure perpendicular to the Au surface, so that the terminal base molecules in the DNA are dissociated from the Au surface, thereby being away from the coupled enhanced electromagnetic field formed in the gap, while the middle base molecules are closer to the Au surface, so that their SERS signals are reduced relative to the middle base molecules;

[0043] Step 2: The ratio of the Raman characteristic peaks of the terminal base molecules and the intermediate base molecules in DNA is enhanced to achieve quantitative and highly selective detection of Hg ions at different concentrations, which can achieve Hg ion concentrations from 10 -3 mol / L to 10 -9mol / L quantitative detection, mainly selected the Raman peak 736cm corresponding to the A sequence and the G sequence -1 and 650cm -1 The ratio I(736cm -1 ) / I(650cm -1 ) and Hg 2+ Ion concentration fitting curve, see Figure 3 shown.

Claims

1. A method for quantitative detection of mercury ions based on surface enhanced Raman scattering technology, characterized in that The following steps are involved: 1) Prepare the ordered array structure of Au nano-finger by nanoimprinting, Au thin film deposition and reactive ion beam etching; 2) The thiolated single-stranded DNA solution was subjected to thiol activation and desalting treatment, and then all DNA samples were Heat in 90℃ hot water for 20 minutes, then cool quickly in an ice water bath; 3) The single-stranded DNA solution treated in step 2) is added dropwise to the Au nano-finger ordered array prepared in step 1) The DNA terminal base molecules have a good affinity with Au, making the single The stranded DNA is mainly attached to the surface of the Au nanofinger in a lying form; 4) Take out the Au nano-finger ordered array structure modified with single-stranded DNA in step 3) and wash it with ultrapure water. Rinse off the unconnected and excess single-stranded DNA solution, soak in ultrapure water for two hours, take out and place in natural Dry conditions; 5) During the evaporation of the solution, the micro-surface tension makes the four adjacent nano-fingers close to each other to form The collapsed Au finger array structure with tetramer as the structural unit forms a double DNA size between the finger structures Sub-nanometer gaps of different sizes can generate enhanced coupled electromagnetic fields regulated by quantum plasmons, and the tetramer coupling The enhanced electromagnetic field in the structure may not depend on the polarization of the incident light; 6) titrating the Au nano-finger closed array structure-based SERS enhanced substrate obtained in step 5) Mercury ion solution, because mercury ions and the middle base molecules in the two adjacent single-stranded DNAs form base separation The Hg-base molecules make the single-stranded DNA lying on the Au surface become a double-stranded DNA spiral space structure perpendicular to the Au surface, so that the terminal base molecules in the DNA dissociate from the Au surface and stay away from the Au surface. The coupling formed in the gap enhances the electromagnetic field, and the intermediate base molecules are closer to the Au surface, so their SERS The signal is reduced relative to the middle base molecule; 7) The ratio of the Raman characteristic peaks is enhanced by the terminal base molecules and the middle base molecules in DNA to achieve non- Quantitative and highly selective detection of Hg ions at the same concentration; The DNA incubation in step 3) is carried out in a refrigerator at 4°C and high humidity for 12 hours overnight, so that one end of the single-stranded DNA is tightly connected to the Au surface by a sulfhydryl bond. Due to the good affinity between the other end base molecule in the DNA and Au, the single-stranded DNA is mainly attached to the surface of the Au nano-finger in a lying form; The terminal base molecule and the middle base molecule in the DNA in step 7) are a G sequence and an A sequence, and the ratio of the two is used to quantitatively measure the concentration of Hg ions.

2. The method for quantitative detection of mercury ions based on surface enhanced Raman scattering technology as claimed in claim 1, characterized in that In the step 1), the height of a single nano-finger in the Au nano-finger ordered array structure is 300 nanometers, including a 250-nanometer polymer column and 50 nanometers of Au on top, with a diameter of 80 nanometers, and the distance between adjacent fingers is 120 nanometers.

3. The method for quantitative detection of mercury ions based on surface enhanced Raman scattering technology as claimed in claim 1, characterized in that The thiolated single-stranded DNA sequence in step 2) is 5'-HS-(CH2)6-TTTTTTTTTTGGGGGGGGGAAAAAAAA, wherein T, G, and A are base molecules with different structures, and is reduced with tri-(2-formylethyl)phosphine hydrochloride (TCEP) reducing agent for later use.

4. The method for quantitative detection of mercury ions based on surface enhanced Raman scattering technology as claimed in claim 1, characterized in that The intermediate base molecule in step 6) is the T sequence in DNA, forming a T-Hg-T structure.

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