CN113295667A

CN113295667A - Detect Cu simultaneously+And Cu2+Surface-enhanced Raman spectrum probe and preparation method and application thereof

Info

Publication number: CN113295667A
Application number: CN202110486085.9A
Authority: CN
Inventors: 田阳; 刘嘉麒; 刘蒙蒙
Original assignee: East China Normal University
Current assignee: East China Normal University
Priority date: 2021-04-30
Filing date: 2021-04-30
Publication date: 2021-08-24
Anticipated expiration: 2041-04-30
Also published as: CN113295667B

Abstract

The invention discloses a method for simultaneously detecting Cu⁺And Cu²⁺The surface enhanced Raman spectrum probe designs and synthesizes Cu containing sulfydryl and having specific recognition group⁺Ligand and Cu²⁺A ligand; then depositing gold nanoparticles on the glass microtube with the tip diameter of 5 mu m by a chemical deposition method to obtain a gold-plated glass microtube; finally, the gold-plated glass microtube is soaked in the solution containing Cu⁺Ligand and Cu²⁺In the ligand solution, the ligand is self-assembled on the gold-plated glass micro-tube through a gold-sulfur bond to obtain the probe. The invention also discloses application of the probe in detection in organisms. The surface-enhanced Raman spectrum probe provided by the invention has the advantages that an external electric field is not required, and multiple substances can be simultaneously analyzed, so that the surface-enhanced Raman spectrum probe can be used for in vivo Cu analysis⁺And Cu²⁺Simultaneous detection to solve Cu problem in vivo detection⁺And Cu²⁺The problem of transformation. The surface-enhanced Raman spectrum probe of the invention is practicalAt present, the simultaneous detection of various electroactive substances in vivo plays an important role.

Description

Detect Cu simultaneously+And Cu2+Surface-enhanced Raman spectrum probe and preparation method and application thereof

Technical Field

The invention belongs to the technical field of analysis, and relates to a method for simultaneously detecting Cu⁺And Cu²⁺The surface enhanced Raman spectroscopy probe and the preparation method and the application thereof.

Background

Real-time monitoring of redox active substances in the brain is of great physiological importance for relieving oxidative stress injuries and for avoiding neurodegenerative diseases. Reduced cuprous (Cu)⁺) And copper (Cu) in the oxidized state²⁺) Is a pair of common redox active substances in organisms. Cu²⁺/Cu⁺The reduction potential of the system is higher, determining Cu⁺And Cu²⁺Can be used as an electron donor or acceptor in various redox reactions (such as mitochondrial respiration and melanin synthesis), and can be used as a component of superoxide dismutase to protect organisms from oxidative stress damage. Furthermore, since Cu⁺And Cu²⁺They also participate in the generation of Reactive Oxygen Species (ROS), and thus Cu⁺And Cu²⁺Can lead to an increase in ROS, further damaging DNA, proteins and lipids, triggering apoptosis, producing neurocognitive disorders, and ultimately leading to alzheimer's disease and other neurodegenerative diseases. Thus, a simultaneous detection of Cu was developed⁺And Cu²⁺The method has important physiological and pathological significance for researching neurodegenerative diseases.

Various methods have been developed to detect Cu in organisms⁺And Cu²⁺Such as fluorescence spectroscopy, electroanalytical methods, and the like. In which various organic fluorescent probes were developed for Cu in cells⁺Or Cu²⁺But not extracellular in vivo; the electroanalytical method has the advantage of high spatial and temporal resolution in a living body, but the method needs an external electric field to generate interference on brain activity, so that the measurement is inaccurate; furthermore, none of these known methods is compatible with Cu⁺And Cu²⁺And realizing simultaneous detection. Therefore, development of a technique capable of detecting Cu simultaneously in a living body⁺And Cu²⁺The method of (2) is imminent.

Disclosure of Invention

In order to solve the defects of the prior art, the invention aims to provide a method for simultaneously detecting Cu⁺And Cu²⁺The surface enhanced Raman spectroscopy probe of (1), Cu on the probe⁺Recognition of ligand and Cu²⁺Recognition of ligand (Cu)⁺Specific recognition molecule of (3) and Cu²⁺Specific recognition molecule) can be stably combined on a gold-plated glass micro-tube with a surface enhanced Raman effect to prepare a compound capable of simultaneously detecting Cu⁺And Cu²⁺The surface enhanced raman spectroscopy probe of (1). The probe can realize Cu in a biological living body⁺And Cu²⁺And simultaneously measuring.

The invention provides a method for detecting Cu simultaneously⁺And Cu²⁺By combining two kinds of Cu which can be respectively combined⁺And Cu²⁺The recognition ligand is modified on a glass micro-tube (gold-plated glass micro-tube) with gold nanoparticles deposited on the surface to form the probe.

Wherein, the Cu⁺The recognition ligand of (2) is Cu⁺Identifying ligands ETMA, bathocuproine and phenanthroline; preferably, ETMA.

Wherein, the Cu²⁺The recognition ligand of (2) is Cu²⁺Identifying ligand PYMA, ethylenediamine tetraacetic acid and bipyridyl; preferably, it is PYMA.

The probe utilizes the specific recognition function of the ligand and the ligand-bound Cu⁺And Cu²⁺The change of the fingerprint spectrum of the rear surface enhanced Raman spectrum realizes the Cu contrast⁺And Cu²⁺While simultaneously detecting. The probe of the invention is used for detecting Cu alone⁺Or Cu²⁺Exhibits a good linear response and detects Cu at the same time⁺And Cu²⁺There is no crosstalk. The probe also has the characteristics of good selectivity, high response speed and the like, and can realize the effect of Cu comparison by combining with an internal reference peak in a surface enhanced Raman spectrum⁺And Cu²⁺Meanwhile, the method can be used for accurately and quantitatively analyzing.

The invention also provides Cu⁺A method for preparing an identified ligand ETMA, said method comprising the steps of:

mixing and stirring 2-methylthio ethanol, thiourea and hydrobromic acid to obtain 2-methylthio ethanethiol; the reaction process is shown as a reaction formula (1):

and (2) mixing and stirring the 2-methylthioethanethiol prepared in the step (1) and bis (2-chloroethyl) amine in absolute ethyl alcohol to obtain bis (2- ((2- (2- (ethylthio) ethyl) thio) ethyl) amine, wherein the reaction process is shown as a reaction formula (2):

step (3) mixing and stirring the bis (2- ((2- (2- (ethylthio) ethyl) thio) ethyl) amine prepared in the step (2), thioglycolic acid, EDC and HOBT in dichloromethane to obtain Cu⁺A ligand ETMA; the reaction process is shown as a reaction formula (3):

the preparation of Cu⁺The overall reaction formula for identifying ligand ETMA is shown in formula (a) below:

in the step (1), the molar ratio of the 2-methylthioethanol to the thiourea to the hydrobromic acid is (0.5-2): (0.5-2): (0.5-2); preferably, 1: 1: 2.

in the step (1), the temperature of the reaction is 100-200 ℃; preferably, it is 100 ℃.

In the step (1), the reaction time is 12-24 hours; preferably, it is 12 hours.

In the step (1), after the reaction, the method further comprises a post-treatment process: after the reaction was completed, dichloromethane was extracted, and then dichloromethane was removed by evaporation.

In the step (2), the molar ratio of the 2-methylthioethanethiol to the bis (2-chloroethyl) amine is (0.5-5): (0.5-2); preferably, 3: 1.

in the step (2), the addition amount of the absolute ethyl alcohol is 50-100 mL; preferably, it is 100 mL.

In the step (2), the reaction temperature is 50-100 ℃; preferably, it is 80 ℃.

In the step (2), the reaction time is 12-24 hours; preferably, it is 24 hours.

In step (3), the molar ratio of the bis (2- ((2- (2- (ethylthio) ethyl) thio) ethyl) amine, thioglycolic acid, EDC and HOBT is (0.5-2): (0.5-2): preferably 1: 1.2: 1.5: 1.5).

In the step (3), the adding amount of the dichloromethane is 50-100 mL; preferably, it is 100 mL.

In the step (3), the reaction temperature is 20-50 ℃; preferably, it is 25 ℃.

In the step (3), the reaction time is 6-12 hours; preferably, it is 12 hours.

The invention also provides Cu²⁺A method of preparing a recognition ligand PYMA, the method comprising the steps of:

mixing and stirring the dimethyl pyridylamine and the N- (2-bromoethyl) phthalimide in anhydrous acetonitrile to obtain 2- (2- (bis (pyridine-2-methyl) amino) ethyl) isoindoline-1, 3-diketone; the reaction process is shown as a reaction formula (1):

mixing and stirring the 2- (2- (bis (pyridine-2-methyl) amino) ethyl) isoindoline-1, 3-dione prepared in the step (1) and hydrazine hydrate in absolute ethyl alcohol to obtain N, N-bis (2-picolyl) ethylenediamine; the reaction process is shown as the reaction formula (2):

step (3) is to use the N, N-di (2-picolyl) ethylenediamine and mercaptoethane prepared in the step (2)Mixing acid, EDC and HOBT in dichloromethane and stirring to obtain Cu²⁺A ligand PYMA; the reaction process is shown as a reaction formula (3):

the preparation of Cu²⁺The general reaction scheme for identifying ligand PYMA is shown in the following formula (B):

in the step (1), the molar ratio of the dimethyl pyridylamine to the N- (2-bromoethyl) phthalimide is (0.5-2): (0.5-2); preferably, 1: 1.

in the step (1), the addition amount of the anhydrous acetonitrile is 50-100 mL; preferably, it is 100 mL.

In the step (1), the reaction temperature is 50-100 ℃; preferably, it is 80 ℃.

In the step (1), the reaction time is 12-24 hours; preferably, it is 24 hours.

In the step (2), the molar ratio of the 2- (2- (bis (pyridine-2-methyl) amino) ethyl) isoindoline-1, 3-dione to the hydrazine hydrate is (0.5-2): (0.5-2); preferably, 1: 1.5.

In the step (2), the reaction time is 12-24 hours; preferably, it is 24 hours.

In the step (3), the molar ratio of the N, N-bis (2-picolyl) ethylenediamine, thioglycolic acid, EDC and HOBT is (0.5-2): (0.5-2): (0.5-2): (0.5-2); preferably, 1: 1.2: 1.5: 1.5.

In the step (3), the reaction time is 6-12 hours; preferably, it is 12 hours.

The invention also provides a preparation method of the gold-plated glass micro-tube, which is characterized by comprising the following steps:

(1) aqua regia (HCl: HNO) was used₃3:1), piranha solution (98% H)₂SO₄:H₂O₂7:3), sequentially cleaning the glass tube, tapering the glass tube by using a microelectrode drawing instrument to obtain a glass micro tube with the tip diameter of 5 mu m;

(2) and soaking the glass microtube in a solution containing chloroauric acid, potassium bicarbonate and glucose, and depositing gold nanoparticles on the surface of the glass microtube to obtain the gold-plated glass microtube.

In the step (1), the glass tube is made of any material with the diameter of 1-2mm, and comprises quartz, borosilicate, calcium silicate and aluminosilicate glass tubes; preferably a 1mm diameter quartz glass tube.

In the step (2), soaking the glass microtube in a mixed solution of chloroauric acid, potassium bicarbonate and glucose to carry out gold plating; the preparation steps of the gold plating of the glass micro-tube comprise chemical deposition, electrodeposition, vapor deposition, magnetron sputtering and the like; preferably, chemical deposition.

In the step (2), the concentration of the chloroauric acid is 0.001-0.1M; preferably, it is 0.012M.

In the step (2), the concentration of the potassium bicarbonate is 0.01-1M; preferably, it is 0.5M.

In the step (2), the concentration of the glucose is 0.001-0.1M; preferably, it is 0.025M.

In the step (2), the deposition temperature is 20-60 ℃; preferably, it is 45 ℃.

In the step (2), the deposition time is 2-8 hours; preferably, it is 4 hours.

The invention also provides a method for simultaneously detecting Cu⁺And Cu²⁺The preparation method of the surface-enhanced Raman spectroscopy probe comprises the step of preparing the surface-enhanced Raman spectroscopy probe at room temperatureSoaking gold-plated glass microtube in Cu-containing solution⁺Recognition of ligands ETMA and Cu²⁺Identifying ligand PYMA in ethanol mixed solution.

Wherein, the Cu⁺The concentration of recognition ligand ETMA is 0.001-1M; preferably, it is 0.01M.

Wherein, the Cu²⁺The concentration of the recognition ligand PYMA is 0.001-1M; preferably, it is 0.01M.

Wherein the soaking time is 12-24 hours; preferably, it is 12 hours.

Cu of the invention⁺Recognition of ligands ETMA and Cu²⁺Identification of ligand PYMA by DFT calculation, Cu⁺And Cu⁺The binding free energy recognizing ligand ETMA was-23.9 kcal/mol, greater than Cu⁺And Cu²⁺Recognition of the binding free energy of ligand PYMA (-12.1kcal/mol), indicating Cu⁺Is easier to be mixed with Cu⁺Recognizing ligand ETMA binding; cu²⁺And Cu²⁺The binding free energy recognizing ligand PYMA was-41.8 kcal/mol, which was greater than Cu²⁺And Cu⁺Recognition of the binding free energy of ligand ETMA (-20.3kcal/mol), indicating Cu²⁺Is easier to be mixed with Cu²⁺Recognition ligand PYMA binding. The surface-enhanced Raman spectrum probe can simultaneously detect Cu⁺And Cu²⁺The reason for (1).

The invention also provides Cu prepared by the preparation method⁺The ligand ETMA was identified.

The invention also provides Cu prepared by the preparation method²⁺The ligand PYMA was identified.

The invention also provides the Cu⁺Recognition of ligands ETMA and Cu²⁺Identification ligand PYMA preparation and Cu detection⁺And Cu²⁺And/or a surface enhanced raman spectroscopy probe.

The invention also provides the Cu prepared by the preparation method and used for detecting Cu at the same time⁺And Cu²⁺The surface enhanced raman spectroscopy probe of (1).

The invention also provides the simultaneous detection of Cu⁺And Cu²⁺The surface enhanced Raman spectrum probe can detect simultaneouslyCu⁺、Cu²⁺And/or monitoring Cu in vivo for non-diagnostic purposes⁺And Cu²⁺Change in concentration, and/or Cu in vivo for non-diagnostic purposes⁺And Cu²⁺And/or real-time detection of Cu in acute models of oxidative stress for non-diagnostic purposes⁺And Cu²⁺The use of (1).

The Cu⁺、Cu²⁺The specific detection steps are as follows:

immersing the surface-enhanced Raman spectroscopy probe prepared in the step into a detection pool containing 10mL of artificial cerebrospinal fluid, and adding 10 mu L of Cu with gradient concentration each time⁺Or Cu²⁺Solution, then surface enhanced raman spectroscopy of the detection probe: with addition of Cu⁺Increase of solution volume, 650cm in surface enhanced Raman spectroscopy^-1Gradually increased in peak intensity of 767cm^-1Almost unchanged in peak intensity of (1), and finally Cu⁺Concentration and peak intensity ratio I₆₅₀/I₇₆₇Exhibits good linearity in the range of 0.5-10 μ M with the linear equation of I₆₅₀/I₇₆₇＝0.081*[Cu⁺]+1.130, limit of detection 0.42 μ M; with addition of Cu²⁺Volume increase of solution, 1018cm in surface enhanced Raman spectrum^-1A new peak appeared and the intensity gradually increased, 767cm^-1Almost unchanged in peak intensity of (1), and finally Cu²⁺Concentration and peak intensity ratio I₁₀₁₈/I₇₆₇Exhibits good linearity in the range of 0.5-10 μ M with the linear equation of I₁₀₁₈/I₇₆₇＝0.214*[Cu²⁺]+0.928 and detection limit of 0.34. mu.M. Then, the surface-enhanced Raman spectroscopy probe prepared in the above step is immersed in a detection pool containing 10mL of artificial cerebrospinal fluid, and 10 μ L of Cu with gradient concentration is added each time⁺And Cu²⁺Solution, then surface enhanced raman spectroscopy of the detection probe: with addition of Cu⁺And Cu²⁺Increase of solution volume, 650cm in surface enhanced Raman spectroscopy^-1Gradually increased in peak intensity of 1018cm^-1A new peak appeared and the intensity gradually increased, 767cm^-1Almost unchanged in peak intensity of (1), and finally Cu⁺And Cu²⁺Concentration and peak intensity ratio I₆₅₀/I₇₆₇、I₁₀₁₈/I₇₆₇The probe shows good linearity in the range of 0.5-10 mu M, and the slope coefficient of a linear curve is consistent with that of the probe when the probe is detected alone, thereby indicating that the surface enhanced Raman spectroscopy probe is used for detecting Cu⁺And Cu²⁺There is no crosstalk. Can be used for Cu⁺And Cu²⁺While simultaneously detecting.

The invention also provides the simultaneous detection of Cu⁺And Cu²⁺The surface enhanced Raman spectroscopy probe detects Cu in the ischemia and reperfusion of the mouse brain⁺And Cu²⁺Use in the variation of concentration.

The beneficial effects of the invention include: the invention firstly prepares a gold-plated glass microtube with gold nanoparticles deposited on the surface, and then synthesizes specific Cu⁺Recognition of ligands ETMA and Cu²⁺Recognizing the ligand PYMA by the addition of specific Cu⁺Recognition of ligands ETMA and Cu²⁺The recognition ligand PYMA is modified on a gold-plated glass micro-tube to construct a structure capable of detecting Cu simultaneously⁺And Cu²⁺The surface enhanced raman spectroscopy probe of (1). The probe firstly provides a method for simultaneously detecting Cu⁺And Cu²⁺The method has the advantages of good selectivity, high response speed and the like. The simultaneous detection of Cu⁺And Cu²⁺The surface enhanced Raman spectrum probe can also be used for detecting Cu in the ischemia and reperfusion of the mouse brain⁺And Cu²⁺The concentration changes. Can also solve the problem of Cu in vivo detection⁺And Cu²⁺The problem of transformation. The surface-enhanced Raman spectrum probe plays an important role in realizing the simultaneous detection of various electroactive substances in a living body.

Drawings

FIG. 1 is a scanning electron microscope image of a glass microtube prepared according to the present invention.

FIG. 2 is a scanning electron microscope image of gold nanoparticles on the surface of a gold-plated glass microtube according to the present invention.

FIG. 3 shows gold-plated glass micro tube (a), Cu prepared by the present invention²⁺Identifying ligand PYMA modified gold-plated glass microtube (b), Cu⁺Recognition ligand ETMA modified gold-plated glass micro-tube (c) and Cu⁺Recognition of ligands ETMA andCu²⁺and (3) identifying a ligand PYMA and modifying X-ray photoelectron spectroscopy (XPS) of the gold-plated glass microtube (d), wherein the high-resolution energy spectrograms of Au element (A), N element (B) and C element (C) are respectively.

FIG. 4 shows that the surface enhanced Raman spectroscopy probe prepared by the invention is used for Cu⁺Different Cu when detected separately⁺Surface enhanced raman spectroscopy at concentration, panel is a linear fit curve (a) to the concentration response curve; cu²⁺Different Cu when detected separately²⁺Surface enhanced raman spectroscopy at concentration, panel is a linear fit curve (B) to the concentration response curve; cu⁺And Cu²⁺While detecting different Cu⁺、Cu²⁺Surface enhanced Raman spectroscopy at concentration (C) and different Cu⁺Concentration, Cu²⁺Concentration to peak intensity ratio I₆₅₀/I₇₆₇、I₁₀₁₈/I₇₆₇Is linearly fitted to the curve (D).

FIG. 5 shows a surface-enhanced Raman spectroscopy probe pair prepared according to the present invention for Cu⁺And Cu²⁺Selective and competitive experiments. Respectively examine common metal ions (Na)⁺、Fe²⁺、Fe³⁺、Zn²⁺) Amino acids (Glu, Cys), neurotransmitters (DA), metabolites (GSH) and common active oxygen (O)₂ ^·-、H₂O₂) Independently of (A) and with Cu⁺、Cu²⁺Interference in the presence of (B).

FIG. 6 shows the surface enhanced Raman spectroscopy signals (A) collected from the surface enhanced Raman spectroscopy probe on the rat brain at different times of ischemia and at reperfusion and the peak intensity ratio I to be obtained from the surface enhanced Raman spectroscopy₆₅₀/I₇₆₇And I₁₀₁₈/I₇₆₇Cu obtained by substituting linear fitting curve calculation⁺、Cu²⁺The concentration change with the ischemia time and the concentration at reperfusion (B).

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

Cu⁺Preparation of recognition ligand ETMA: 2-methylthioethanol (4.25g, 40mmol), thiourea (3.05g, 40mmol), 48% hydrobromic acid (8.5mL, 75mmol) were mixed, stirred at 100 ℃ for 12 hours, extracted with dichloromethane and the solvent was evaporated to give 2-methylthioethanethiol (3.91g) in 80% yield. 2-Methylthioethanethiol (3.91g, 32mmol) and bis (2-chloroethyl) amine (1.90g, 11mmol) were mixed in 100mL of absolute ethanol and stirred at 80 ℃ for 24 hours to give bis (2- ((2- (2- (ethylthio) ethyl) thio) ethyl) amine (2.69g), 78% yield bis (2- ((2- (2- (ethylthio) ethyl) thio) ethyl) amine (2.69g, 8.5mmol), thioglycolic acid (0.92g, 10.2mmol), EDC (2.44g, 12.8mmol) and HOBT (1.73g, 12.8mmol) were mixed in 100mL of dichloromethane and stirred at 25 ℃ for 12 hours to give Cu⁺Ligand ETMA (1.77g), 54% yield.

¹H NMR(500MHz,CDCl₃)δ(ppm):3.54(dt,4H),3.38(d,2H),2.86–2.69(m,12H),2.57(q,4H),2.16(t,1H),1.26(dt,6H)。

¹³C NMR(500MHz,CDCl₃)δ(ppm):170.32,49.38,47.10,32.78,32.31,31.89,31.73,31.15,29.38,26.24,26.18,26.06,14.81。

Example 2

Cu²⁺Preparation of recognition ligand PYMA: dimethylpyridine (4.68g, 23mmol) and N- (2-bromoethyl) phthalimide (5.92g, 23mmol) were mixed in 100mL of anhydrous acetonitrile and stirred at 80 ℃ for 24 hours to give 2- (2- (bis (pyridine-2-methyl) amino) ethyl) isoindoline-1, 3-dione (5.23g) in 61% yield. 2- (2- (bis (pyridin-2-methyl) amino) ethyl) isoindoline-1, 3-dione (5.23g, 14mmol) and hydrazine hydrate (0.70g, 14mmol) were mixed in 100mL absolute ethanol and stirred at 80 ℃ for 24 hours to give N, N-bis (2-picolyl) ethylenediamine (2.23g) in 64% yield. N, N-bis (2-picolyl) ethylenediamine (2.23g, 9.2mmol), mercaptoacetic acid (1.02g, 11mmol), EDC (2.26g, 13.8mmol) and HOBT (1.60g, 13.8mmol) were mixed in 100mL of dichloromethane and stirred at 25 ℃ for 12 hours to give Cu²⁺Ligand PYMA.

¹H NMR(500MHz,CDCl₃)δ(ppm):8.70(d,2H),8.52(t,1H),7.87(t,2H),7.60(d,2H),7.41(t,2H),4.29(s,4H),3.50(q,2H),3.17(m,4H)。

¹³C NMR(500MHz,CDCl₃)δ(ppm):170.94,153.26,147.61,139.56,125.48,124.21,57.85,53.50,36.52,27.97。

Example 3

Preparing a gold-plated glass micro-tube: queen water (HCl: HNO) for quartz glass tube with diameter of 1mm₃3:1), piranha solution (98% H)₂SO₄:H₂O₂And 7:3), and tapering the glass tube by using a microelectrode drawing instrument to obtain the glass microtube with the tip diameter of 5 microns. Then, the glass microtube was immersed in 0.012M chloroauric acid, 0.5M potassium bicarbonate and 0.025M glucose at 45 ℃ for about 4h until a clearly visible gold layer was formed on the surface of the quartz tube, indicating that the gold-plated glass microtube had been successfully constructed.

FIG. 1 is a scanning electron microscope photograph of a glass microtube prepared in example 3 of the present invention.

Fig. 2 is a scanning electron microscope image of gold nanoparticles on the surface of a gold-plated glass microtube prepared in example 3 of the present invention.

Example 4

Simultaneous detection of Cu⁺And Cu²⁺The preparation of the surface enhanced Raman spectroscopy probe comprises the following steps: after washing the gold-plated glass microtube with deionized water and absolute ethanol, the gold-plated glass microtube was immersed in an ethanol solution of 0.01M ETMA and 0.01M PYMA at room temperature for 12h to form a simultaneous Cu detection⁺And Cu²⁺The surface enhanced raman spectroscopy probe of (1).

FIG. 3 shows gold-plated glass microtubes (a), Cu prepared in example 1 of the present invention²⁺Identifying ligand PYMA modified gold-plated glass microtube (b), Cu⁺Recognition ligand ETMA modified gold-plated glass micro-tube (c) and Cu⁺Recognition of ligands ETMA and Cu²⁺And (3) identifying a ligand PYMA and modifying X-ray photoelectron spectroscopy (XPS) of the gold-plated glass microtube (d), wherein the high-resolution energy spectrograms of Au element (A), N element (B) and C element (C) are respectively. Wherein the C element signal and the N element signal indicate the pairingThe ETMA and the PYMA ligand are successfully modified on the surface of the gold-plated glass micro-tube.

Example 5

Surface enhanced Raman spectroscopy probe for Cu⁺And Cu²⁺And simultaneously detecting: the surface enhanced Raman spectroscopy probe was immersed in a detection cell containing 10mL of artificial cerebrospinal fluid, to which 10. mu.L of Cu was added in a gradient concentration⁺Or Cu²⁺Solution, then surface enhanced raman spectroscopy of the detection probe: with addition of Cu⁺Increase of solution volume, 650cm in surface enhanced Raman spectroscopy^-1Gradually increased in peak intensity of 767cm^-1Almost unchanged in peak intensity of (1), and finally Cu⁺Concentration and peak intensity ratio I₆₅₀/I₇₆₇Exhibits good linearity in the range of 0.5-10 μ M with a detection limit of 0.42 μ M; with addition of Cu²⁺Volume increase of solution, 1018cm in surface enhanced Raman spectrum^-1A new peak appeared and the intensity gradually increased, 767cm^-1Almost unchanged in peak intensity of (1), and finally Cu²⁺Concentration and peak intensity ratio I₁₀₁₈/I₇₆₇Exhibits good linearity in the range of 0.5-10. mu.M, with a detection limit of 0.34. mu.M. Then, the surface-enhanced Raman spectroscopy probe prepared in the above step is immersed in a detection pool containing 10mL of artificial cerebrospinal fluid, and 10 μ L of Cu with gradient concentration is added each time⁺And Cu²⁺Solution, then surface enhanced raman spectroscopy of the detection probe: with addition of Cu⁺And Cu²⁺Increase of solution volume, 650cm in surface enhanced Raman spectroscopy^-1Gradually increased in peak intensity of 1018cm^-1A new peak appeared and the intensity gradually increased, 767cm^-1Almost unchanged in peak intensity of (1), and finally Cu⁺And Cu²⁺Concentration and peak intensity ratio I₆₅₀/I₇₆₇、I₁₀₁₈/I₇₆₇Exhibits good linearity in the range of 0.5-10 μ M, and the linear curve slope coefficient is consistent with that of the detection alone.

FIG. 4 shows a surface enhanced Raman spectroscopy probe for Cu⁺Different Cu when detected separately⁺Surface enhanced Raman Spectroscopy at concentration (A), Final Cu⁺Concentration and peak intensity ratio I₆₅₀/I₇₆₇Exhibit good performance in the range of 0.5-10 μ MLinear, linear equation of I₆₅₀/I₇₆₇＝0.081*[Cu⁺]+1.130, limit of detection 0.42 μ M; cu²⁺Different Cu when detected separately²⁺Surface enhanced Raman spectroscopy at concentration (B), final Cu²⁺Concentration and peak intensity ratio I₁₀₁₈/I₇₆₇Exhibits good linearity in the range of 0.5-10 μ M with the linear equation of I₁₀₁₈/I₇₆₇＝0.214*[Cu²⁺]+0.928, limit of detection 0.34 μ M; cu⁺And Cu²⁺While detecting different Cu⁺、Cu²⁺Surface enhanced Raman spectroscopy at concentration (C) and different Cu⁺Concentration, Cu²⁺Concentration to peak intensity ratio I₆₅₀/I₇₆₇、I₁₀₁₈/I₇₆₇Is linearly fitted to the curve (D). Cu⁺And Cu²⁺The slope coefficient of a linear fitting curve has no obvious difference between the simultaneous detection and the single detection, and the simultaneous detection of Cu by the surface-enhanced Raman spectrum probe is proved⁺And Cu²⁺Crosstalk is not present and can be used for simultaneous detection.

Example 6

Selective study of surface enhanced raman spectroscopy probes: in order to explore the common interferent to simultaneously detect Cu in the invention⁺And Cu²⁺The influence of the surface enhanced Raman spectroscopy probe of (1) is that common metal ions (Na) are respectively inspected⁺、Fe²⁺、Fe³⁺、Zn² ⁺) Amino acids (Glu, Cys), neurotransmitters (DA), metabolites (GSH) and common active oxygen (O)₂ ^·-、H₂O₂) Independently of and with Cu⁺、Cu²⁺Interference when co-existing. Wherein Na⁺The concentration of (2) was 100mM, the concentration of DA was 1. mu.M, and the concentrations of the other substances were all 10. mu.M. As can be seen from FIG. 5, A shows that when an interfering substance is present alone, the interfering substance is responsible for Cu⁺And Cu²⁺Less than 4.1%, B indicates interferents with Cu⁺And Cu²⁺In coexistence, interferents to Cu⁺And Cu²⁺Interference of less than 4.7%. The dark gray blocks in FIG. 5A represent the interference capability of the current interferent alone, and the dark gray blocks in FIG. 5B represent the current interferent with Cu⁺Or Cu²⁺Interference capability when present. The percentages in FIG. 5A represent the I obtained with the addition of the interferent alone₆₅₀/I₇₆₇With addition of Cu alone⁺Obtained of₆₅₀/I₇₆₇Ratio of the two or I obtained when an interfering substance is added alone₁₀₁₈/I₇₆₇With addition of Cu alone²⁺Obtained of₁₀₁₈/I₇₆₇The ratio of (A) to (B); the percentages in FIG. 5B represent the simultaneous addition of interferents and Cu⁺Is obtained by₆₅₀/I₇₆₇With addition of Cu alone⁺Obtained of₆₅₀/I₇₆₇In a ratio of, or in combination with, Cu²⁺Is obtained by₁₀₁₈/I₇₆₇With addition of Cu alone²⁺Obtained of₁₀₁₈/I₇₆₇The ratio of.

Example 7

Simultaneous detection of Cu⁺And Cu²⁺The surface-enhanced Raman spectroscopy probe is used for Cu in the cerebral ischemia and reperfusion period of mice⁺And Cu²⁺And simultaneously detecting: the preparation of the animal model was approved by the public animal care and use committee of the university of east china. The C57BL/6 mice used in the experiments were purchased from Shanghai Biomodel Biotech development, Inc. Adult male C57 mice (20-25g) were anesthetized with 50mg/kg pentobarbital and mounted in a brain stereotaxic apparatus and a heating pad was placed under the mice to maintain body temperature at 37 ℃. The skull was drilled with a cranial drill, exposing the cerebral cortex and covered with a cotton ball soaked with saline to keep the cortex moist. Then, the mice were subjected to Middle Cerebral Artery Occlusion (MCAO): the Common Carotid Artery (CCA), Internal Carotid Artery (ICA) and External Carotid Artery (ECA) were surgically isolated and the ICA and CCA were temporarily clamped with artery clamps, and then a small incision was made in the ECA and a wire plug was inserted into the ICA. Thereafter, the arterial clamp on the ICA was removed and the plug was inserted approximately 10mm continuously from the arterial bifurcation. The arterial clamp on the common carotid artery was then removed to complete the procedure. Then, Cu will be detected simultaneously by the stereotaxic apparatus⁺And Cu²⁺The surface enhanced raman spectroscopy probe of (1.8 mm, 1.9mm, 0.8mm) was inserted into the cerebral cortex of mice. Finally, the mouse is placed on the movable platform of the confocal Raman spectrometer and observed through the ocular lensAnd observing and collecting the surface enhanced Raman spectrum signals by operating the movable platform. And drawing out the line plug to perform reperfusion on the rat brain after 60 minutes of ischemia, and collecting the surface enhanced Raman spectrum signal.

FIG. 6 shows the surface enhanced Raman spectroscopy signals (A) collected from the surface enhanced Raman spectroscopy probe on the rat brain at different times of ischemia and at reperfusion and the peak intensity ratio I to be obtained from the surface enhanced Raman spectroscopy₆₅₀/I₇₆₇And I₁₀₁₈/I₇₆₇Cu obtained by substituting linear fitting curve calculation⁺、Cu²⁺The concentration change with the ischemia time and the concentration at reperfusion (B). The figure shows that Cu in cerebral cortex of normal mice⁺And Cu²⁺The levels of (A) were 1.76. + -. 0.43. mu.M and 2.16. + -. 0.45. mu.M, respectively. Within the first ten minutes of ischemia, Cu⁺The concentration of (A) is increased from 1.76 +/-0.43 mu M to 3.16 +/-0.49 mu M by 1.80 times; cu²⁺The concentration of (A) increased from 2.16. + -. 0.45. mu.M to 9.21. + -. 0.49. mu.M by a factor of 4.26. Within the next 50 minutes, Cu⁺And Cu²⁺The concentration of (A) is almost constant (<2.8%). After reperfusion, Cu⁺The concentration is recovered to 2.23 +/-0.43 mu M, which is 1.26 times of that before ischemia, and Cu is added²⁺The concentration was restored to 3.75. + -. 0.54. mu.M, which was 1.73 times that before ischemia. These results indicate that ischemia causes Cu in the brain⁺And Cu²⁺Increase of (2), especially of Cu²⁺A large increase also indicates that reperfusion can restore ischemia-induced Cu⁺And Cu²⁺But fails to return to the pre-ischemic basal level.

The protection of the present invention is not limited to the above embodiments. Variations and advantages that may occur to those skilled in the art may be incorporated into the invention without departing from the spirit and scope of the inventive concept, which is set forth in the following claims.

Claims

1. Detect Cu simultaneously⁺And Cu²⁺The surface-enhanced Raman spectroscopy probe of (1), wherein the surface-enhanced Raman spectroscopy probe is prepared by mixing Cu⁺And Cu²⁺The specific recognition ligand is modified on a gold-plated glass micro-tube to prepare the gold-plated glass micro-tubeTo the probe;

wherein, the Cu⁺The recognition ligand of (A) includes Cu⁺Identifying ligands ETMA, bathocuproine and phenanthroline;

the Cu²⁺The recognition ligand of (A) includes Cu²⁺Identifying ligand PYMA, ethylenediamine tetraacetic acid and bipyridyl.

2. Cu⁺A process for the preparation of an identified ligand ETMA, said process comprising the steps of:

mixing and stirring 2-methylthio ethanol, thiourea and hydrobromic acid to obtain a product 12-methylthio ethanethiol;

mixing and stirring the 2-methylthioethanethiol prepared in the step (1) and bis (2-chloroethyl) amine in absolute ethyl alcohol to obtain a product 2 bis (2- ((2- (2- (ethylthio) ethyl) thio) ethyl) amine;

step (3) mixing and stirring the bis (2- ((2- (2- (ethylthio) ethyl) thio) ethyl) amine prepared in the step (2), thioglycolic acid, EDC and HOBT in dichloromethane to obtain Cu⁺A ligand ETMA;

the reaction process of the preparation method is shown as a reaction formula (A):

3. the method according to claim 2, wherein in the step (1), the molar ratio of the 2-methylthioethanol to the thiourea to the hydrobromic acid is (0.5-2): (0.5-2): (0.5-2); the reaction temperature is 100-200 ℃; the reaction time is 12-24 hours; and/or the presence of a gas in the gas,

in the step (2), the molar ratio of the 2-methylthioethanethiol to the bis (2-chloroethyl) amine is (0.5-5): (0.5-2); the addition amount of the absolute ethyl alcohol is 50-100 mL; the reaction temperature is 50-100 ℃; the reaction time is 12-24 hours; and/or the presence of a gas in the gas,

in the step (3), the molar ratio of the bis (2- ((2- (2- (ethylthio) ethyl) thio) ethyl) amine to the mercaptoacetic acid to the EDC to the HOBT is (0.5-2): 0.5-2), the addition amount of the dichloromethane is 50-100mL, the reaction temperature is 20-50 ℃, and the reaction time is 6-12 hours.

4. Cu prepared by the method of claim 2 or 3⁺The ligand ETMA was identified.

5. Cu²⁺The preparation method of the identification ligand PYMA is characterized by comprising the following steps:

mixing and stirring the dimethyl pyridylamine and the N- (2-bromoethyl) phthalimide in anhydrous acetonitrile to obtain a product 12- (2- (bis (pyridine-2-methyl) amino) ethyl) isoindoline-1, 3-diketone;

step (2), mixing and stirring the 2- (2- (bis (pyridine-2-methyl) amino) ethyl) isoindoline-1, 3-dione prepared in the step (1) and hydrazine hydrate in absolute ethyl alcohol to obtain a product 2N, N-bis (2-picolyl) ethylenediamine;

step (3) mixing and stirring the N, N-di (2-picolyl) ethylenediamine prepared in the step (2), thioglycolic acid, EDC and HOBT in dichloromethane to obtain Cu²⁺A ligand PYMA;

the reaction process of the preparation method is shown as a reaction formula (B):

6. the method according to claim 5, wherein in the step (1), the molar ratio of the lutidine amine to the N- (2-bromoethyl) phthalimide is (0.5-2): (0.5-2); the addition amount of the anhydrous acetonitrile is 50-100 mL; the reaction temperature is 50-100 ℃; the reaction time is 12-24 hours; and/or the presence of a gas in the gas,

in the step (2), the molar ratio of the 2- (2- (bis (pyridine-2-methyl) amino) ethyl) isoindoline-1, 3-dione to the hydrazine hydrate is (0.5-2): (0.5-2); the addition amount of the absolute ethyl alcohol is 50-100 mL; the reaction temperature is 50-100 ℃; the reaction time is 12-24 hours; and/or the presence of a gas in the gas,

in the step (3), the molar ratio of the N, N-bis (2-picolyl) ethylenediamine, thioglycolic acid, EDC and HOBT is (0.5-2): (0.5-2): (0.5-2): (0.5-2); the addition amount of the dichloromethane is 50-100 mL; the reaction temperature is 20-50 ℃; the reaction time is 6-12 hours.

7. Cu prepared by the method of claim 5 or 6²⁺The ligand PYMA was identified.

8. The preparation method of the gold-plated glass microtube is characterized by comprising the following steps of:

step (1), aqua regia HCl: HNO is used₃Mermaid solution 98% H, 3:1₂SO₄:H₂O₂Sequentially cleaning the glass tube in a ratio of 7:3, tapering the glass tube by using a microelectrode drawing instrument to obtain a glass micro tube with the tip diameter of 5 mu m;

and (2) soaking the glass microtube in a solution containing chloroauric acid, potassium bicarbonate and glucose, and depositing gold nanoparticles on the surface of the glass microtube to obtain the gold-plated glass microtube.

9. The method according to claim 8, wherein in the step (1), the glass tube is a glass tube of any material having a diameter of 1 to 2mm, including quartz, borosilicate, calcium silicate and aluminosilicate glass tubes.

10. The method according to claim 8, wherein in the step (2), the glass microtube is dipped in a mixed solution of chloroauric acid, potassium bicarbonate and glucose to perform gold plating; the preparation steps of the gold plating of the glass micro-tube comprise chemical deposition, electrodeposition, vapor deposition and magnetron sputtering; the concentration of the chloroauric acid is 0.001-0.1M; the concentration of the potassium bicarbonate is 0.01-1M; the concentration of the glucose is 0.001-0.1M; the deposition temperature is 20-60 ℃; the deposition time is 2-8 hours.

11. A gold-plated glass microtube with gold nanoparticles deposited on the surface, prepared by the method according to any one of claims 8 to 10.

12. Detect Cu simultaneously⁺And Cu²⁺The method for preparing the surface-enhanced Raman spectroscopy probe is characterized in that the simultaneous detection of Cu is carried out⁺And Cu²⁺The surface-enhanced Raman spectroscopy probe of (1) by immersing the gold-plated glass microtube of claim 11 in Cu at a concentration of 0.001-1M, respectively⁺Recognition of ligands ETMA and Cu²⁺Soaking the ligand PYMA in ethanol mixed solution for 12-24 hours.

13. Simultaneous detection of Cu prepared by the preparation method according to claim 12⁺And Cu²⁺The surface enhanced raman spectroscopy probe of (1).

14. Simultaneous detection of Cu as claimed in claim 1 or 13⁺And Cu²⁺Surface Enhanced Raman Spectroscopy (SERS) probe of Cu outside cells⁺And Cu²⁺Simultaneous detection, and/or monitoring of Cu in vivo for non-diagnostic purposes⁺And Cu²⁺Change in concentration, and/or Cu in vivo for non-diagnostic purposes⁺And Cu²⁺And/or real-time detection of Cu in acute models of oxidative stress for non-diagnostic purposes⁺And Cu²⁺The use of (1).