A surface-enhanced Raman spectroscopy probe for specific detection of cysteine ​​and its preparation method and application

Abstract

The present invention discloses a surface enhanced Raman spectroscopy probe GNS@CRP@EBN (CEG) for detecting cysteine ​​Cys based on a reaction type and a preparation method and application thereof. First, a ligand CRP containing a terminal alkynyl group that can specifically recognize cysteine ​​is designed and synthesized, and then a gold nanostar (GNS) is synthesized as a Raman enhancement substrate by a seed growth method; finally, CRP and a probe p-alkynylbenzonitrile (EBN) providing a reference signal are modified onto the gold nanostar through a gold acetylene bond to form a surface enhanced Raman spectroscopy probe GNS@CRP@EBN (CEG) that specifically recognizes Cys; CEG has obvious changes in Raman spectral peaks before and after detecting Cys. The Raman probe described in the present invention has the advantages of no need for an external electric field and resistance to photobleaching, and has the capabilities of high selectivity, accuracy, anti-interference and low toxicity; the built-in reference signal peak appears in the "cell Raman silent zone" (1800‑2800cm ‑1 ) makes CEG suitable for the detection and imaging of cysteine ​​in living cells.

Description

Technical Field

[0001] The invention belongs to the field of analytical technology and relates to a surface enhanced Raman spectroscopy probe CEG for specifically detecting cysteine, a preparation method and an application thereof. Background Art

[0002] Cysteine ​​(Cys), one of the three thiols in the human body and the only one of the twenty naturally occurring amino acids to contain a reduced sulfhydryl group, plays a crucial role in regulating redox homeostasis in animal cells, in addition to its role in protein synthesis. Cysteine ​​is not only a major component of the antioxidant glutathione (GSH), but is also a key antioxidant itself, protecting cells and tissues from endogenous reactive oxygen species (ROS) and free radicals. Furthermore, cysteine ​​undergoes various post-translational modifications, regulating diverse physiological processes such as cell growth and apoptosis, signal transduction, and immune system regulation. Dysregulated cysteine ​​metabolism is also a manifestation of certain diseases, such as several neurodegenerative disorders, and restoring cysteine ​​homeostasis has therapeutic benefits for these conditions. Therefore, developing strategies for accurately measuring cysteine ​​concentrations is of great significance and value for studying various physiological and pathological processes and for the diagnosis of diseases.

[0003] Due to the presence of two other thiol molecules that are structurally highly similar to cysteine—glutathione (GSH) and homocysteine ​​(Hcy), only a few probes or methods can currently accurately detect cysteine. Known methods developed for cysteine ​​detection include gel electrophoresis, mass spectrometry, chromatography, electrochemistry, and in-cell nuclear magnetic resonance spectroscopy, but these methods generally require specialized equipment or complex sample pretreatment. Therefore, the design of a convenient and highly selective probe and method for intracellular cysteine ​​detection is crucial. Summary of the Invention

[0004] The present invention aims to provide a surface-enhanced Raman spectroscopy probe for the specific detection of cysteine, namely, a gold nanostar@cysteine ​​ligand@reference molecule (GNS@CRP@EBN), a preparation method thereof, and an application thereof in the detection of intracellular cysteine. The method comprises: first designing and synthesizing a ligand with high selectivity for cysteine, then using the prepared gold nanostar as a Raman enhancement substrate, and modifying the synthesized cysteine ​​ligand and the selected reference molecule onto the surface of the gold nanostar to form a surface-enhanced Raman spectroscopy probe for the specific detection of cysteine, thereby realizing accurate quantitative analysis of cysteine.

[0005] The GNS@CRP@EBN surface-enhanced Raman spectroscopy (CEG) probe proposed in this paper has advantages such as good selectivity and strong anti-interference ability. It comprises a gold nanostar substrate (GNS), and a Cys-recognizing ligand (CRP) and a reference molecule (p-alkynylbenzonitrile (EBN)) modified on the substrate. The GNS substrate serves as a Raman enhancement substrate, and the Cys-recognizing ligand (CRP) and the reference molecule (EBN) are modified onto the GNS substrate via gold-acetylene bonds.

[0006] In the innovative design of the present invention, the design concept of the ligand molecule mainly includes three parts: first, the recognition group part, the acrylate group can undergo nucleophilic addition with the thiol group of the thiol in the solution, and the entire recognition group falls off after the amino and carbonyl groups undergo intramolecular cyclization, resulting in a change in the Raman signal of the probe. In addition, since cysteine ​​has higher nucleophilic ability and smaller steric hindrance than homocysteine ​​and glutathione, highly selective detection of cysteine ​​is achieved; second, the connecting group part, the molecule is connected to the substrate through the gold acetylene bond, which can not only achieve the purpose of enhancing the Raman signal, but also the stability of the gold acetylene bond itself can help the ligand molecule resist the attack of other thiol and amino molecules in the biological environment, thereby improving the stability of the probe; finally, the main part contributes to the main Raman spectrum. When the recognition group of the strong electron-withdrawing group falls off, the electron binding ability of each part of the main body changes due to the conjugated structure system and the push-pull electron effect, thereby leading to changes in the Raman spectrum after light excitation.

[0007] The present invention proposes a preparation method of the surface-enhanced Raman spectroscopy probe CEG for specific detection of Cys. The method comprises the following steps: firstly synthesizing a Cys recognition ligand CRP containing a terminal alkynyl group and capable of specifically recognizing cysteine; synthesizing a gold nanostar (GNS) Raman enhancement substrate by a seed growth method; then adding a mixed solution of the Cys recognition ligand CRP and p-alkynylbenzonitrile (EBN) to an aqueous solution of the gold nanostars, modifying the gold nanostars onto the gold nanostar (GNS) Raman enhancement substrate via a gold-alkyne bond, and performing a mixing reaction followed by centrifugation to obtain the surface-enhanced Raman spectroscopy probe CEG for specific recognition of Cys, namely, GNS@CRP@EBN.

[0008] In the method for preparing the surface-enhanced Raman spectroscopy probe CEG for specific detection of Cys of the present invention, the method for preparing the cysteine ​​recognition ligand CRP is the inventive point of the present invention, and the preparation method comprises the following steps:

[0009] Step (1), 4-trimethylsilylethynylbenzaldehyde and potassium carbonate are mixed and stirred in anhydrous methanol, filtered and purified by column chromatography to obtain the product 4-ethynylbenzaldehyde; the reaction process is shown in reaction formula (1):

[0010]

[0011] Step (2), mixing the 4-ethynylbenzaldehyde, 2-hydroxyacetophenone and barium hydroxide octahydrate prepared in step (1) in anhydrous methanol to obtain the product 3-(4-ethynylphenyl)-1-(2-hydroxyphenyl)-2-propen-1-one; the reaction process is shown in reaction formula (2):

[0012]

[0013] Step (3), mixing the 3-(4-ethynylphenyl)-1-(2-hydroxyphenyl)-2-propen-1-one prepared in step (2), acryloyl chloride and triethylamine in anhydrous dichloromethane to obtain a Cys ligand CRP; the reaction process is shown in reaction formula (3):

[0014]

[0015] The overall reaction formula for preparing the Cys recognition ligand CRP is shown below:

[0016]

[0017] In step (1), the molar ratio of 4-trimethylsilylethynylbenzaldehyde to potassium carbonate is 1:(1-2); preferably, 1:1.1.

[0018] In step (1), the volume / molar ratio of the added amount of anhydrous methanol to the raw material 1 (4-trimethylsilylethynylbenzaldehyde) is (75-150 mL): (20-40 mmol); preferably, it is 100 mL:37 mmol.

[0019] In step (1), the reaction temperature is 25-40°C; preferably, 25°C.

[0020] In step (1), the reaction time is 2-6 hours; preferably, 3 hours.

[0021] In step (1), the column chromatography purification eluent is a mixed solvent of ethyl acetate and petroleum ether; the volume ratio of ethyl acetate to petroleum ether in the mixed solvent is 1:(3-5); preferably, ethyl acetate:petroleum ether=1:4.

[0022] In step (2), the molar ratio of 4-ethynylbenzaldehyde, 2-hydroxyacetophenone and barium hydroxide octahydrate is 1:(1-1.5):(1-2); preferably, 1:1.1:1.2.

[0023] In step (2), the volume / molar ratio of the added amount of anhydrous methanol to the raw material 1 (4-trimethylsilylethynylbenzaldehyde) is (75-150 mL): (20-40 mmol); preferably, it is 100 mL:37 mmol.

[0024] In step (2), the reaction temperature is 25-67°C; preferably, 60°C.

[0025] In step (2), the reaction time is 8-16 hours; preferably, 12 hours.

[0026] In step (2), after the reaction, a post-treatment process is also included: filtering while hot after the reaction is completed, dissolving the solid with dichloromethane and adjusting the pH to neutral, evaporating the solvent after extraction, and finally recrystallizing with methanol to purify the product.

[0027] In step (3), the molar / volume ratio of the product 2 (3-(4-ethynylphenyl)-1-(2-hydroxyphenyl)-2-propen-1-one), acryloyl chloride and triethylamine is 1:(1-2):(1.2-2); preferably, it is 1:1.2:1.5.

[0028] In step (3), the triethylamine and acryloyl chloride are added in stages to the anhydrous dichloromethane solution of the product 2 (3-(4-ethynylphenyl)-1-(2-hydroxyphenyl)-2-propen-1-one). Anhydrous triethylamine is added first under ice bath conditions, and the ice bath mixing time is 20-60 minutes; preferably, 30 minutes.

[0029] In step (3), the acryloyl chloride is added dropwise to a solution of the product 2 (3-(4-ethynylphenyl)-1-(2-hydroxyphenyl)-2-propen-1-one) and triethylamine in anhydrous dichloromethane at a dropping speed of 4-10 seconds per drop; preferably, 6 seconds per drop.

[0030] In step (3), the volume / molar ratio of the added amount of anhydrous dichloromethane to the product 2 (3-(4-ethynylphenyl)-1-(2-hydroxyphenyl)-2-propen-1-one) is (50-100 mL): (5-10 mmol); preferably, it is 50 mL: 5 mmol.

[0031] In step (3), the reaction temperature is 25-40°C; preferably, 25°C.

[0032] In step (3), the reaction time is 8-16 hours; preferably, 12 hours.

[0033] In step (3), after the reaction, a post-treatment process is also included: after the reaction is completed, water is added for quenching, extraction is performed, drying is performed, the solvent is evaporated, and then the product is purified by column chromatography (ethyl acetate: petroleum ether = 1:12) to obtain the product.

[0034] The present invention also provides a method for preparing a gold nanostar substrate GNS, characterized in that the preparation method can be slightly modified with reference to the method mentioned in NanoLett.2008,2,2473-2480. Specifically, in the preparation method of the surface-enhanced Raman spectroscopy probe CEG for specific detection of Cys of the present invention, the preparation of the gold nanostar substrate GNS comprises the following steps: adding 15 mL of sodium citrate solution to 100 mL of boiling chloroauric acid solution, and continuously stirring vigorously to obtain a gold seed solution; mixing the gold seed solution obtained in step (1), chloroauric acid solution, silver nitrate solution and ascorbic acid solution, and dilute hydrochloric acid (1M) and stirring, and centrifuging to obtain gold nanostars.

[0035] The mass fraction / molar concentration ratio of sodium citrate and chloroauric acid is (0.5-2%): (0.5-2 mM), preferably 1%: 1 mM, and the reaction time is 5-20 min, preferably 10 min.

[0036] The volume / molar ratio of the gold seed solution, chloroauric acid solution, dilute hydrochloric acid, silver nitrate solution and ascorbic acid solution is 0.5 mL: (10-15 μmol): (25-100 μmol): (1-2 μmol): (20-40 μmol); preferably, 0.5 mL: 12.5 μmol: 50 μmol: 1.5 μmol: 25 μmol.

[0037] All the glassware used were soaked in aqua regia (concentrated hydrochloric acid:concentrated nitric acid=3:1) for 24 h in advance.

[0038] The stirring and mixing time is 20-60s; preferably, 30s.

[0039] The ratio of the centrifugal speed to time is (3000-6000 rpm): (20-10 min); preferably, 5000 rpm: 15 min.

[0040] All the glassware used were soaked in aqua regia (concentrated hydrochloric acid:concentrated nitric acid=3:1) for 24 h in advance.

[0041] The present invention also provides a method for preparing a surface-enhanced Raman spectroscopy probe for quantitatively detecting cysteine. The preparation method comprises mixing a gold nanostar solution with a dimethyl sulfoxide solution containing a cysteine ​​recognition ligand CRP and a reference molecule EBN under heating conditions.

[0042] The molar concentration ratio of the cysteine ​​recognition ligand CRP and the reference molecule EBN is (0.2-0.5 mM): (0.1-0.25 mM); preferably, 0.5 mM: 0.25 mM.

[0043] Wherein, the heating temperature is set to 40-60°C; preferably, 60°C.

[0044] The heating and mixing time is 6-12 hours, preferably 6 hours.

[0045] Before heating, the mixed solution is subjected to an operation of passing nitrogen through the solution to remove oxygen for 20-60 minutes, preferably 30 minutes.

[0046] The present invention also provides a cysteine ​​recognition ligand CRP prepared by the above preparation method. By the preparation method of the Cys recognition ligand CRP provided by the present invention, a Cys recognition ligand CRP containing a terminal alkyne group that can specifically recognize cysteine ​​is obtained.

[0047] The present invention also provides the use of the cysteine ​​recognition ligand CRP in preparing a cysteine-responsive surface-enhanced Raman spectroscopy probe (GNS@CRP@EBN).

[0048] The present invention also provides a GNS@CRP@EBN surface enhanced Raman spectroscopy probe prepared by the above preparation method.

[0049] The present invention also provides the use of the cysteine ​​ligand or the GNS@CRP@EBN cysteine-responsive surface-enhanced Raman spectroscopy probe in detecting cysteine.

[0050] The present invention also provides a method for detecting cysteine, using the surface-enhanced Raman spectroscopy probe CEG for specific detection of Cys. The detection method comprises the following specific steps:

[0051] Take 1 mL of the GNS@CRP@EBN cysteine-responsive surface-enhanced Raman spectroscopy probe prepared in the above steps, add 10 μL of gradient concentrations of cysteine ​​diluted with phosphate buffer solution each time, and then measure the surface-enhanced Raman spectrum of the probe at an excitation wavelength of 785 nm: as the volume of cysteine ​​solution added increases, the surface-enhanced Raman spectrum at 1624 cm -1 The peak intensity of 2228cm -1 The peak intensity of cysteine ​​is almost unchanged, and the final cysteine ​​concentration and peak intensity are higher than I 1624 / I 2228 It exhibits good linearity in the range of 5-150 μM, and the linear equation is I 1624 / I 2228 =-0.0123*[Cys]+3.2181, the detection limit is 1.16 μM.

[0052] The concentration of the phosphate buffer solution is preferably 100 mM, and the pH is preferably 7.4.

[0053] The present invention also provides application of the surface-enhanced Raman spectroscopy probe for specifically detecting cysteine ​​in intracellular cysteine ​​concentration detection and Raman imaging.

[0054] The beneficial effects of the present invention are as follows: the present invention first synthesizes a ligand CRP for specific detection of cysteine ​​and a gold nanostar substrate material GNS for enhancing Raman signals, and then utilizes a gold acetylene bond to assemble the ligand CRP and the reference probe 4-alkynylbenzonitrile EBN onto the surface of the gold nanostar to construct a new type of cysteine ​​Raman probe. This probe simultaneously displays the signal for recognizing cysteine ​​and the reference probe signal, can correct the interference caused by the environment and eliminate the signal fluctuation caused by the excitation light intensity or other reasons, thereby improving the accuracy of quantitative analysis of cysteine, and is applied to the detection and imaging of intracellular cysteine ​​content. The surface-enhanced Raman spectroscopy probe of the present invention has the advantages of high selectivity, accuracy and biocompatibility. The built-in reference signal peak appears in the "cell Raman silent zone" (1800-2800cm -1 ) makes CEG suitable for widespread application in the detection and imaging of cysteine ​​in living cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 The figure shows the H-NMR spectrum of the cysteine ​​ligand synthesized by the present invention.

[0056] Figure 2 This is the C-NMR spectrum of the cysteine ​​ligand synthesized by the present invention.

[0057] Figure 3 This is a transmission electron microscope image of the gold nanostar material prepared by the present invention. The inset in the figure is a high-resolution transmission electron microscope image and particle size distribution of the gold nanostar.

[0058] Figure 4 Ultraviolet absorption spectra of the gold seed (A) and gold nanostar material (B) prepared in the present invention.

[0059] Figure 5 Fourier transform infrared spectra of cysteine ​​ligand CRP (A), reference molecule p-alkynylbenzonitrile EBN (B) and GNS@CRP@EBN (C).

[0060] Figure 6 Raman spectra of cysteine ​​ligand CRP (A), reference molecule p-alkynylbenzonitrile EBN (B), GNS@CRP@EBN (C), and GNS@CRP@EBN after Cys recognition (D).

[0061] Figure 7 The surface enhanced Raman spectra of the cysteine ​​Raman probe prepared by the present invention at different concentrations when cysteine ​​is detected alone. The small figure shows the concentration and peak intensity ratio I1624 / I 2228 The linear fitting curve of .

[0062] Figure 8 The selectivity of the surface-enhanced Raman spectroscopy probe prepared by the present invention for cysteine ​​was investigated. Common amino acids and proteins containing cysteine ​​residues (1-5: Cys, GSH, Hcy, BSA, Glu), neurotransmitters (6-8: DA, Ach, Glucose), common reactive oxygen species (9-11: H2O2, H2S, NO) and common metal ions (12-14: Ca 2+ , K + 、Na + ) when it exists alone.

[0063] Figure 9 The prepared surface-enhanced Raman spectroscopy probe for detecting cysteine ​​was used in cell cytotoxicity analysis experiments.

[0064] Figure 10 The surface-enhanced Raman spectroscopy probes prepared for detecting cysteine ​​were used for Raman imaging of cysteine ​​in normal cells, cells treated with N-ethylmaleimide (NEM), and cells treated with N-acetylcysteine ​​(NAC). (A, B, C) are bright-field images of normal cells, cells treated with NEM, and cells treated with NAC, respectively; (A1, B1, C1) are the peak intensity ratios of the cells in (A, B, and C), respectively. 1624 / I 2228 The resulting Raman imaging image shows that the color changes from blue to red, indicating that the Raman signal intensity at the corresponding position is getting stronger and stronger. DETAILED DESCRIPTION

[0065] The present invention is further described in detail with reference to the following specific examples and accompanying drawings. The processes, conditions, experimental methods, etc. for implementing the present invention, except for those specifically mentioned below, are common knowledge and common common sense in the art and are not particularly limited by the present invention.

[0066] Example 1

[0067] Preparation of cysteine ​​ligand: 4-trimethylsilylethynylbenzaldehyde (7.50 g, 37.07 mmol) was dissolved in methanol (100 mL) and potassium carbonate (5.10 g, 37.31 mmol) was added. The reaction mixture was stirred at room temperature for 3 hours and then filtered to remove the potassium carbonate. The combined filtrate was vacuum-solventized and purified by column chromatography (ethyl acetate: petroleum ether = 1:4) to obtain a yellow solid product, 4-ethynylbenzaldehyde (4.58 g, yield: 95.02%).

[0068] 4-Ethynylbenzaldehyde (1.91 g, 14.69 mmol) and 2-hydroxyacetophenone (2.02 g, 14.85 mmol) were dissolved in methanol (60 mL). After stirring at 60°C for 10 minutes, barium hydroxide octahydrate (4.64 g, 15.52 mmol) was added to the mixture, causing the solution to rapidly turn ochre yellow. The reaction was then stirred at 60°C under nitrogen overnight, resulting in the formation of a brick-red precipitate. The precipitate was washed with cold methanol and the pH adjusted to ~7 with 0.5 M hydrochloric acid. The resulting bright yellow precipitate was extracted with dichloromethane and washed with water and a saturated saline solution. The organic phase was separated, dried over anhydrous sodium sulfate, filtered, and the solvent removed in vacuo. Finally, the product was purified by recrystallization from methanol to obtain CRP-OH (2.33 g, 64.01% yield) as an orange solid.

[0069] To a solution of CRP-OH (1.86 g, 7.50 mmol) in anhydrous dichloromethane (75.00 mL) was added triethylamine (1.60 mL), and the solution was stirred in an ice bath for 30 minutes. Acryloyl chloride (0.81 g, 9.00 mmol) was then added dropwise to the solution. The mixture was stirred at room temperature and reacted overnight. The mixture was extracted with dichloromethane. The combined organic layers were dried over anhydrous sodium sulfate and the solvent was removed in vacuo. The crude product was purified by column chromatography (petroleum ether: ethyl acetate = 12: 1) to give CRP (2.35 g, yield: 84.66%) as a yellow solid.

[0070] 1 HNMR (600MHz, DMSO-d6) δ (ppm): 7.89-7.87 (dd, 1H), 7.80-7.79 (d, 2H), 7.70-7.67 (m, 1H), 7.55-7.53 (m, 3H), 7.49-7.44(m, 2H), 7.35-7.33(d, 1H), 6.50-6.47(dd, 1H), 6.36-6.31(m, 1H), 6.10-6.08(dd, 1H), 4.39(s, 1H).

[0071] 13 C NMR (600MHz, DMSO-d6) δ (ppm): 190.55,164.42,148.68,143.80,135.19,134.39,133.61, 132.66,132.24,130.51,129.40,127.78,126.90,126.40,124.24,124.08,83.67,83.53.

[0072] Figure 1 This is the H-NMR spectrum of the cysteine ​​ligand synthesized in Example 1 of the present invention.

[0073] Figure 2 This is the C-NMR spectrum of the cysteine ​​ligand synthesized in Example 1 of the present invention.

[0074] Figure 1 and Figure 2 This indicates that the cysteine ​​ligand CRP has been successfully synthesized.

[0075] Example 2

[0076] Preparation of gold nanostars: Gold nanostars (GNS) were prepared by an improved seed growth method. First, 15.00 mL of 1.00% sodium citrate solution was added to 100.00 mL of boiling 1.00 mM chloroauric acid solution under vigorous stirring to prepare the seed solution. After addition, the solution quickly changed from light yellow to dark blue, and then slowly changed to wine red. After boiling for 10 minutes, the solution was cooled to room temperature, and the prepared gold seeds were stored at 4°C. The maximum ultraviolet absorption of the synthesized gold seeds was at 521 nm. Then, at room temperature, 500.00 μL of gold seeds were added. The gold nanostars were obtained by adding the gold nanostar solution to 50.00 mL of a 0.25 mM chloroauric acid solution containing 50.00 μL of 1.00 M HCl. 750.00 μL of 2.00 mM silver nitrate solution and 250.00 μL of 100 mM ascorbic acid solution were then added simultaneously. The solution rapidly changed from light red to blue. After stirring at medium speed for 30 seconds, the solution was immediately centrifuged (5000 rpm for 15 minutes) and the supernatant removed to obtain the gold nanostars. All glassware used in the reaction was first soaked in aqua regia (concentrated hydrochloric acid:concentrated nitric acid = 3:1) for 24 hours, then rinsed with ultrapure water and IR-dried.

[0077] Figure 3 This is a transmission electron microscopy image of the gold nanostars prepared in Example 2 of the present invention, which proves that gold nanoparticles with an average size of 66±9 nm and an irregular spike structure on the surface are successfully prepared.

[0078] Figure 4 The ultraviolet absorption spectra of the gold seeds (A) and gold nanostars (B) prepared in Example 2 of the present invention show that the maximum absorption wavelength of the gold nanostars is around 756 nm, which can match the excitation light of 785 nm to produce a better Raman enhancement effect.

[0079] Example 3

[0080] Preparation of GNS@CRP@EBN: A DMSO mixture consisting of CRP prepared in Example 1 and 4-ethynylbenzonitrile (EBN) (500 μM:200 μM) was added to an aqueous solution of GNS (1 mg / mL). The mixture was deoxygenated with nitrogen for 30 minutes and then stored at 60°C under sealed conditions for 6 hours. The resulting mixture was centrifuged at 4500 rpm for purification, and the supernatant was removed to obtain GNS@CRP@EBN.

[0081] Figure 5 The infrared spectrum of the preparation process of Example 3 of the present invention, where A, B, and C are the infrared spectra of CRP, EBN, and GNS@CRP@EBN, respectively. -1 and 3233cm -1 The vibration peak at 2104cm-1 on A and B belongs to the carbon-hydrogen bond on the terminal alkyne group. When the molecule forms a gold-alkyne bond with the gold nanostar, the carbon-hydrogen bond breaks and the vibration peak disappears (as shown in C). -1 and 2105cm -1 The vibration peak at 65cm belongs to the carbon-carbon triple bond of the alkynyl group. After the gold-alkyne bond is formed, the change of the connecting groups at both ends of the bond causes charge transfer between the bond and gold and affects the vibration of the chemical bond itself, resulting in the vibration peak of the alkynyl group at about 65cm -1 The red shift (as shown in C) indicates that the molecules are assembled onto the gold nanostars.

[0082] Figure 6 This is the Raman spectrum of the preparation process of Example 3 of the present invention, where A, B, and C are the Raman spectra of CRP, EBN, and GNS@CRP@EBN, respectively. The 1624 cm-1 peak attributable to A can be clearly observed on C. -1 The characteristic peaks of 2228cm and 2228cm -1 The characteristic peak is located at 2100 cm -1 The Raman peak of the alkynyl group finally occurs at about 50 cm -1 The redshift of Figure 5 The results were consistent, indicating that the surface enhanced Raman spectroscopy probe GNS@CRP@EBN for cysteine ​​was successfully constructed. When the probe recognized cysteine, the peak at 1624 cm -1 The Raman peak intensity at 2228 cm -1 The Raman peak intensity at the position remains stable, indicating that the probe has the ability to detect cysteine ​​and has a certain self-correction ability in complex environments.

[0083] Example 4

[0084] Take 1 mL of the GNS@CRP@EBN cysteine-responsive surface-enhanced Raman spectroscopy probe prepared in Example 3 of the present invention, add 10 μL of gradient concentrations of cysteine ​​diluted with phosphate buffer solution each time, and then measure the surface-enhanced Raman spectrum of the probe at an excitation wavelength of 785 nm: Figure 7 It can be seen that with the increase of the volume of cysteine ​​solution, the 1624 cm -1 The peak intensity of 2228cm -1 The peak intensity is almost unchanged, and the final peak intensity is greater than I 1624 / I 2228 The concentration ratio of cysteine ​​showed good linearity in the range of 5-150 μM, and the linear equation was I 1624 / I 2228 =-0.0123*[Cys]+3.2181, the detection limit is 1.16 μM.

[0085] In order to explore the effects of common interfering substances on the surface-enhanced Raman spectroscopy probe for the specific detection of cysteine ​​in the present invention, common amino acids and proteins containing cysteine ​​residues (1-5: Cys, GSH, Hcy, BSA, Glu), neurotransmitters (6-8: DA, Ach, Glucose), common reactive oxygen species (9-11: H2O2, H2S, NO) and common metal ions (12-14: Ca 2+ , K + 、Na + ) when it exists alone. Figure 8 It can be seen that when interfering substances exist alone, including glutathione (GSH), homocysteine ​​(Hcy) and proteins containing cysteine ​​residues (BSA), which have similar structures to cysteine, interfere with cysteine ​​detection by less than 8.1%, indicating that the probe has good selectivity. Figure 8 The percentages represent the I obtained when the interferent was added alone. 1624 / I 2228 Compared with the I obtained by adding cysteine ​​alone 1624 / I 2228 The ratio.

[0086] Example 5

[0087] Cytotoxicity study of the GNS@CRP@EBN surface-enhanced Raman spectroscopy probe prepared in Example 3 of the present invention: In order to apply the Raman probe of the present invention to cell imaging research, the cytotoxicity study of the probe was carried out. Figure 9The neuronal cells were incubated with different concentrations of the probe (0, 10, 50, 100, 500 and 1000 μg / mL) at 37°C for 24 hours, and then cytotoxicity analysis was performed. As can be seen from the figure, after 24 hours of incubation, the neuronal cell survival rate was still higher than 89% when the probe concentration was 1 mg / mL. This concentration is much higher than the concentration we actually used, thus proving that the probe of the present invention has very low cytotoxicity.

[0088] Example 6

[0089] The GNS@CRP@EBN surface enhanced Raman spectroscopy probe was applied to Raman imaging of intracellular cysteine: 100 μg / mL of GNS@CRP@EBN prepared in Example 3 of the present invention was incubated with neuronal cells for 1 hour, and then the culture dish was washed three times with HBSS to remove the probe that did not enter the cells, and the cells were subjected to Raman imaging (A1) under 785 nm excitation, including a bright field image (A); the neuronal cells were then stimulated with 50 μM N-acetylcysteine ​​(NAC) for 30 minutes, and then the cells were subjected to Raman imaging (B1), including a bright field image (B), wherein NAC, as a cysteine ​​supplement, can quickly increase the content of intracellular cysteine; another group first incubated the neuronal cells with N-ethylmaleimide (NEM) for 30 minutes, wherein NEM is generally used as a thiol scavenger to reduce the content of intracellular cysteine, and then GNS@CRP@EBN was added for incubation, and the subsequent steps were repeated to obtain the bright field image (C) and Raman imaging image (C1) of the cells. Figure 10 As shown in Figure 2, the color distribution and depth can prove that the probe GNS@CRP@EBN successfully enters the cell and is distributed in the cytoplasm. 1624 / I 2228 The value of is positively correlated with the intracellular cysteine ​​concentration, demonstrating that the surface-enhanced Raman spectroscopy probe GNS@CRP@EBN for specific detection of cysteine ​​in the present invention is successfully used for imaging of intracellular cysteine.

[0090] The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present invention and are protected by the appended claims.

Claims

1. A surface enhanced Raman spectroscopy probe CEG for specific detection of Cys, characterized in that: The probe CEG includes a gold nanostar substrate GNS, and a Cys recognition ligand CRP and a reference molecule 4-alkynylbenzonitrile EBN modified on the gold nanostar substrate GNS through a gold acetylene bond; wherein the structure of the CRP is as follows:

2. A method for preparing a surface enhanced Raman spectroscopy probe CEG for specific detection of Cys, characterized in that: First, a recognition ligand CRP containing a terminal alkynyl group that can specifically recognize cysteine ​​Cys is synthesized; then a gold nanostar GNS is synthesized as a Raman enhancement substrate by a seed growth method; then a mixed solution of the Cys recognition ligand CRP and p-alkynylbenzonitrile EBN is added to the gold nanostar aqueous solution, and modified onto the gold nanostar substrate GNS through a gold acetylene bond, and after a mixed reaction, centrifugation is performed to obtain the surface enhanced Raman spectroscopy probe CEG that specifically recognizes Cys, namely GNS@CRP@EBN.

3. The preparation method according to claim 2, characterized in that: The concentration ratio of the gold nanostar substrate GNS, the Cys recognition ligand CRP and the reference molecule EBN is (0.5-2 mg / mL): (0.2-0.5 mM): (0.1-0.25 mM); the reaction temperature is 40-60° C.; and the reaction time is 6-12 h.

4. The preparation method according to claim 2, characterized in that: The method further includes performing a deoxygenation operation on the mixed solution for 20-60 minutes.

5. The preparation method according to claim 2, characterized in that: The preparation process of the Cys recognition ligand CRP is shown in reaction formula (A): Reaction formula (A) The preparation method comprises the following steps: Step (1): 4-trimethylsilylethynylbenzaldehyde and potassium carbonate are mixed and stirred in anhydrous methanol, filtered, and purified by column chromatography to obtain the product 4-ethynylbenzaldehyde; Step (2): mixing and stirring the 4-ethynylbenzaldehyde, 2-hydroxyacetophenone and barium hydroxide octahydrate prepared in step (1) in anhydrous methanol to obtain the product 3-(4-ethynylphenyl)-1-(2-hydroxyphenyl)-2-propene-1-one; Step (3): Mix and stir the 3-(4-ethynylphenyl)-1-(2-hydroxyphenyl)-2-propene-1-one prepared in step (2), acryloyl chloride and triethylamine in anhydrous dichloromethane to obtain Cys ligand CRP.

6. The preparation method according to claim 5, characterized in that: In the step (1), the molar ratio of 4-trimethylsilylethynylbenzaldehyde to potassium carbonate is 1:(1-2); the volume / molar ratio of the amount of anhydrous methanol added to 4-trimethylsilylethynylbenzaldehyde is (75-150mL):(20-40mmol); the reaction temperature is 25-40°C; the reaction time is 2-6 hours; the eluent of the column chromatography purification method is a mixed solvent of ethyl acetate and petroleum ether; the volume ratio of ethyl acetate to petroleum ether in the mixed solvent is 1:(3-5); and / or, In the step (2), the molar ratio of 4-ethynylbenzaldehyde, 2-hydroxyacetophenone and barium hydroxide octahydrate is 1:(1-1.5):(1-2); the volume ratio of the amount of anhydrous methanol added to 4-trimethylsilylethynylbenzaldehyde is (75-150 mL):(20-40 mmol); the reaction temperature is 25-67° C.; the reaction time is 8-16 h; and / or, In the step (3), the molar / volume ratio of the 3-(4-ethynylphenyl)-1-(2-hydroxyphenyl)-2-propene-1-one, acryloyl chloride and triethylamine is 1:(1-2):(1.2-2); the volume / molar ratio of the amount of anhydrous dichloromethane added to 3-(4-ethynylphenyl)-1-(2-hydroxyphenyl)-2-propene-1-one is (50-100 mL):(5-10 mmol); the reaction temperature is 25-40°C; the reaction time is 8-16 h; the eluent for the column chromatography purification method is a mixed solvent of ethyl acetate and petroleum ether; the volume ratio of ethyl acetate to petroleum ether in the mixed solvent is 1:(10-15).

7. The preparation method according to claim 2, characterized in that: The preparation method of the gold nanostar GNS is as follows: adding a sodium citrate aqueous solution to a boiling chloroauric acid aqueous solution and vigorously stirring to obtain a gold seed solution; mixing the gold seed solution, chloroauric acid aqueous solution, silver nitrate aqueous solution and ascorbic acid aqueous solution, adjusting the pH with 1M dilute hydrochloric acid, stirring and mixing, and centrifuging to obtain the gold nanostar GNS.

8. The preparation method according to claim 7, characterized in that: The mass fraction / molar concentration ratio of the sodium citrate and chloroauric acid is (0.5-2%): (0.5-2mM); the mass fraction of the added sodium citrate aqueous solution is 0.5-2%; the reaction time is 5-20min; The volume / molar ratio of the volume of the gold seed solution and the added chloroauric acid, dilute hydrochloric acid, silver nitrate and ascorbic acid is 0.5 mL: (10-15 μmol): (25-100 μmol): (1-2 μmol): (20-40 μmol); the stirring time is 20-60 s; and the centrifugation condition is 3000-6000 rpm for 10-20 min.

9. A method for preparing a Cys recognition ligand CRP, characterized in that: The preparation method comprises the following steps: Step (1): 4-trimethylsilylethynylbenzaldehyde and potassium carbonate are mixed and stirred in anhydrous methanol, filtered, and purified by column chromatography to obtain the product 4-ethynylbenzaldehyde; Step (2): mixing the 4-ethynylbenzaldehyde, 2-hydroxyacetophenone and barium hydroxide octahydrate prepared in step (1) in anhydrous methanol, filtering, adjusting pH, extracting, drying, evaporating the solvent and recrystallizing to obtain the product 3-(4-ethynylphenyl)-1-(2-hydroxyphenyl)-2-propene-1-one; Step (3): The 3-(4-ethynylphenyl)-1-(2-hydroxyphenyl)-2-propene-1-one, acryloyl chloride and triethylamine prepared in step (2) are mixed and stirred in anhydrous dichloromethane, and the Cys recognition ligand CRP is obtained by extraction, drying, evaporation of the solvent and purification by column chromatography.

10. The preparation method according to claim 9, characterized in that: In the step (1), the molar ratio of 4-trimethylsilylethynylbenzaldehyde to potassium carbonate is 1:(1-2); the volume / molar ratio of the amount of anhydrous methanol added to 4-trimethylsilylethynylbenzaldehyde is (75-150mL):(20-40mmol); the reaction temperature is 25-40°C; the reaction time is 2-6 hours; the eluent of the column chromatography purification method is a mixed solvent of ethyl acetate and petroleum ether; the volume ratio of ethyl acetate to petroleum ether in the mixed solvent is 1:(3-5); and / or, In the step (2), the molar ratio of 4-ethynylbenzaldehyde, 2-hydroxyacetophenone and barium hydroxide octahydrate is 1:(1-1.5):(1-2); the volume ratio of the amount of anhydrous methanol added to 4-trimethylsilylethynylbenzaldehyde is (75-150 mL):(20-40 mmol); the reaction temperature is 25-67° C.; the reaction time is 8-16 h; and / or, In the step (3), the molar / volume ratio of the 3-(4-ethynylphenyl)-1-(2-hydroxyphenyl)-2-propene-1-one, acryloyl chloride and triethylamine is 1:(1-2):(1.2-2); the volume / molar ratio of the amount of anhydrous dichloromethane added to 3-(4-ethynylphenyl)-1-(2-hydroxyphenyl)-2-propene-1-one is (50-100 mL):(5-10 mmol); the reaction temperature is 25-40°C; the reaction time is 8-16 h; the eluent for the column chromatography purification method is a mixed solvent of ethyl acetate and petroleum ether; the volume ratio of ethyl acetate to petroleum ether in the mixed solvent is 1:(10-15).

11. A surface enhanced Raman spectroscopy probe CEG for specific detection of Cys prepared by the preparation method according to any one of claims 2 to 8, and / or a Cys recognition ligand CRP prepared by the method according to claim 9 or 10, and / or a gold nanostar substrate GNS prepared by the method according to claim 10.

12. Use of the surface enhanced Raman spectroscopy probe CEG for specific detection of Cys as claimed in claim 1, the surface enhanced Raman spectroscopy probe CEG for specific detection of Cys prepared by the preparation method according to any one of claims 2 to 8, the Cys recognition ligand CRP prepared by the method according to claim 9 or 10, and the gold nanostar substrate GNS prepared by the method according to claim 10 in detecting Cys, and in detecting changes in intracellular Cys concentration and Raman imaging.

13. A method for detecting cysteine, characterized in that: In the detection method, the surface enhanced Raman spectroscopy probe CEG for specific detection of Cys as described in claim 1 is used and the surface enhanced Raman spectroscopy probe CEG for specific detection of Cys is prepared by the preparation method according to any one of claims 2 to 8.

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