CN114994011A - Surface-enhanced Raman spectrum probe for specifically detecting cysteine, and preparation method and application thereof - Google Patents

Surface-enhanced Raman spectrum probe for specifically detecting cysteine, and preparation method and application thereof Download PDF

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CN114994011A
CN114994011A CN202210549715.7A CN202210549715A CN114994011A CN 114994011 A CN114994011 A CN 114994011A CN 202210549715 A CN202210549715 A CN 202210549715A CN 114994011 A CN114994011 A CN 114994011A
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crp
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cysteine
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田阳
张思涵
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East China Normal University
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Abstract

The invention discloses a reaction type-based surface-enhanced Raman spectrum probe GNS @ CRP @ EBN (CEG) for detecting cysteine Cys and a preparation method and application thereof. Firstly, a ligand CRP containing terminal alkynyl and capable of specifically recognizing cysteine is designed and synthesized, and then Gold Nanostar (GNS) is synthesized by a seed growth method and used as RamanA reinforcing substrate; finally, modifying the CRP and a probe pair alkynyl benzonitrile (EBN) for providing a reference signal to the gold nanostar through a gold-acetylene bond to form a surface enhanced Raman spectroscopy probe GNS @ CRP @ EBN (CEG) for specifically identifying Cys; CEG has obvious changes of Raman spectrum peaks before and after Cys detection. The Raman probe has the advantages of no need of an external electric field, photobleaching resistance and the like, and has the capabilities of high selectivity, accuracy, anti-interference performance, low toxicity and the like; the built-in reference signal peak appears in the "cell Raman silence region" (1800- ‑1 ) CEG is made suitable for use in the detection and imaging of cysteine in living cells.

Description

Surface-enhanced Raman spectrum probe for specifically detecting cysteine, and preparation method and application thereof
Technical Field
The invention belongs to the technical field of analysis, and relates to a surface enhanced Raman spectroscopy (CEG) probe for specifically detecting cysteine, and a preparation method and application thereof.
Background
Cysteine (Cys) is one of three kinds of thiols in human body, is the only amino acid containing reduction type sulfhydryl in twenty kinds of natural amino acids, and plays an important role in regulating redox balance in animal cells besides being applied to protein synthesis. In addition to being a major component of the important antioxidant Glutathione (GSH), cysteine itself is also an important antioxidant that protects cells and tissues from the oxidation of endogenous Reactive Oxygen Species (ROS) and free radicals. In addition, cysteine undergoes various post-translational modifications and can regulate various physiological processes, such as cell growth, apoptosis, signal transduction, and immune system regulation. Dysregulated cysteine metabolism is also a manifestation of certain diseases, e.g. a close association with several neurodegenerative diseases; while restoration of cysteine balance has therapeutic benefits for these diseases. Therefore, the research of a strategy for accurately detecting the concentration of cysteine is of great significance and value for the research of various physiological and pathological processes and the diagnosis of diseases.
Due to the presence of two other thiol molecules, Glutathione (GSH) and homocysteine (Hcy), which are structurally highly similar to cysteine, there are currently very few probes or methods available to achieve accurate detection of cysteine. Known methods developed for cysteine detection include gel electrophoresis, mass spectrometry, chromatography, electrochemistry, intracellular magnetic resonance spectroscopy, and the like, but these methods generally require professional equipment techniques or complicated sample pretreatment. Therefore, it is crucial to design a convenient and highly selective probe and method for intracellular cysteine detection.
Disclosure of Invention
The invention aims to provide a surface-enhanced Raman spectroscopy probe for specifically detecting cysteine, namely a gold nano star @ cysteine ligand @ reference molecule (GNS @ CRP @ EBN), a preparation method thereof and application thereof in detecting intracellular cysteine, wherein the method comprises the following steps: firstly, a ligand with high selectivity on cysteine is designed and synthesized, then the prepared gold nano star is used as a Raman enhancement substrate, and the synthesized cysteine ligand and the selected reference molecule are modified on the surface of the gold nano star to form a surface enhanced Raman spectrum probe for specifically detecting cysteine, so that accurate quantitative analysis on cysteine can be realized.
The GNS @ CRP @ EBN surface-enhanced Raman spectrum probe CEG provided by the invention has the advantages of good selectivity, strong anti-interference capability and the like. The gold nano-star substrate GNS and the Cys recognition ligand CRP and the reference molecule para-alkynyl benzonitrile EBN which are modified on the substrate are included; wherein the gold nanostar substrate GNS serves as a raman-enhanced substrate; wherein the Cys recognition ligand CRP and the reference molecule EBN are modified onto the gold nanostar substrate GNS by a gold acetylenic bond.
In the innovative design of the invention, the design concept of ligand molecules mainly comprises three parts: firstly, a recognition group part is adopted, an acrylate group can perform nucleophilic addition with a sulfhydryl group of mercaptan in a solution, and the whole recognition group falls off after intramolecular cyclization of amino and carbonyl, so that a Raman signal of the probe is changed, and cysteine has higher nucleophilic ability and smaller steric hindrance compared with homocysteine and glutathione, so that high-selectivity detection of cysteine is realized; the molecules are connected with the substrate through the gold acetylene bonds, so that the purpose of enhancing Raman signals can be achieved, meanwhile, the stability of the gold acetylene bonds can help ligand molecules resist attack of other molecules with sulfydryl and amino in a biological environment, and the stability of the probe is improved; and finally, the main body part contributes to the main Raman spectrum, and when the recognition group of the strong electron-withdrawing group falls off, the electron binding capacity of each part of the main body is changed due to a conjugated structure system and a push-pull electron effect, so that the Raman spectrum is changed after light excitation.
The invention provides a preparation method of a surface enhanced Raman spectroscopy probe CEG for specifically detecting Cys, which comprises the steps of firstly synthesizing a Cys recognition ligand CRP containing terminal alkynyl and capable of specifically recognizing cysteine; synthesizing a gold nano star GNS Raman enhanced substrate by a seed growth method; and then adding the mixed solution of the Cys recognition ligand CRP and the alkynyl benzonitrile EBN into the aqueous solution of the gold nano star, modifying the gold nano star GNS Raman enhanced substrate through a gold-acetylene bond, mixing, reacting and centrifuging to obtain the surface-enhanced Raman spectrum probe CEG (GNS CRP @ EBN) for specifically recognizing the Cys.
In the preparation method of the surface enhanced Raman spectroscopy probe CEG for specifically detecting Cys, the preparation method of the cysteine recognition ligand CRP is the invention point of the invention, and the preparation method comprises the following steps:
mixing and stirring 4-trimethylsilylacetylbenzaldehyde and potassium carbonate in anhydrous methanol, filtering, and purifying by column chromatography to obtain a product 4-acetylenyl benzaldehyde; the reaction process is shown as a reaction formula (1):
Figure BDA0003654303860000021
mixing and stirring the 4-ethynylbenzaldehyde, the 2-hydroxyacetophenone and the barium hydroxide octahydrate prepared in the step (1) in anhydrous methanol to obtain a product 3- (4-ethynylphenyl) -1- (2-hydroxyphenyl) -2-propylene-1-one; the reaction process is shown as a reaction formula (2):
Figure BDA0003654303860000022
step (3), mixing and stirring the 3- (4-ethynylphenyl) -1- (2-hydroxyphenyl) -2-propen-1-one prepared in the step (2), acryloyl chloride and triethylamine in anhydrous dichloromethane to obtain a Cys ligand CRP; the reaction process is shown as a reaction formula (3):
Figure BDA0003654303860000031
the general reaction formula for preparing the Cys recognition ligand CRP is shown as the following formula:
Figure BDA0003654303860000032
in the step (1), the molar ratio of the 4-trimethylsilylacetylbenzaldehyde to the potassium carbonate is 1: (1-2); preferably, it is 1: 1.1.
In the step (1), the volume/mol ratio of the added amount of the anhydrous methanol to the raw material 1 (4-trimethylsilylacetylbenzaldehyde) is (75-150 mL): (20-40 mmol); preferably, 100 mL: 37 mmol.
In the step (1), the reaction temperature is 25-40 ℃; preferably, it is 25 ℃.
In the step (1), the reaction time is 2-6 h; preferably, it is 3 h.
In the step (1), 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: (3-5); preferably, the ratio of ethyl acetate: petroleum ether is 1: 4.
In the step (2), the molar ratio of the 4-acetylenyl benzaldehyde to the 2-hydroxyacetophenone to the barium hydroxide octahydrate is 1: (1-1.5) and (1-2); preferably, it is 1:1.1: 1.2.
In the step (2), the volume/mol ratio of the added amount of the anhydrous methanol to the raw material 1 (4-trimethylsilylacetylbenzaldehyde) is (75-150 mL): (20-40 mmol); preferably, 100 mL: 37 mmol.
In the step (2), the reaction temperature is 25-67 ℃; preferably, it is 60 ℃.
In the step (2), the reaction time is 8-16 h; preferably 12 h.
In the step (2), after the reaction, the method further comprises a post-treatment process: and filtering while hot after the reaction is finished, dissolving the solid by using dichloromethane, adjusting the pH value to be neutral, extracting, evaporating the solvent, and finally recrystallizing and purifying methanol to obtain the product.
In the 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, 1: 1.2: 1.5.
in the step (3), the triethylamine and the acryloyl chloride are added into an anhydrous dichloromethane solution of a product 2(3- (4-ethynylphenyl) -1- (2-hydroxyphenyl) -2-propylene-1-one) in sections, anhydrous triethylamine is added firstly under the ice bath condition, and the ice bath mixing time is 20-60 min; preferably, it is 30 min.
In the step (3), the acryloyl chloride is added into an anhydrous dichloromethane solution of a product 2(3- (4-ethynylphenyl) -1- (2-hydroxyphenyl) -2-propen-1-one) and triethylamine in a dropwise manner, wherein the dropwise adding speed is 4-10 seconds per drop; preferably 6 seconds per drop.
In step (3), the volume/mole ratio of the added amount of the anhydrous dichloromethane to the product 2(3- (4-ethynylphenyl) -1- (2-hydroxyphenyl) -2-propen-1-one) is (50-100 mL): (5-10 mmol); preferably, 50 mL: 5 mmol.
In the step (3), the reaction temperature is 25-40 ℃; preferably, it is 25 ℃.
In the step (3), the reaction time is 8-16 h; preferably 12 h.
In the step (3), after the reaction, the method further comprises a post-treatment process: after the reaction is finished, water is added for quenching, extraction and drying are carried out, a solvent is evaporated, and then the product is purified by column chromatography (ethyl acetate: petroleum ether: 1: 12).
The invention also provides a preparation method of the gold nanostar substrate GNS, which is characterized in that the preparation method can be slightly modified by referring to the method mentioned in NanoLett.2008,2, 2473-2480, and specifically, in the preparation method of the surface enhanced Raman spectroscopy probe CEG for specifically detecting Cys, the preparation method of the gold nanostar substrate GNS comprises the following steps: adding 15mL of sodium citrate solution into 100mL of boiled chloroauric acid solution, and continuously and violently stirring to obtain gold seed solution; and (2) mixing and stirring the gold seed solution obtained in the step (1), a chloroauric acid solution, a silver nitrate solution and an ascorbic acid solution with diluted hydrochloric acid (1M), and centrifuging to obtain the gold nano-star.
Wherein the mass fraction/molar concentration ratio of the sodium citrate to the chloroauric acid is (0.5-2%): (0.5-2 mM); preferably, 1%: 1 mM. The reaction time is 5-20 min; preferably, it is 10 min.
Wherein the volume/mole ratio of the gold seed solution to the chloroauric acid solution to the dilute hydrochloric acid, silver nitrate solution and ascorbic acid solution is 0.5 mL: (10-15. mu. mol): (25-100. mu. mol): (1-2. mu. mol): (20-40. mu. mol); preferably, 0.5 mL: 12.5. mu. mol: 50 mu mol: 1.5. mu. mol: 25. mu. mol.
All the glassware used was previously soaked with aqua regia (concentrated hydrochloric acid: concentrated nitric acid: 3:1) for 24 h.
Wherein, the stirring and mixing time is 20-60 s; preferably 30 s.
Wherein the rotation speed and time ratio of the centrifugation is (3000-6000 rpm): (20-10 min); preferably, 5000 rpm: and 15 min.
All the glassware used was soaked in aqua regia (concentrated hydrochloric acid: concentrated nitric acid: 3:1) for 24 h.
The invention also provides a preparation method of the surface-enhanced Raman spectrum probe for quantitatively detecting cysteine, which is obtained by mixing the gold nano-star solution with a dimethyl sulfoxide solution containing a cysteine recognition ligand CRP and a reference molecule EBN under the heating condition.
Wherein the molar concentration ratio of the cysteine recognition ligand CRP to the reference molecule EBN is (0.2-0.5 mM): (0.1-0.25 mM); preferably, it is 0.5 mM: 0.25 mM.
Wherein the heating temperature is set to be 40-60 ℃; preferably, it is 60 ℃.
Wherein the heating and mixing time is 6-12 h; preferably, it is 6 h.
Wherein, the mixed solution is also subjected to nitrogen gas aeration and oxygen gas removal operation before being heated, and the time is 20-60 min; preferably, it is 30 min.
The invention also provides the cysteine recognition ligand CRP prepared by the preparation method. The Cys recognition ligand CRP containing terminal alkynyl and capable of specifically recognizing cysteine is obtained by the preparation method of the Cys recognition ligand CRP provided by the invention.
The invention also provides application of the cysteine recognition ligand CRP in preparation of a cysteine-responsive surface-enhanced Raman spectroscopy probe (GNS @ CRP @ EBN).
The invention also provides the GNS @ CRP @ EBN surface-enhanced Raman spectrum probe prepared by the preparation method.
The invention also provides application of the cysteine ligand or the GNS @ CRP @ EBN cysteine-responsive surface-enhanced Raman spectrum probe in cysteine detection.
The invention also provides a cysteine detection method, and the surface enhanced Raman spectroscopy probe CEG for detecting Cys by utilizing the specificity. The detection method comprises the following specific steps:
taking 1mL of the GNS @ CRP @ EBN cysteine-responsive surface-enhanced Raman spectrum probe prepared in the above step, adding 10 μ L of cysteine with gradient concentration diluted by phosphate buffer solution each time, and then measuring the surface-enhanced Raman spectrum of the probe with an excitation wavelength of 785 nm: 1624cm in surface enhanced Raman spectroscopy with increasing volume of cysteine solution added -1 Gradually decreases in peak intensity of 2228cm -1 Almost unchanged in peak intensity, final cysteine concentration and peak intensity ratio I 1624 /I 2228 Exhibits good linearity in the range of 5-150 μ M, and the linear equation is I 1624 /I 2228 ＝-0.0123*[Cys]+3.2181, limit of detection 1.16. mu.M.
The phosphate buffer solution is preferably at a concentration of 100mM and a pH of 7.4.
The invention also provides application of the surface enhanced Raman spectroscopy probe for specifically detecting cysteine in intracellular cysteine concentration detection and Raman imaging.
The invention has the beneficial effects that: the method comprises the steps of firstly synthesizing a ligand CRP for specifically detecting cysteine and a gold nano-star substrate material GNS for enhancing Raman signals, and then assembling the ligand CRP and a reference probe to alkynyl benzonitrile EBN by utilizing a gold-acetylene bond to the surface of a gold nano-star to construct a novel cysteine Raman probe. The probe simultaneously displays a signal for identifying cysteine and a signal of a reference probe, can correct the interference generated by the environment and eliminate signal fluctuation caused by excitation light intensity or other reasons, improves the accuracy of quantitative analysis of cysteine, and is applied to detection and imaging of the content of cysteine in cells. The invention relates to a watchThe surface enhanced Raman spectrum probe has the advantages of high selectivity, accuracy, biocompatibility and the like. The built-in reference signal peak appears in the cell Raman silence region (1800- -1 ) The CEG is suitable for being widely applied to the detection and imaging of cysteine in living cells.
Drawings
FIG. 1 shows nuclear magnetic hydrogen spectra of cysteine ligands synthesized in the present invention.
FIG. 2 is a nuclear magnetic carbon spectrum of a cysteine ligand synthesized in the present invention.
FIG. 3 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 and particle size distribution diagram of the gold nano-star.
Fig. 4 is an ultraviolet absorption spectrum of the gold seed (a) and the gold nanostar material (B) prepared by the present invention.
FIG. 5 is a Fourier transform infrared spectrum of cysteine ligand CRP (A), reference molecule p-alkynylbenzonitrile EBN (B) and GNS @ CRP @ EBN (C).
FIG. 6 is a Raman spectrum of cysteine ligand CRP (A), alkynyl benzonitrile EBN (B), GNS @ CRP @ EBN (C) and GNS @ CRP @ EBN after Cys recognition (D).
FIG. 7 shows the surface enhanced Raman spectra of cysteine Raman probe prepared according to the present invention at different concentrations when used for detecting cysteine alone, where the small graph is the concentration to peak intensity ratio I 1624 /I 2228 Is linearly fitted to the curve.
FIG. 8 is a selective test of surface enhanced Raman spectroscopy probes prepared according to the present invention for cysteine. Common amino acids and proteins containing cysteine residues (1-5: Cys, GSH, Hcy, BSA, Glu), neurotransmitters (6-8: DA, Ach, Glucose), common active oxygen species (9-11: H), respectively, were examined 2 O 2 、H 2 S, NO) and common metal ions (12-14: ca 2+ 、K + 、Na + ) Interference when present alone.
FIG. 9 is a surface enhanced Raman spectroscopy probe prepared to detect cysteine for cellular cytotoxicity assay experiments.
FIG. 10 is a preparation methodThe cysteine-detecting surface-enhanced raman spectroscopy probe of (a) was used for raman imaging of intracellular cysteine after normal, N-ethylmaleimide (NEM) and N-acetylcysteine (NAC) treatment, respectively. (A, B, C) brightfield images of normal, NEM and NAC treated cells, respectively; (A1, B1, C1) are each the peak intensity ratio I of (A, B, C) 1624 /I 2228 The obtained Raman imaging graph shows that the intensity of the Raman signal at the corresponding position is stronger and stronger from blue to red in color.
Detailed Description
The present invention will be described in further detail with reference to the following specific examples and the accompanying drawings. The procedures, conditions, experimental methods and the like for carrying out the present invention are general knowledge and common general knowledge in the art except for the contents specifically mentioned below, and the present invention is not particularly limited.
Example 1
Preparation of cysteine ligand: 4-trimethylsilylacetylbenzaldehyde (7.50g, 37.07mmol) was dissolved in methanol (100mL) and potassium carbonate (5.10g, 37.31mmol) was added. The reaction mixture was stirred at room temperature for 3 hours, and then filtered to remove potassium carbonate. The combined filtrates were subjected to solvent removal in vacuo and purified by column chromatography (ethyl acetate: petroleum ether ═ 1:4), to collect 4-ethynylbenzaldehyde (4.58g, yield: 95.02%) as a yellow solid product.
4-ethynylbenzaldehyde (1.91g, 14.69mmol) and 2-hydroxyacetophenone (2.02g, 14.85mmol) were dissolved in methanol (60 mL). After stirring for 10min at 60 ℃, barium hydroxide octahydrate (4.64g, 15.52mmol) was added to the mixture and the solution quickly turned to a yellowish brown color, then the reaction was stirred overnight at 60 ℃ under nitrogen to form a brick red precipitate. The precipitate was washed with cold methanol solution and the pH adjusted to-7 with 0.5M hydrochloric acid. The bright yellow precipitate formed was extracted with dichloromethane and washed with water and saturated salt solution. The organic phase was separated, dried over anhydrous sodium sulfate, filtered and the solvent removed in vacuo. Finally, it was purified by recrystallization from methanol to collect CRP-OH (2.33g, yield: 64.01%) as an orange solid.
To a solution of CRP-OH (1.86g, 7.50mmol) in anhydrous dichloromethane (75.00mL) was added triethylamine (1.60mL) and the solution was stirred in an ice bath for 30 min. Acryloyl chloride (0.81g, 9.00mmol) was then added to the solution by a dropwise addition method. The mixture was stirred at room temperature for reaction 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 using column chromatography (petroleum ether: ethyl acetate ═ 12:1) to finally obtain CRP (2.35g, yield: 84.66%) as a yellow solid.
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)。
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。
FIG. 1 is a nuclear magnetic hydrogen spectrum of a cysteine ligand synthesized in example 1 of the present invention.
FIG. 2 is a nuclear magnetic carbon spectrum of the synthesized cysteine ligand of example 1 of the present invention.
FIGS. 1 and 2 illustrate that the cysteine ligand CRP has been successfully synthesized.
Example 2
Preparing gold nano star: gold Nanostars (GNS) were prepared by an improved seed growth method. Firstly, 15.00mL of 1.00 percent sodium citrate solution is added into 100.00mL of boiling 1.00mM chloroauric acid solution under vigorous stirring to prepare a seed solution, the added solution is quickly changed from light yellow to dark blue and then slowly changed into wine red, after boiling for 10min, the solution is cooled to room temperature, then the prepared gold seeds are stored at 4 ℃, and the maximum ultraviolet absorption of the synthesized gold seeds is at 521 nm; then, at room temperature, 500.00. mu.L of gold seed solution was added to 50.00mL of chloroauric acid (0.25mM) solution containing 50.00. mu.L of LHCl (1.00M), and 750.00. mu.L of silver nitrate solution (2.00mM) and 250.00. mu.L of ascorbic acid solution (100.00mM) were rapidly added to the above solution at the same time, and the solution rapidly changed from pale red to blue, and immediately centrifuged (5000rpm, 15 minutes) after stirring at medium speed for 30 seconds, and the supernatant was removed to obtain gold nanostars. All glass instruments used in the reaction process are soaked in aqua regia (concentrated hydrochloric acid: concentrated nitric acid: 3:1) for 24 hours, washed with ultrapure water and then dried by infrared rays.
Fig. 3 is a transmission electron microscope image of the gold nanostars prepared in example 2 of the present invention, which demonstrates that gold nanoparticles having an irregular spiked structure on the surface and an average size of 66 ± 9nm were successfully prepared.
Fig. 4 shows the ultraviolet absorption spectra of the gold seed (a) and the gold nano-star (B) prepared in example 2 of the present invention, wherein the maximum absorption wavelength of the gold nano-star is about 756nm, which can match the excitation light of 785nm to generate a better raman enhancement effect.
Example 3
Preparation of GNS @ CRP @ EBN: to an aqueous solution of GNS (1mg/mL) was added a DMSO mixed solution composed of CRP prepared in inventive example 1 and 4-Ethynylbenzonitrile (EBN) (500. mu.M: 200. mu.M). The mixed solution was deaerated with nitrogen for 30min and then allowed to stand at 60 ℃ for 6 hours under a sealed condition. And centrifuging and purifying the reacted mixed solution at 4500rpm, and removing a supernatant to obtain the GNS @ CRP @ EBN.
FIG. 5 is an IR spectrum of the preparation of example 3 of the present invention, wherein A, B and C are the IR spectra of CRP, EBN and GNS @ CRP @ EBN, respectively. A and B are positioned at 3261cm -1 And 3233cm -1 The vibration peak at the position belongs to a carbon-hydrogen bond on a terminal alkynyl, and when the molecule and the gold nano star form gold-acetylene bond connection, the carbon-hydrogen bond is broken to cause the vibration peak at the position to disappear (as shown in C); while A and B are located at 2104cm -1 And 2105cm -1 The vibration peak at (A) belongs to the carbon-carbon triple bond of alkynyl, and after a gold-acetylene bond is formed, due to the change of connecting groups at two ends of the bond, charge transfer occurs between the bond and gold, and the vibration of the chemical bond is influenced, so that the vibration peak of alkynyl occurs about 65cm -1 Red-shifted (as shown by C), indicating assembly of the molecule to the gold nanostar.
FIG. 6 shows Raman spectra of the preparation process of example 3 of the present invention, wherein A, B and C are Raman spectra of CRP, EBN and GNS @ CRP @ EBN, respectively. A apparent 1624cm assignment to A was observed on C -1 Is characterized byPeak and 2228cm assigned to B -1 While originally locating at 2100cm -1 The Raman peak at alkynyl finally occurs about 50cm -1 The result is consistent with that of FIG. 5, which shows that the surface enhanced Raman spectroscopy probe GNS @ CRP @ EBN of cysteine is successfully constructed; when the probe recognized cysteine, it was located at 1624cm -1 The intensity of the Raman peak at (A) is reduced, and 2228cm as reference signal -1 The intensity of the Raman peak is stable and unchanged, which shows that the probe has the capability of detecting cysteine and certain self-correction capability in a complex environment.
Example 4
Taking 1mL of the GNS @ CRP @ EBN cysteine-responsive surface-enhanced Raman spectrum probe prepared in the embodiment 3 of the invention, adding 10 μ L of cysteine with gradient concentration diluted by phosphate buffer solution each time, and measuring the surface-enhanced Raman spectrum of the probe with an excitation wavelength of 785 nm: as can be seen from FIG. 7, the volume of cysteine solution added increases, and 1624cm in surface enhanced Raman spectrum -1 Gradually decreases in peak intensity of 2228cm -1 Almost constant in peak intensity of (a), final peak intensity ratio I 1624 /I 2228 And cysteine concentration ratio in the range of 5-150. mu.M, and the linear equation is I 1624 /I 2228 ＝-0.0123*[Cys]+3.2181, limit of detection 1.16. mu.M.
In order to explore the influence of common interferents on the surface-enhanced Raman spectroscopy probe for specifically detecting cysteine in the invention, common amino acids and proteins containing cysteine residues (1-5: Cys, GSH, Hcy, BSA, Glu), neurotransmitters (6-8: DA, Ach, Glucose), common active oxygen (9-11: H) were respectively examined 2 O 2 、H 2 S, NO) and common metal ions (12-14: ca 2+ 、K + 、Na + ) Interference when present alone. As can be seen from FIG. 8, in the presence of the interferents alone, the interferents including Glutathione (GSH), homocysteine (Hcy) and protein containing cysteine residues (BSA) which have similar structures to cysteine, interfered less than 8.1% with respect to the detection of cysteine, indicating that the probes have good selectivity. The percentages in FIG. 8 represent the results obtained when the interferents were added aloneObtained I 1624 /I 2228 With addition of cysteine alone to obtain I 1624 /I 2228 The ratio of.
Example 5
The cytotoxicity research of the GNS @ CRP @ EBN surface-enhanced Raman spectroscopy probe prepared in the embodiment 3 of the invention comprises the following steps: in order to apply the raman probe of the present invention to cell imaging studies, cytotoxicity studies of the probe were performed. FIG. 9 shows the cytotoxicity assay after incubation of neuronal cells with different concentrations of probes (0, 10, 50, 100, 500 and 1000. mu.g/mL) for 24h at 37 ℃; as can be seen from the figure, after 24h of incubation, the survival rate of the neuron cells is still higher than 89% when the concentration of the probe is 1mg/mL, and the concentration is far higher than the concentration actually applied by us, so that the cytotoxicity of the probe is very low.
Example 6
The GNS @ CRP @ EBN surface-enhanced Raman spectroscopy probe is applied to Raman imaging of intracellular cysteine: incubating 100 mu g/mL of GNS @ CRP @ EBN prepared in the embodiment 3 of the invention with neuronal cells for 1h, washing the culture dish three times with HBSS, removing probes which do not enter the cells, and performing Raman imaging on the cells under 785nm excitation (A1), including a bright field image (A); stimulating the neuronal cells with 50 μ M N-acetylcysteine (NAC) for 30min, and performing raman imaging (B1) on the cells, including bright field imaging (B), wherein NAC as a cysteine supplement rapidly increases the intracellular cysteine content; and in the other group, the neuron cells are firstly incubated with N-ethylmaleimide (NEM) for 30min, wherein the NEM is generally used as a thiol scavenger and can reduce the intracellular cysteine content, then GNS @ CRP @ EBN is added for incubation, and the subsequent steps are repeated to obtain a bright field image (C) and a Raman imaging image (C1) of the cells. As shown in FIG. 10, the color distribution and intensity can demonstrate that the probe GNS @ CRP @ EBN successfully enters the cell and is distributed in the cytoplasm, and I 1624 /I 2228 The value of (A) is in positive correlation with the concentration of the intracellular cysteine, and the surface-enhanced Raman spectroscopy probe GNS @ CRP @ EBN for specifically detecting the cysteine is proved to be successfully used for imaging the intracellular cysteine.
The protection content of the present invention is not limited to the above embodiments. Variations and advantages that may occur to those skilled in the art are intended to be included within the invention without departing from the spirit and scope of the inventive concept, and the scope of the invention is to be determined by the appended claims.

Claims (13)

1. A surface enhanced raman spectroscopy probe CEG for specifically detecting Cys, comprising a gold nanostar substrate GNS, and a Cys recognition ligand CRP and a reference molecule p-alkynylbenzonitrile EBN modified on the gold nanostar substrate GNS by a gold acetylenic bond.
2. A preparation method of a surface enhanced Raman spectroscopy probe CEG for specifically detecting Cys is characterized in that a recognition ligand CRP containing terminal alkynyl and capable of specifically recognizing cysteine Cys is synthesized; synthesizing gold nano star GNS as a Raman enhancement substrate by a seed growth method; and then adding the mixed solution of the Cys recognition ligand CRP and the p-alkynyl benzonitrile EBN into the gold nano-star aqueous solution, modifying the gold nano-star substrate GNS through a gold-acetylene bond, mixing, reacting and centrifuging to obtain the surface enhanced Raman spectrum probe CEG (GNS @ CRP @ EBN) for specifically recognizing the Cys.
3. The method according to claim 2,
the concentration ratio of the gold nano-star substrate GNS to the Cys recognition ligand CRP to 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 ℃; the reaction time is 6-12 h.
4. The method according to claim 2, further comprising subjecting the mixed solution to an oxygen removal operation for 20 to 60 min.
5. The method of claim 2, wherein the Cys recognition ligand CRP is prepared according to the following reaction scheme (A):
Figure FDA0003654303850000011
the preparation method comprises the following steps:
step (1): mixing and stirring 4-trimethylsilylacetylbenzaldehyde and potassium carbonate in anhydrous methanol, filtering, and purifying by column chromatography to obtain a product, namely 4-ethynylbenzaldehyde;
step (2): mixing and stirring the 4-ethynylbenzaldehyde, the 2-hydroxyacetophenone and the barium hydroxide octahydrate obtained in the step (1) in anhydrous methanol to obtain a product 3- (4-ethynylphenyl) -1- (2-hydroxyphenyl) -2-propylene-1-one;
and (3): and (3), (4-ethynylphenyl) -1- (2-hydroxyphenyl) -2-propen-1-one prepared in the step (2), acryloyl chloride and triethylamine are mixed and stirred in anhydrous dichloromethane to obtain Cys ligand CRP.
6. The method according to claim 5,
in the step (1): the molar ratio of the 4-trimethylsilylacetylbenzaldehyde to the potassium carbonate is 1: (1-2); the volume/mol ratio of the added anhydrous methanol to the 4-trimethylsilylacetylbenzaldehyde is (75-150 mL): (20-40 mmol); the reaction temperature is 25-40 ℃; 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 the presence of a gas in the gas,
in the step (2): the molar ratio of the 4-acetylenyl benzaldehyde to the 2-hydroxyacetophenone to the barium hydroxide octahydrate is 1: (1-1.5): (1-2); the volume ratio of the addition amount of the anhydrous methanol to the 4-trimethylsilylacetylbenzaldehyde is (75-150 mL): (20-40 mmol); the reaction temperature is 25-67 ℃; the reaction time is 8-16 h; and/or the presence of a gas in the gas,
in the step (3): the mol/volume ratio of the 3- (4-ethynylphenyl) -1- (2-hydroxyphenyl) -2-propen-1-one, the acryloyl chloride and the triethylamine is 1: (1-2): (1.2-2); the volume/mole ratio of the anhydrous dichloromethane added to 3- (4-ethynylphenyl) -1- (2-hydroxyphenyl) -2-propen-1-one is (50-100 mL): (5-10 mmol); the reaction temperature is 25-40 ℃; the reaction time is 8-16 h; 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: (10-15).
7. The method of claim 2, wherein the gold nanostar GNS is prepared by: adding the sodium citrate aqueous solution into the boiled chloroauric acid aqueous solution, and violently stirring to obtain a gold seed solution; and mixing the gold seed solution, the chloroauric acid aqueous solution, the silver nitrate aqueous solution and the ascorbic acid aqueous solution, adjusting the pH value with 1M dilute hydrochloric acid, uniformly stirring, and centrifuging to obtain the gold nano-star GNS.
8. The method according to claim 7,
the mass fraction/molar concentration ratio of the sodium citrate to the chloroauric acid is (0.5-2%): (0.5-2 mM); the mass fraction of the added sodium citrate aqueous solution is 0.5-2%; the reaction time is 5-20 min;
the volume/mol ratio of the gold seed solution to the added chloroauric acid, dilute hydrochloric acid, silver nitrate and ascorbic acid is 0.5 mL: (10-15. mu. mol): (25-100. mu. mol): (1-2. mu. mol): (20-40. mu. mol); the stirring time is 20-60 s; the centrifugation condition is 3000-6000rpm for 10-20 min.
9. A preparation method of Cys recognition ligand CRP, which is characterized by comprising the following steps:
step (1): mixing and stirring 4-trimethylsilylacetylbenzaldehyde and potassium carbonate in anhydrous methanol, filtering, and purifying by column chromatography to obtain a product, namely 4-ethynylbenzaldehyde;
step (2): mixing and stirring the 4-ethynylbenzaldehyde, the 2-hydroxyacetophenone and the barium hydroxide octahydrate prepared in the step (1) in anhydrous methanol, filtering, adjusting the pH value, extracting, drying, evaporating a solvent and recrystallizing to obtain a product 3- (4-ethynylphenyl) -1- (2-hydroxyphenyl) -2-propylene-1-one;
and (3): and (3), (4-ethynylphenyl) -1- (2-hydroxyphenyl) -2-propen-1-one obtained in the step (2), acryloyl chloride and triethylamine are mixed and stirred in anhydrous dichloromethane, and the Cys recognition ligand CRP is obtained through extraction, drying, solvent evaporation and column chromatography purification.
10. The method according to claim 9,
in the step (1): the molar ratio of the 4-trimethylsilylacetylbenzaldehyde to the potassium carbonate is 1: (1-2); the volume/mol ratio of the added anhydrous methanol to the 4-trimethylsilylacetylbenzaldehyde is (75-150 mL): (20-40 mmol); the reaction temperature is 25-40 ℃; 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 the presence of a gas in the gas,
in the step (2): the molar ratio of the 4-acetylenyl benzaldehyde to the 2-hydroxyacetophenone to the barium hydroxide octahydrate is 1: (1-1.5): (1-2); the volume ratio of the addition amount of the anhydrous methanol to the 4-trimethylsilylacetylbenzaldehyde is (75-150 mL): (20-40 mmol); the reaction temperature is 25-67 ℃; the reaction time is 8-16 h; and/or the presence of a gas in the gas,
in the step (3): the mol/volume ratio of the 3- (4-ethynylphenyl) -1- (2-hydroxyphenyl) -2-propen-1-one, the acryloyl chloride and the triethylamine is 1: (1-2): (1.2-2); the volume/mole ratio of the anhydrous dichloromethane added to 3- (4-ethynylphenyl) -1- (2-hydroxyphenyl) -2-propen-1-one is (50-100 mL): (5-10 mmol); the reaction temperature is 25-40 ℃; the reaction time is 8-16 h; 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: (10-15).
11. Surface-enhanced Raman spectroscopy (CEG) for obtaining a probe specific for Cys detection prepared by the preparation method of any one of claims 2 to 8, and/or Cys recognition ligand (CRP) prepared by the method of claim 9 or 10, and/or Gold Nanostar Substrate (GNS) prepared by the method of claim 10.
12. The surface-enhanced Raman spectroscopy (CEG) for specifically detecting Cys according to claim 1, the surface-enhanced Raman spectroscopy (CEG) for specifically detecting 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, the application of the Gold Nanostar Substrate (GNS) prepared by the method according to claim 10 in detection of Cys, and the application in detection of intracellular Cys concentration change and Raman imaging.
13. A cysteine detection method, wherein a surface enhanced Raman spectroscopy (CEG) probe for specifically detecting Cys is used in the detection method, and the CEG probe is prepared according to any one of claims 2 to 8.
CN202210549715.7A 2022-05-20 2022-05-20 Surface-enhanced Raman spectrum probe for specifically detecting cysteine, and preparation method and application thereof Pending CN114994011A (en)

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Publication number Priority date Publication date Assignee Title
CN115531539A (en) * 2022-09-19 2022-12-30 海南大学 Near-infrared SERS signal enhanced nano-probe, preparation method thereof and application thereof in integrated diagnosis and treatment of infectious bacteria

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115531539A (en) * 2022-09-19 2022-12-30 海南大学 Near-infrared SERS signal enhanced nano-probe, preparation method thereof and application thereof in integrated diagnosis and treatment of infectious bacteria
CN115531539B (en) * 2022-09-19 2023-09-15 海南大学 Near-infrared SERS signal enhanced nano probe, preparation method thereof and application thereof in integrated diagnosis and treatment of infectious bacteria

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