Method for enhancing the SERS signal intensity of proteins by introducing ionic liquids

Abstract

The present invention discloses a method for improving the SERS signal intensity of proteins by introducing ionic liquids. The method immerses a titanium dioxide nanotube array in a mixed solution containing a protein to be detected and an ionic liquid [P 6,6,6,14 [FuA] to prepare a SERS active substrate. The present invention introduces the ionic liquid [P 6,6,6,14 [FuA] into the SERS active substrate, enhances the interaction between the protein and the substrate, increases the ionic conductivity, increases the overall polarizability of the detection system, and improves the SERS performance of the protein. The method of the present invention has high sensitivity and detectability, and has broad application prospects in the field of trace detection of proteins.

Description

Technical Field

[0001] The present invention belongs to the technical field of surface-enhanced Raman spectroscopy, and relates to a method for improving the SERS signal intensity of proteins by introducing ionic liquids. Background Art

[0002] Surface-enhanced Raman scattering (SERS), as a highly sensitive spectroscopic technique, has been widely used to study the interfacial behavior of biomolecules on solid surfaces. By enhancing the signal of the analyte through chemical and physical enhancement mechanisms, rich information about the structure of the substance can be obtained at the molecular level. However, the SERS signal of the analyte is extremely dependent on the physicochemical properties of the SERS substrate. When using SERS for trace detection of biological proteins, how to establish a detection system that can generate high-intensity signals with only a small amount of protein is a current research difficulty.

[0003] Traditional research on enhancing the SERS signal mainly focuses on optimizing the active substrate. For example, noble metals such as gold and silver nanoparticles are used for modification to obtain surface-covered or core-shell substrates, thereby improving the effect of electromagnetic field enhancement. However, when the interaction between protein molecules and the active substrate is weak, optimizing the substrate itself does not achieve an ideal enhancement effect on the SERS signal. Therefore, how to strengthen the interaction between protein molecules and the active substrate, increase the polarizability of the system, and thus enhance the SERS effect remains a difficult problem to be solved urgently.

[0004] As a type of salt substance, ionic liquids have unique physicochemical properties, including adjustable chemical structures, thermal stability, chemical stability, excellent ionic conductivity, etc. Bai et al. successfully synthesized chiral ionic liquid monolayer-stabilized gold nanoparticles in a two-phase liquid-liquid system, which can self-assemble into a ring structure at the air / water interface. The molecular structure of chiral ionic liquids has an important influence on the formation of the ring structure of gold nanoparticles. The aggregated ring structure formed by the self-assembly of gold nanoparticles as an SERS substrate greatly enhances the signal intensity of rhodamine 6G (R6G) (BAI X, LI X, ZHENGL. Chiral Ionic Liquid Monolayer-Stabilized Gold Nanoparticles: Synthesis, Self-Assembly, and Application to SERS[J]. Langmuir, 2010, 26(14):12209–12214.). Summary of the Invention

[0005] The object of the present invention is to provide a method for improving the SERS signal intensity of proteins by introducing ionic liquids. This method enhances the interaction between protein molecules and the active substrate by introducing ionic liquids, and improves the electron transfer ability, thereby improving the SERS performance and detection sensitivity.

[0006] The technical solution for achieving the object of the present invention is as follows:

[0007] The method for improving the SERS signal intensity of proteins by introducing ionic liquids includes the following steps:

[0008] (1) Synthesize a titanium dioxide nanotube array with a three-dimensional stereoscopic tubular structure on the surface of a clean titanium sheet by electrochemical anodization.

[0009] (2) Fully dissolve the protein to be detected and the ionic liquid [P 6,6,6,14 [FuA] in a buffer solution to obtain a uniform mixed solution, then immerse the titanium dioxide nanotube array in the mixed solution, and after taking it out, dry it for SERS detection.

[0010] Preferably, in step (1), the titanium dioxide nanotube array is prepared by an existing electrochemical anodization method, and the specific steps are as follows:

[0011] (a) Use the clean titanium sheet as the anode and the platinum sheet as the cathode, use ethylene glycol added with the corrosion inhibitor ammonium fluoride and deionized water as the electrolyte, and adopt the electrochemical anodization method to react at a voltage of 15 - 55V for 12 - 36h to obtain a titanium dioxide nanotube array;

[0012] (b) Clean the titanium dioxide nanotube array, sinter it at a high temperature of 300 - 600°C, and keep it warm for 20 - 60min to obtain a titanium dioxide nanotube array with a three-dimensional stereoscopic tubular structure through coarse crystallization.

[0013] Preferably, in step (a), in the electrolyte, ammonium fluoride accounts for 0.1 - 0.5wt% of the mass of ethylene glycol, and deionized water accounts for 0.5 - 5wt% of the mass of ethylene glycol.

[0014] Preferably, in step (b), the cleaning time is 5 - 30min.

[0015] Preferably, in step (2), before immersing the titanium dioxide nanotube array in the mixed solution, cut the titanium dioxide nanotube as needed to obtain a titanium dioxide nanotube array substrate with appropriate size.

[0016] Preferably, in step (2), the buffer solution is a phosphate buffer solution with a concentration of 0.1 - 0.4mol / L and a pH value of 5.8 - 7.5.

[0017] Preferably, in step (2), the concentration of the ionic liquid in the mixed solution is 10 -3 g / L.

[0018] In step (2), the protein to be detected includes but is not limited to bovine serum albumin, cytochrome c, lysozyme, collagenase, human serum albumin, etc.

[0019] The ionic liquid [P 6,6,6,14 [FuA] introduced in the present invention can enhance the interaction between protein molecules and the SERS active substrate and improve the electron transfer ability. The ionic liquid is partially dissociated, and the free cations and anions are hydrated in the mixed solution. Among them, the cations approach the protein with balanced negative charges around, and the anions approach the TiO2 nanotube array substrate with balanced positive charges around. The strong electrostatic interaction occurring between them increases the charge transfer ability and effectively improves the SERS performance.

[0020] Compared with the traditional SERS enhancement technology, the present invention has the following advantages:

[0021] Compared with the traditional method of optimizing the substrate to enhance the SERS signal, introducing ionic liquid for Raman enhancement detection does not rely on the electromagnetic enhancement effect brought by the substrate itself, but enhances the SERS effect by enhancing the interaction between the protein and the active substrate and increasing the polarizability of the system. After introducing the ionic liquid, stronger SERS signals can be excited by fewer target molecules, realizing trace detection and being applicable to various protein molecule SERS detection systems. Brief Description of the Drawings

[0022] Figure 1 is the schematic diagram of the SERS active substrate detection principle of the present invention;

[0023] Figure 2 is the structural diagram of the ionic liquid [P 6,6,6,14 [FuA];

[0024] Figure 3 is the microscopic morphology diagram of Example 1 under the atomic force microscope (AFM);

[0025] Figure 4 is the microscopic morphology diagram of Example 1 under the field emission scanning electron microscope (FESEM);

[0026] Figure 5 is the surface Raman enhanced scattering test result diagram of Example 1, Comparative Example 1, and Comparative Example 2. Detailed Embodiments

[0027] The present invention will be further described in detail below in conjunction with specific embodiments and drawings.

[0028] In the following examples, the preparation of the titanium dioxide nanotube array refers to the reference [DONG Y, WU N, JI X, et al. Excellent Trace Detection of Proteins on TiO 2 Nanotube Substrates through Novel Topography Optimization[J]. The Journal of Physical Chemistry C, 2020, 124(50):27790–27800.].

[0029] The preparation of the ionic liquid refers to the references [SHAH F U, GNEZDILOV O I, FILIPPOV A. Ion dynamics in halogen-free phosphonium bis(salicylato)borate ionic liquid electrolytes for lithium-ion batteries[J]. Physical Chemistry Chemical Physics, 2017, 19(25):16721–16730] and [LEE J Y, SELFRIDGE K M, KOHN E M, et al. Effects of Ionic Liquid Alkyl Chain Length on Denaturation of Myoglobin by Anionic, Cationic, and Zwitterionic Detergents[J]. Biomolecules, 2019, 9(7):264.].

[0030] Example 1

[0031] Step 1: Prepare the TiO2 nanotube array, which specifically includes the following steps:

[0032] The TiO2 nanotube array was prepared by electrochemical anodic oxidation. The main solvent of the electrolyte was ethylene glycol, with 1 wt% deionized water and 0.2 wt% ammonium fluoride added as the etchant. The anode was a clean titanium sheet. The cathode was a platinum sheet. The power supply was turned on and the variable voltage method was used. First, the reaction was carried out at a relatively low voltage of 5V for 12h, and then the voltage was increased to 35V and the reaction continued for 12h. Finally, the prepared TiO2 nanotube array was placed in deionized water and ultrasonically cleaned for 10min to remove the residual covering layer on the surface of the nanotubes during etching, obtaining a TiO2 nanotube array with a uniform and clean surface. After drying with nitrogen, it was sintered at a high temperature of 500°C for 2h to promote crystallization, and then naturally cooled to 60°C and taken out, and cut into a TiO2 nanotube array of 1cm x 1cm.

[0033] Step 2: Prepare a SERS active substrate containing an ionic liquid using the TiO2 nanotube array, which specifically includes the following steps:

[0034] 33.6 mg of cytochrome c was added to 10 mL of phosphate buffer solution with pH = 7.2, and then 0.01 g of ionic liquid [P 6,6,6,14 [FuA] was added. After ultrasonic oscillation at 50 KHz for 15 min, a uniformly mixed solution of the ionic liquid and cytochrome c buffer solution was obtained. Then, the TiO2 nanotube array substrate was immersed in the mixed solution and taken out after being stored at 4°C for 12h. The TiO2 nanotube array substrate was rinsed three times with phosphate buffer solution with pH = 7.2 and then dried with nitrogen to obtain a SERS active substrate containing an ionic liquid.

[0035] Comparative Example 1

[0036] In this comparative example, the introduction method of the ionic liquid was changed, and the rest was the same as in Example 1. It specifically includes the following steps:

[0037] Step 1 was the same as Step 1 in the example.

[0038] Step 2: Prepare a TiO2 nanotube array with an ionic liquid immobilized on its surface, which specifically includes the following steps:

[0039] 0.01 g of ionic liquid [P 6,6,6,14 [FuA] was added to 60 mL of ethanol. After ultrasonic oscillation at 50 KHz for 20 min to ensure complete dissolution, the TiO2 nanotube array was immersed in the ethanol solution of the ionic liquid and heated in an oil bath at 60°C for 24h. Subsequently, the TiO2 nanotube array was placed in a vacuum drying oven and dried at 35°C for 12h to ensure complete volatilization of ethanol, obtaining a TiO2 nanotube array with an ionic liquid immobilized on its surface.

[0040] Step 3: Prepare a SERS active substrate, which specifically includes the following steps:

[0041] 33.6 mg of cytochrome c was added to 10 mL of phosphate buffer solution with pH = 7.2 and sonicated at 50 KHz for 15 min to obtain a uniformly mixed cytochrome c buffer solution. The TiO₂ nanotube array with ionic liquid immobilized on its surface was immersed in the mixed solution and taken out after being stored at 4 °C for 12 h. After rinsing the TiO₂ nanotube array substrate three times with phosphate buffer solution with pH = 7.2 and drying it with nitrogen, the TiO₂ nanotube array SERS active substrate with ionic liquid immobilized on its surface was obtained.

[0042] Comparative Example 2

[0043] This comparative example is basically the same as Example 1, except that no ionic liquid is introduced, and the SERS active substrate is only composed of TiO₂ nanotube array and cytochrome c.

[0044] Surface Raman enhanced scattering experiment:

[0045] Using the SERS active substrates prepared in Example 1, Comparative Example 1, and Comparative Example 2 to conduct Raman enhancement experiments on cytochrome c, which specifically includes the following steps:

[0046] Step 1: The SERS active substrates (1 cm x 1 cm) prepared in Example 1, Comparative Example 1, and Comparative Example 2 were respectively adhered to clean glass slides, and Raman enhancement experiments were carried out on a Raman Spectrometer (HORIBA JOBIN YVON).

[0047] Step 2: Set the laser wavelength to 532 nm, the laser power to 5 mW, the exposure time to 20 s, the number of cycles to 2 times, and the test range to 900 - 1800 cm -1 ;

[0048] Step 3: Four measurements were carried out at multiple sample positions to avoid errors.

[0049] Figure 4 are the SERS signal maps obtained with the active substrates of Example 1, Comparative Example 1, and Comparative Example 2. It can be seen from Figure 4 the distribution and band positions of the Raman spectrum of cytochrome c. The υ4(A 1g ) mode of the spectral characteristic peak is at 1364 cm -1 and the υ 10 (B 1g ) mode is at 1637 cm -1 corresponding to the oxidized native state cytochrome c, indicating that after introducing the ionic liquid into the research system, the cytochrome c molecules still maintain good biological activity on each substrate (Example 1, Comparative Example 1, Comparative Example 2), which also proves that the ionic liquid [P used in this study6,6,6,14 [FuA] biocompatibility. Compared with Comparative Example 2, the signal intensity of cytochrome c on Example 1 was significantly enhanced, indicating that the presence of ionic liquid indeed improved the SERS performance. In Example 1, the strongest peak of Cyt c was mainly at 1172 cm -1 of υ 30 (B 2g ) mode, 1314 cm of υ 22 (A 2g ) mode, 1407 cm -1 of υ 29 (B 2g ) mode and 1586 cm -1 of υ 19 (A 2g ) mode, corresponding to the vibrations of the semi-ring, C-H, quarter-ring, and C-C bonds respectively, which is consistent with the experimental results of Comparative Example 1.

[0050] In addition, the signal enhancement effect of Comparative Example 1 was not as good as that of Example 1, indicating that immobilizing the ionic liquid on the surface of the TiO2 nanotube array is not the best way to introduce the ionic liquid. It can be seen from Figure 4 that the signal intensity and the fluctuations of the peaks and valleys at the key positions of Comparative Example 1 are not as good as those of Example 1.

[0051] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for improving the SERS signal intensity of proteins by introducing ionic liquids, characterized in that, It includes the following steps: (1) Synthesize a titanium dioxide nanotube array with a three-dimensional stereoscopic tubular structure on the surface of a clean titanium sheet by electrochemical anodic oxidation; (2) Dissolve the protein to be detected and the ionic liquid [P 6,6,6,14 [FuA] sufficiently in a buffer solution to obtain a homogeneous mixed solution, then immerse the titanium dioxide nanotube array in the mixed solution, take it out and dry it for SERS detection.

2. The method according to claim 1, wherein In step (1), the titanium dioxide nanotube array is prepared by the following steps: (a) Use the clean titanium sheet as the anode and the platinum sheet as the cathode. Take ethylene glycol added with the corrosion inhibitor ammonium fluoride and deionized water as the electrolyte, and adopt the electrochemical anodic oxidation method to react at a voltage of 15 - 55V for 12 - 36h to obtain the titanium dioxide nanotube array; (b) Clean the titanium dioxide nanotube array, perform high-temperature sintering at 300 - 600°C, and keep the temperature for 20 - 60min to obtain a titanium dioxide nanotube array with a three-dimensional stereoscopic tubular structure through coarse crystallization.

3. The method according to claim 2, characterized in that, In step (a), in the electrolyte, ammonium fluoride accounts for 0.1 - 0.5wt% of the mass of ethylene glycol, and deionized water accounts for 0.5 - 5wt% of the mass of ethylene glycol.

4. The method according to claim 2, wherein In step (b), the cleaning time is 5 - 30min.

5. The method according to claim 1, characterized in that, In step (2), before immersing the titanium dioxide nanotube array in the mixed solution, cut the titanium dioxide nanotubes as needed to obtain a titanium dioxide nanotube array substrate with appropriate size.

6. The method according to claim 1, wherein In step (2), the buffer solution is a phosphate buffer solution with a concentration of 0.1 - 0.4mol / L and a pH value of 5.8 - 7.

5.

7. The method according to claim 1, characterized in that In step (2), the concentration of the ionic liquid in the mixed solution is 10 -3 g / L.

8. The method according to claim 1, wherein In step (2), the protein to be detected is bovine serum albumin, cytochrome c, lysozyme, collagenase or human serum albumin.

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