Surface enhanced Raman spectroscopy method for enhancing protein by using halogen-free ionic liquid

Abstract

The invention discloses a surface enhanced Raman spectroscopy method for enhancing protein by using halogen-free ionic liquid. According to the method, a halogen-free ionic liquid with biocompatibility is introduced into a trace protein solution to be detected, so that a Raman detection signal on the surface of an amorphous alloy nanotube array substrate is enhanced, and an enhancement factor on the protein detection signal is enhanced by about 40-50 times; the enhancement amplitude is obviously higher than that of an enhancement factor on a protein detection signal on the surface of the TiO2 nanotube array substrate. The method is simple and low in cost, and has a wide application prospect in the field of protein trace detection.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of surface-enhanced Raman spectroscopy detection, and relates to a method for enhancing the surface-enhanced Raman spectroscopy of proteins by using a halogen-free ionic liquid. BACKGROUND

[0002] Trace detection of proteins refers to the detection of proteins in extremely low content (ng / mL or femtomole / L), which has very important significance in many fields. For example, in the biomedical field, trace protein detection can be used to study protein interactions, cell signaling, metabolic pathways, and other biological processes; in the screening and diagnosis of disease markers, by detecting trace proteins (such as glycated hemoglobin, carcinoembryonic antigen, troponin, etc.) in urine, blood or other body fluids, early diagnosis, disease monitoring and treatment feedback information can be provided; in the field of food safety detection, it can be used to detect potential allergens, toxins and pollutants in food, and plays an important role in ensuring food safety, complying with food regulations and improving product quality. However, due to the low sensitivity, poor specificity, and complex sample processing of current common protein detection techniques such as enzyme-linked immunosorbent assay and mass spectrometry, these techniques are difficult to apply to trace protein detection. Therefore, it is urgent to develop new detection techniques to realize trace protein detection.

[0003] Surface-enhanced Raman scattering technology (SERS) has ultra-high sensitivity, greatly reducing the detection limit of proteins, and can realize trace detection of proteins. In addition, SERS technology can directly use the characteristic vibration of proteins itself without external labeling, simplifying the experimental steps and reducing the damage to the sample. It has the characteristics of rapidity, non-destructiveness and multi-information acquisition, and can be used for analysis and quantification of proteins in complex samples, overcoming the shortcomings of high antibody dependence of immunological methods, multiple interference of electrochemical methods, and high sample requirements of mass spectrometry. It can realize rapid and high-sensitivity trace detection of proteins in different environments. Common SERS substrate materials include metal nanoparticles or nanostructures such as gold, silver and copper, and the shape and structure of the SERS substrate have important influence on the enhancement effect. Currently, scholars mainly regulate the SERS substrate to improve the SERS performance.

[0004] In SERS tests, trace additives can be added as connecting molecules to effectively enhance the interaction between proteins and SERS substrates, thereby improving SERS performance. Common trace additives such as reducing agents are usually used to reduce disulfide bonds, allowing proteins to dissociate from their native conformation into monomer form, which can improve detection sensitivity. They can reduce disulfide bonds in proteins to sulfhydryl groups, but the spatial structure of proteins will change due to the addition of reducing agents, thereby changing the original protein spectrum and having irreversible effects on subsequent analysis and research. Another common trace additive is a protein dye, which aims to increase the visibility of proteins or enhance the interaction between proteins and other reagents, but cannot effectively enhance the interaction between proteins and SERS substrates, so it is less commonly used in SERS detection. Therefore, selecting trace additives with stable chemical properties, controllable structures, and biocompatibility is the key to enhancing the interaction between proteins and SERS substrates.

[0005] Ionic liquids are ionic compounds with a melting point close to room temperature, composed of asymmetric organic cations and organic or inorganic anions, which are combined by ionic bonds to form stable liquid structures. Compared with traditional molecular liquids, the structure of ionic liquids can be precisely controlled by selecting different cations and anions. This makes ionic liquids have wide controllability in solubility, polarity, acidity, surface tension, conductivity, and other aspects, adapting to the needs of various applications. In recent years, ionic liquids have been widely used in biological research due to their good biocompatibility and strong controllability. Studies have shown that ionic liquids can effectively enhance the interaction between proteins and substrates, such as [MimV 11 ,V 11 ]Br and [MimA 11 ,A 11 ]Cl ionic liquids improve the sensitivity of SERS, but these halogen-containing ionic liquids have poor biocompatibility and limited enhancement range, etc. (Duo Zhang, Hairan Zhang, Highly sensitive SERS platform on isotropic ionic liquid-based liposome, Journal of Molecular Liquids, Volume 391, Part A, 2023).

[0006] Amorphous alloy is a special type of metal alloy, which is characterized by having a disordered atomic structure, different from the crystal structure. In amorphous alloys, the atomic structure lacks long-range periodic order, and presents a disordered or nearly disordered structure. This disordered structure makes amorphous alloys have a series of special properties and advantages. For example, the disordered atomic structure can lead to more local electric field enhancement effect, which is crucial for the enhancement of SERS signal. At the same time, it also has a higher scattering cross section, which can effectively scatter the incident laser light and improve the acquisition efficiency of SERS signal. In addition, the high hardness and strength, excellent elasticity and toughness, excellent corrosion resistance, high electrical conductivity and magnetic properties of amorphous alloy also help to improve the strength, stability and reliability of SERS signal, which is conducive to its wide application in environmental monitoring, analytical chemistry and clinical medicine (F. U. Shah, O. I. Gnezdilov, I. A. Khan, A. Filippov, N. A. Slad, P. Johansson, Structural and ion dynamics in fluorine-free oligoether carboxylate ionic liquid-based electrolytes, J. Phys. Chem. B. 124 (2020) 9690-9700.). SUMMARY

[0007] The purpose of the present application is to provide a method for enhancing the surface-enhanced Raman spectroscopy of proteins using halogen-free ionic liquids. This method introduces ionic liquids with different structures as trace additives into the original SERS system, enhances the adhesion between proteins and SERS substrates, and thus improves the SERS performance and increases the SERS detection sensitivity.

[0008] The technical solution to achieve the purpose of the present application is as follows:

[0009] The method for enhancing the surface-enhanced Raman spectroscopy of proteins using halogen-free ionic liquids comprises the following steps:

[0010] The protein solution and halogen-free ionic liquid are mixed uniformly by ultrasonic mixing according to the ratio of 1-100 mL: 0.001-0.5 g, then the ZrAlCo-O (ZACO) nanotube array substrate is immersed in the mixed solution at 0-10℃, after the immersion is completed, the substrate is washed and dried with nitrogen, and then Raman measurement is carried out to obtain the SERS spectrum.

[0011] Further, the protein is an immunoglobulin, a cytokine, a protein marker, an enzyme, a peptide, a nucleic acid binding protein, a viral protein, a plasma protein, a cell signaling protein, a structural protein, a signal molecule, a tissue protein, a glycoprotein, a toxin protein, a membrane protein, a heat shock protein, a binding protein, a tumor-related protein, a metabolic regulatory protein, etc. In the specific embodiment of the present application, cytochrome c (Cyt c) is taken as an example.

[0012] Further, the halogen-free ionic liquid is 1-butyl-3-methylimidazole acetate, 1-hexyl-3-methylimidazole methanesulfonate, 2-2-(2-methoxyethoxy)ethoxy tetrabutylphosphonium salt, 2-2-(2-methoxyethoxy)ethoxy trihexyl(tetradecyl)phosphonium salt, trihexyl(tetradecyl)phosphonium thien-2-carboxylate, trihexyl(tetradecyl)phosphonium furoate, triethylphosphonium tetraethylphosphonic acid hydrochloride, trimethylphosphonium hexadecylphosphonic acid hydrochloride, tributylphosphonium octamethylphosphonic acid hydrochloride, dimethylphosphonium dodecylphosphonic acid hydrochloride, 1-octyl-3-methylimidazole nitrate, etc.

[0013] Further, the protein solution is prepared by dissolving the protein in the PBS buffer.

[0014] Further, the ZrAlCo-O nanotube array substrate is prepared by an electrochemical anodic oxidation method.

[0015] Further, the ratio of the protein solution and the halogen-free ionic liquid is 10 mL:0.01 g.

[0016] Further, the soaking time is 10-15 h.

[0017] Further, the PBS buffer is used for washing.

[0018] Further, the nitrogen blowing drying time is 1-2 h.

[0019] The present application compares the detection system with the introduction of halogen-free ionic liquid with the control system without the introduction of halogen-free ionic liquid, and finds that the resonance Raman spectrum peak intensity of the detection system with the introduction of halogen-free ionic liquid is obviously enhanced. At the same time, after the introduction of halogen-free ionic liquid, the N element content of the ZACO nanotube array substrate surface is greatly improved, and through calculation, the enhancement factor EFs of Cyt c on the ZACO substrate surface is increased by 2.5-41.2 times, which proves that the introduction of halogen-free ionic liquid can effectively enhance the adsorption of protein on the SERS substrate surface. Further, the enhancement intensity of the characteristic peak of Cyt c on the ZACO substrate is strongly related to the length of the cation in the halogen-free ionic liquid. When the halogen-free ionic liquid with a long cation chain length is introduced, the SERS effect is more obvious. Through AFM, the adhesion between Cyt c and the ZACO substrate is measured by using Cyt c grafted probes, and the results show that in the presence of long-chain halogen-free ionic liquid, the ZACO substrate shows stronger adhesion to Cyt c, which promotes the adsorption of Cyt c on the substrate surface, thereby enhancing the SERS signal. In addition, in the system containing long-chain halogen-free ionic liquid, the calculated Also stronger, and the van der Waals force is the main contribution force, which is derived from the interaction between the long alkyl chain on the cation and Cyt c. The above results show that the long-chain halogen-free ionic liquid anchors the Cyt c molecules on the ZACO substrate, and the bridge attraction generated thereby enhances the SERS signal of Cyt c and improves the SERS detection sensitivity.

[0020] Compared with the prior art, the present application has the following advantages:

[0021] (1) The present application uses halogen-free ionic liquid as a trace additive, which has stable chemical properties, good biocompatibility, strong controllability, and can effectively enhance the interaction between protein and substrate.

[0022] (2) Compared with the traditional method of enhancing the surface enhanced Raman spectrum of protein, by introducing halogen-free ionic liquid, the EF value of the ZrAlCo-O nanotube array substrate surface is enhanced by about 40-50 times, which is significantly higher than the enhancement multiple of the EF value on the TiO2 nanotube array substrate surface (about 4-8 times), effectively improving and enhancing the SERS performance of protein on the substrate surface.

[0023] (3) The surface enhanced Raman spectrum method of the present application is simple in process and low in cost, and has wide application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1Raman spectra (a) obtained for Example 1, Example 2, Example 3 and Example 4, Raman spectra (b) obtained for Example 4, Comparative Example 1 and Comparative Example 2, Raman spectra (c) obtained for Example 1, Example 4, Comparative Example 3, Comparative Example 4.

[0025] Figure 2 Molecular structures and sizes of the cations and anions of the three ionic liquids.

[0026] Figure 3 Typical AFM retraction force curves obtained under Example 4, Example 3, Example 2 and Example 1, respectively. DETAILED DESCRIPTION

[0027] The application will be further described in conjunction with the examples and the accompanying drawings.

[0028] In the following examples, the preparation of 2-2-(2-methoxyethoxy)ethoxy tetrabutylphosphonium salt ([P 4,4,4,4 ][MEEA], P4M) is referred to (Wang X, Guo L. SERS Activity of Semiconductors: Crystalline and Amorphous Nanomaterials. Angewandte Chemie International Edition, 2020, 59(11): 4231-4239.), the preparation of 2-2-(2-methoxyethoxy)ethoxy trihexyl(tetradecyl)phosphonium salt ([P 6,6,6,14 ][MEEA], P6M) and thiophene-2-carboxylic acid trihexyl(tetradecyl)phosphonium salt ([P 6,6,6,14 ][TpA], P4T) is referred to (X. Wang, M. Zhang, L. Li, F. U. Shah, R. An, Supported fluorine-free ionic liquids with highly sensitive gas-sensing performance. Mol. Liq. 390 (2023) 123122.), and the preparation of ZACO nanotube arrays is referred to (Wu Z Q F. Ni-decorated ZrAlCo-O nanotube arrays with ultrahigh sensitivity for non-enzymatic glucose sensing. Electrochimica Acta, 2019, 311.).

[0029] Preparation of PBS buffer: NaH2PO4 and Na2HPO4 solutions with a concentration of 0.1 M were mixed in a volume ratio of 28:72 to prepare a PBS buffer with a pH of 7.2 and a concentration of 0.01 M.

[0030] Preparation of Cyt c solution: a 5×10 -4 M Cyt c solution was prepared in a PBS buffer with a pH of 7.2 and a concentration of 0.01 M.

[0031] Example 1

[0032] A 0.01 g halogen-free ionic liquid [P 6,6,6,14 ][MEEA] was added to 10 mL of the Cyt c solution, and ultrasonic oscillation was used to fully dissolve the ionic liquid to form a mixed solution. The ZACO nanotube array substrate was immersed in the Cyt c solution and the mixed solution, respectively, for 15 h. After being taken out, the substrate was rinsed with a PBS solution to remove the Cyt c molecules physically adsorbed on the surface of the substrate, and was fully dried with nitrogen, and then was subjected to Raman measurement to obtain a SERS spectrum.

[0033] The specific method of Raman measurement was as follows: a Raman spectrometer (HORIBA JOBIN YVON, Aramis) was used, an AR + light source with a wavelength of 532 nm was used, the laser power was controlled at 5.4 mW, the exposure time was set to 10 s, and the cycle was 2 times. Three points of each sample were selected for testing under the same test conditions to ensure the reliability of the data. The SERS signal and the intensity were obtained through the processing of LabSpec 5 software.

[0034] Example 2

[0035] This example is basically the same as Example 1, and the only difference is that a halogen-free ionic liquid [P 6,6,6,14 ][TpA] is used.

[0036] Example 3

[0037] This example is basically the same as Example 1, and the only difference is that a halogen-free ionic liquid [P 4,4,4,4 ][MEEA] is used.

[0038] Example 4

[0039] This example is basically the same as Example 1, and the only difference is that no ionic liquid is added.

[0040] Comparative Example 1

[0041] This example is basically the same as Example 1, and the only difference is that a halogen-free ionic liquid [P 6,6,6,14 ][MEEA] is used.

[0042] Comparative Example 2

[0043] This example is basically the same as Example 1, the only difference is that the amount of [P 6,6,6,14 ] added is 0.005 g.

[0044] Comparative Example 3

[0045] This example is basically the same as Comparative Example 1, the only difference is that TiO2nanotubes (TNA) are used as the substrate. The TiO2nanotubes are prepared by electrochemical anodic oxidation method, and the specific method is described in the reference (An R, Dong Y, Zhu J, Rao C. Adhesion and friction forces in biofouling attachments to nanotube-and PEG-patterned TiO2surfaces. Colloids and surfaces B: Biointerfaces, 2017, 159.).

[0046] Comparative Example 4

[0047] This comparative example is basically the same as Comparative Example 3, the only difference is that no ionic liquid is added.

[0048] Performance characterization

[0049] (1) SERS test:

[0050] SERS tests were performed on Examples 1-4 and Comparative Examples 1-4, which included the following steps:

[0051] In SERS technology, the enhancement factor (EF) is an important parameter for evaluating the degree of signal enhancement of the probe molecule, which refers to the multiple of the signal enhancement of the probe molecule in SERS test compared with conventional Raman detection. As a widely accepted standard for evaluating SERS performance, the SERS effect can be further quantified by calculating EF to evaluate the surface enhancement effect: the larger the value of the enhancement factor, the more significant the SERS effect, i.e. the better the surface enhancement effect.

[0052] Figure 1The (a) figure in the figure is the Raman spectrum obtained in Example 1, Example 2, Example 3 and Example 4, respectively. The ratio of the EF value of Cyt c on ZACO after introducing halogen-free ionic liquid P6M, P6T, P4M to the EF value of the control system (Cyt c-TNA) without introducing halogen-free ionic liquid is 41.2:1, 31.4:1, 2.5:1, respectively, which can be calculated from the data in the figure. It can be seen that the order of the degree of improvement of SERS performance of each ionic liquid is P6M>P6T>P4M. In addition, it can be seen from the figure that the ZACO substrate and the three halogen-free ionic liquids (P4M, P6M and P6T) have good biocompatibility, and the characteristic peak enhancement intensity of Cyt c on the ZACO substrate is strongly related to the length of the cation of the IL, and the order is P6M>P6T>P4M. Further, according to the XDLVO theory, compared with not introducing halogen-free ionic liquid and introducing P4M, the presence of P6M and P6T is more conducive to enhancing the interaction force between Cyt c and the ZACO substrate, which is due to the bridging effect mediated by the long cation "tail" of the ionic liquid.

[0053] Figure 1 The (b) figure in the figure is the Raman spectrum obtained in Example 4, Comparative Example 1 and Comparative Example 2, respectively. The SERS signal of Cyt c adsorbed on the ZACO substrate by Cyt c-P6M(0.005)-ZACO and Cyt c-P6M(0.05)-ZACO is much weaker than that of Cyt c adsorbed on the ZACO substrate by Cyt c-P6M-ZACO, which is due to the different degrees of ion dissociation of the ionic liquid. The degree of dissociation of the ionic liquid in the environment with more water phase is greater than that in the environment with more ionic liquid phase. Therefore, in the Cyt c solution rich in water and low in P6M amount (0.005g), both the cations and the anions are highly dissociated, and the degree of dissociation is greater than that with a higher concentration of P6M (0.01g). It should be noted that in the environment with a lower concentration of ionic liquid, fewer free ions in the system result in low ionic conductivity, which is not conducive to the charge transfer process, thereby affecting the SERS performance.

[0054] Figure 1The (c) figure in the figure is the Raman spectrum obtained under the conditions of Example 1, Example 4 and Comparative Example 3, Comparative Example 4, respectively. It can be calculated from the data in the figure that even if 0.01 g of P6M (Cyt c-P6M-TNA) is added, the EF value of Cyt c introducing P6M on the surface of the TNA substrate is only 3.0:1 compared with the condition without introduction, which is much smaller than the EF value of P6M on the surface of the ZACO substrate (41.2:1). At the same time, it can be seen from the figure that the ionic liquid P6M is better combined with the ZACO substrate than with the TNA substrate, and no matter whether the ionic liquid is introduced or not, the SERS performance of the Cyt c-ZACO system is better than that of the Cyt c-TNA system. This shows that as a SERS substrate, ZACO has better SERS performance than TNA.

[0055] (2) Adhesion test:

[0056] Adhesion tests were performed on Example 4, Example 3, Example 2 and Example 1, and the specific steps are as follows:

[0057] Step 1: First, correct the position of the laser on the probe cantilever beam, and then focus the clear probe.

[0058] Step 2: Before measuring the force-distance curve, the elastic coefficient of the protein-modified probe needs to be corrected using sapphire. The protein-modified probe is XNC12 / CRX-Au (B cantilever beam), and the reference elastic coefficient is 0.32 N / m<1 N / m. For probes with an elastic coefficient <1 N / m, the actual elastic coefficient is directly corrected by thermal calibration (LIU W, BONIN K, GUTHOLD M. Easy and direct method for calibrating atomic force microscopy lateral force measurements. Review of Scientific Instruments, 2007, 78(6): 063707.; SADEGHHASSANI S, DARAEE M, SOBAT Z. Application of atomic force microscopy in adhesion force measurements. Journal of Adhesion Science and Technology, 2021, 35(3): 221-241.).

[0059] Step 3: After correction, the force-distance curve of the Cyt c-modified probe on the surface of the TNA and ZACO is obtained by a single needle, respectively, to obtain the adhesion force F ATwenty force curves were performed on each sample surface to calculate the average adhesion force.

[0060] Figure 3 Typical AFM retraction force curves obtained under Example 4, Example 3, Example 2 and Example 1, respectively. Without the introduction of ionic liquid, the interaction force between ZACO substrate and Cyt c is the weakest. With the introduction of halogen-free ionic liquid (P4M, P6M, P6T), the interaction force between Cyt c and ZACO substrate is enhanced.

Claims

1. A method for enhancing surface enhanced Raman spectroscopy of proteins using a halogen-free ionic liquid, characterized in that, The method comprises the following steps: The protein solution and the halogen-free ionic liquid are mixed uniformly by ultrasonic, and then the ZrAlCo-O nanotube array substrate is soaked in the mixed solution at 0-10 DEG C, after the soaking, the ZrAlCo-O nanotube array substrate is washed, dried by nitrogen blowing, and then Raman measurement is carried out to obtain the SERS spectrum.

2. The surface-enhanced Raman spectroscopy method according to claim 1, characterized by, The protein is immunoglobulin, cytokine, protein marker, enzyme, peptide, nucleic acid binding protein, virus protein, plasma protein, cell signal transmission protein, structural protein, signal molecule, tissue protein, glycoprotein, toxin protein, membrane protein, heat shock protein, binding protein, tumor related protein or metabolic regulation protein.

3. The surface enhanced Raman spectroscopy method according to claim 1, wherein The protein is cytochrome c .

4. The surface enhanced Raman spectroscopy method according to claim 1, wherein The halogen-free ionic liquid is 1-butyl-3-methylimidazole acetate, 1-hexyl-3-methylimidazole methanesulfonate, 2-2- (2-methoxyethoxy) ethoxytetrabutylphosphonium salt, 2-2- (2-methoxyethoxy) ethoxytrihexyl (tetradecyl) phosphonium salt, tricontyl hexamethylphosphoric acid trimethylamine hydrochloride, thiophene-2-carboxylic acid trihexyl (tetradecyl) phosphonium salt, furan acid trihexyl tetradecyl phosphonium salt, tetraethylphosphoric acid trimethylamine hydrochloride, hexadecylphosphoric acid trimethylamine hydrochloride, octamethylphosphoric acid tributylamine hydrochloride, dodecylphosphoric acid dimethylamine hydrochloride or 1-octyl-3-methylimidazole nitrate.

5. The surface enhanced Raman spectroscopy method according to claim 1, wherein The protein solution is prepared by dissolving the protein in PBS buffer.

6. The surface enhanced Raman spectroscopy method according to claim 1, wherein The ZrAlCo-O nanotube array substrate is prepared by electrochemical anodic oxidation method.

7. The surface-enhanced Raman spectroscopy method according to claim 1, characterized by, The ratio of the protein solution to the halogen-free ionic liquid is 10 mL:0.01 g.

8. The surface-enhanced Raman spectroscopy method according to claim 1, characterized by, The soaking time is 10-15 h.

9. The surface enhanced Raman spectroscopy method according to claim 1, wherein The washing is carried out by using PBS buffer.

10. The surface-enhanced Raman spectroscopy method according to claim 1, characterized by, The nitrogen blowing drying time is 1-2 h.

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