CN114034679B - Construction and application of high-reproducibility surface-enhanced Raman spectrum platform - Google Patents
Construction and application of high-reproducibility surface-enhanced Raman spectrum platform Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/65—Raman scattering
- G01N21/658—Raman scattering enhancement Raman, e.g. surface plasmons
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- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Physics & Mathematics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
The invention discloses a construction method of a high-reproducibility surface-enhanced Raman spectrum platform, which comprises the following steps: designing and synthesizing a rigid probe molecule RP1 which can react with norepinephrine; secondly, modifying two rigid probe molecules of RP1 and commercial MBA on gold nanoparticles through Au-S to form a functionalized SERS substrate; and finally, adding oleic acid to form a liquid/liquid interface, and finally constructing the high-reproducibility surface-enhanced Raman spectrum detection platform. The method also provides a method for detecting in-vivo/in-vitro norepinephrine NE by the high-reproducibility surface-enhanced Raman spectrum platform: reacting the functionalized gold nanoparticles modified with rigid probe molecules with norepinephrine; and measuring the Raman spectrum, and measuring the content of the norepinephrine by measuring the relation between the peak intensity of the characteristic Raman spectrum and the norepinephrine. By monitoring the concentration changes in vivo and in vitro, the method has important significance for researching the behavior of NE in organisms and the relationship with diseases.
Description
Technical Field
The invention belongs to the technical field of surface-enhanced Raman spectroscopy analysis and detection, and relates to construction of a high-reproducibility surface-enhanced Raman spectroscopy platform, which is used for detecting norepinephrine in vivo/in vitro.
Background
Surface Enhanced Raman Spectroscopy (SERS) is a fingerprint spectrum, has the characteristics of high sensitivity and nondestructive detection, is widely applied to the fields of substance structure and identification, biological detection and diagnosis, intracellular imaging and molecules and the like, and becomes the most promising method for biological system analysis and detection. The generation of "hot spots" by electromagnetic field coupling during the approach of nanoparticles to each other results in an increase in electromagnetic field strength, which is the main reason for the high sensitivity of raman spectroscopy. However, the electromagnetic field intensity strongly depends on the spacing of the nanoparticles, and the analyte cannot fall exactly at the "hot spot", resulting in poor reproducibility of detection, which severely hampers the development of SERS technology.
Aiming at the problem of poor reproducibility of SERS detection, the current solution mainly comprises the following steps: the method has the advantages that the substrate material is prefabricated, the reference molecules are introduced, the interface Raman and the like are realized, but the targeting efficiency of the prefabricated substrate is low, the additional reference molecules and the substance to be detected cannot be positioned at the same 'hot spot', the selectivity of the interface Raman is poor and the like, and the problem of reproducibility of SERS faces a great challenge.
Disclosure of Invention
In order to solve the defects in the prior art, the invention aims to provide a construction method of a high-reproducibility surface-enhanced Raman spectrum platform, and a ternary regulation system is constructed by combining probe molecules with a liquid/liquid interface detection platform, and the specific method is shown in figure 7. Firstly, a liquid/liquid interface is used as a detection substrate, nano particles are uniformly distributed on the interface under the action of interfacial tension, so that a multi-hot-spot SERS substrate is formed, and unitary regulation and control are realized. Secondly, in order to further control the spacing of the nano particles, rigid probe molecules are designed and synthesized, and the spacing of the nano particles is precisely fixed so as to realize binary regulation and control. Finally, the alkynyl peak of the rigid molecule at the same 'hot spot' with the object to be detected is used as a reference peak to further increase reproducibility and realize ternary regulation. Therefore, the invention constructs a ternary regulation strategy and realizes high reproducibility detection of SERS. Finally, the high-reproducibility surface-enhanced Raman spectrum platform is applied to detection of norepinephrine in mouse cerebrospinal fluid.
The invention provides a construction method of a high-reproducibility surface-enhanced Raman spectrum platform, which comprises the following specific steps:
step (1), designing and synthesizing a rigid probe molecule RP1 which specifically reacts with norepinephrine;
step (2), synthesizing gold nanoparticles as a Raman enhancement substrate;
step (3), modifying the rigid probe molecule RP1 and the rigid probe molecule MBA obtained in the step (1) on the gold nanoparticle obtained in the step (2) through Au-S bond;
the rigid probe molecule MBA is commercially available MBA, available from TCI company.
And (4) adding a second organic solvent to form a liquid/liquid interface Raman spectrum detection platform.
The rigid probe molecules RP1 and MBA have structures shown in a formula (A) and a formula (B) respectively:
in the step (1) of the present invention, the preparation method of the rigid probe molecule RP1 comprises the following steps: taking p-iodobenzaldehyde and ethynyl thiophene as raw materials, and reacting in a dry first organic solvent under the catalysis of cuprous iodide and bis (triphenylphosphine) palladium dichloride to generate a rigid probe molecule RP1, wherein the reaction process is shown as a reaction formula (I):
wherein the molar ratio of the p-iodobenzaldehyde to the ethynyl thiophene is (5-6): (5-6); preferably 5:6;
wherein the total addition of the mixed catalyst of cuprous iodide and bis (triphenylphosphine) palladium dichloride is 2-3 times of the total amount of the reaction raw materials; preferably 2.7 times; the reaction raw materials are p-iodobenzaldehyde and ethynyl thiophene;
wherein the molar ratio of the cuprous iodide to the bis (triphenylphosphine) palladium dichloride in the catalyst is (1-2): (1-2); preferably 1:2.
Wherein the first organic solvent comprises one or more of triethylamine, tetrahydrofuran, acetonitrile, acetone and the like; preferably, the solution is a mixed solution of triethylamine and tetrahydrofuran; the mixing proportion of the two components in the mixed solution is (1-2): (1-3), preferably 1:3
Wherein the dosage volume of the first organic solvent is 20-30mL; preferably 20mL.
Wherein the temperature of the reaction is 10-40 ℃; preferably 25 ℃.
Wherein the reaction time is 12-20h; preferably 16h.
In the step (2) of the invention, the preparation method of the gold nanoparticles comprises the following steps: mixing chloroauric acid solution into ultrapure water, heating to boil, adding 0.1-2mL of sodium citrate solution, and heating to reflux to generate gold nanoparticles; the specific particle size of the gold nanoparticles is determined by ultraviolet absorption.
Wherein the molar concentration of the chloroauric acid solution is 8-12mM; preferably 10mM.
Wherein the chloroauric acid solution accounts for 1-1.5% of the volume of the ultrapure water; preferably 1.2%.
Wherein the mass concentration of the sodium citrate solution is 0.7-2.5%; preferably 1%.
Wherein, the volume of the sodium citrate is 0.3mL, 0.5mL, 1.0mL, 1.5mL and 2mL; preferably 0.3mL.
Wherein, the diameter of the gold nano-particles synthesized in the invention is 15-23nm; preferably 20nm.
In one embodiment, 49mL of ultrapure water and 0.58mL of a 10mM chloroauric acid solution are taken and placed in a 100mL round bottom flask, after heating to boil, 0.58mL of 1% wt sodium citrate is rapidly added, and then heated to reflux, to obtain gold nanoparticles having a diameter of 20nm.
In the step (3) of the invention, the total concentration of the probe molecules is 1-20 mu M; preferably 10. Mu.M; the RP1 and MBA solutions were at the same concentration. The oscillation modification time is 0.5-8 hours; preferably 4h. The volume ratio of the probe solution to the gold nanoparticle solution is (1-10): (20-30), preferably 1:20.
In the step (4), the second organic solvent is one or more selected from oleic acid, n-butanol, n-hexane, 1, 2-dichloroethane, acetone and the like; preferably oleic acid. Adding a second organic solvent, and standing for 1-10 minutes; preferably 2 minutes. The volume of the second organic solvent is 100-200 mu L; preferably 100 μl.
The invention also provides a high-reproducibility surface-enhanced Raman spectrum platform constructed by the method.
The invention also provides application of the high-reproducibility surface-enhanced Raman spectrum platform in-vitro detection of norepinephrine.
The invention also provides application of the high-reproducibility surface-enhanced Raman spectrum platform in-vivo detection of norepinephrine in the cerebrospinal fluid of the mice.
The end aldehyde group of the probe molecule RP1 reacts with the amino alcohol group of norepinephrine to generate oxazolidine, and the borane group of the probe MBA reacts with the o-diphenol of the norepinephrine to generate borate group. When the invention uses surface enhanced Raman spectrum to detect in vivo/in vitro norepinephrine, the reaction time of the functionalized gold nanoparticles modified by the rigid probe molecules and the norepinephrine is 0.5-10 minutes; preferably 6 minutes.
By utilizing the characteristic that the rigid probe molecules can react with NE specifically, the probe molecules react with NE with different concentrations, thereby obtaining a SERS curve and a linear range thereof, and further obtaining the change of the concentration of NE of a sample to be detected according to the Raman spectrum of norepinephrine to be detected.
The invention also provides a method of detecting NE in vitro by SERS, the method comprising:
step a, jointly modifying RP1 rigid probe molecules shown in a formula (A) and MBA rigid probe molecules shown in a formula (B) on the surfaces of gold nanoparticles to form functionalized gold nanoparticles;
step b, reacting the functionalized gold nanoparticles obtained in the step a with norepinephrine with the content to be measured in vitro;
step c, detection by Raman spectroscopy, by measuring I 868 /I 2207 To obtain the content of norepinephrine NE to be detected in vitro.
Wherein the concentration of the rigid probe suitable for the method is 1-20 mu M; preferably 10. Mu.M (probe concentration in the whole reaction system).
Wherein the reaction time from the modification of the probe molecules to the gold nanoparticles is 0.5-8h; preferably 4h.
Wherein the reaction time of the functionalized gold nanoparticles and the NE is 0.5-10 minutes; preferably 6 minutes.
Wherein the method is suitable for detecting NE in a linear range of 0.6nM to 40nM.
Wherein the method is suitable for detecting NE with a minimum limit of detection of 0.025nM.
The linear equation is y=0.036x+0.81, r 2 ＝0.999。
In one embodiment of the invention, the method for detecting NE in vitro by SERS comprises:
(1) And (3) making a standard curve:
a10. Mu.M rigid probe (RP 1, MBA) was reacted with solutions containing different concentrations of NE (0.6, 2,4,10,20,30,40,45 nM) at normal temperature and pressure, raman spectra were recorded at different concentrations, and then a correlation curve was made for each set of Raman intensities and NE concentrations, giving a linear range of 0.6-40nM.
(2) Determination of NE content in samples
A10. Mu.M total concentration of rigid probe (RP 1, MBA) was reacted with NE in the sample at normal temperature and pressure, the Raman spectrum was measured, and the NE content in the sample was calculated from the relationship between NE concentration and Raman intensity.
(3) Determination of reproducibility of detection of NE
The total concentration of 10. Mu.M of rigid probe (RP 1, MBA) was reacted with 4nM NE at normal temperature and pressure, the Raman intensity of the 50 results was recorded and measured, and then the reproducibility of the detection was calculated.
The Raman spectrum obtained by the method and the linear range thereof can be used for detecting the change of the concentration of the NE in vitro.
The invention also provides a method for detecting NE in the cerebrospinal fluid of the mouse by SERS, which comprises the following specific steps:
step a, jointly modifying RP1 rigid probe molecules shown in a formula (a) and MBA rigid probe molecules shown in a formula (B) on the surfaces of gold nanoparticles to form functionalized gold nanoparticles;
step b, reacting the functionalized gold nanoparticles obtained in the step a with norepinephrine in cerebrospinal fluid;
step c, raman spectrum detection, by measuring I 868 /I 2207 To determine the NE content.
Wherein the concentration of the rigid probe suitable for the method is 1-20 mu M; preferably 10. Mu.M (probe concentration in the whole reaction system).
Wherein the reaction time from the modification of the probe molecules to the gold nanoparticles is 0.5-8h; preferably 4h.
Wherein the mouse cerebrospinal fluid is obtained from a wistar male mouse, and the weight of the mouse is 200-250g.
Wherein, the reaction time of the functionalized gold nano particles and norepinephrine in the cerebrospinal fluid of the mice is 0.5-10 minutes; preferably 6 minutes.
In one embodiment of the invention, the method for detecting the concentration of NE in cerebrospinal fluid of a mouse by SERS comprises:
adding oleic acid into the gold particle solution of the modified probe molecules, and standing for 2 minutes;
transferring the solution to a 96-well plate, adding fresh taken mouse cerebrospinal fluid into gold particle solution modified with rigid probe molecules, reacting with the gold particle solution at 37 ℃ for 6 minutes, and carrying out Raman detection by using a 785nm light source;
and (5) according to the intensity of the Raman spectrum, comparing the working curve to obtain the concentration of NE in cerebrospinal fluid.
The linear equation of the working curve is y=0.036x+0.81, r 2 ＝0.999。
In the invention, the rigid probe is respectively reacted with the cerebrospinal fluid of the mice before and after administration in the stress stimulation, and then the detection of Raman spectrum is carried out. The intensity of Raman spectrum is used for judging the difference of the content of NE in the cerebrospinal fluid of the mice before and after administration, and then the method can be used for exploring the relation of the drug to the release amount of NE caused by acute stress.
Compared with similar related methods, the method has the following parts with outstanding innovation: 1. and 2, fixing the distance between the nanoparticles by using rigid probe molecules, 3, using alkynyl peaks as references, and triple regulating the reproducibility of SERS detection to realize high-reproducibility detection.
The invention has the advantages that the high-reproducibility SERS platform is constructed through ternary regulation, compared with the detection of the same probe molecule at a non-interface, the detection limit is reduced by three orders of magnitude, the linear range is 0.6-40nM, the lowest detection limit is 0.025nM (S/N=3), the reproducibility is increased by 5%, compared with the flexible molecule positioned on the same detection platform, the reproducibility is increased by 9%, compared with the reference without alkynyl peak, the reproducibility is increased by 2%, and the detection reproducibility can be greatly increased by the visible ternary regulation strategy. In addition, the dual recognition probe can specifically bind to NE, and has good selectivity for neurotransmitters, amino acids, metal ions and other bioactive substances. Therefore, the high-reproducibility SERS platform can meet the detection of NE in vitro or in vivo, and can explore the influence of drugs on the concentration change of NE in mouse cerebrospinal fluid caused by acute stress.
Drawings
Fig. 1: (a) gold particle SEM and mapping characterization; (B) uv absorption spectrum of gold nanoparticle solution; (C) particle size distribution of gold particles; (D) enhancement factor of gold particles.
Fig. 2: (a) SERS peaks of gold particles at the interface; (B) SERS peaks of functionalized gold nanoparticles at the interface; (C) A SERS peak at the interface after the NE reacts with the functionalized gold nanoparticles; (D-E) XPS data, wherein a: au/RP1, b: au/rp1+mba, c: au/RP1+NE +MBA.
Fig. 3: (A) the RP1, MBA probes detect the Raman spectrum of NE at the interface; (B) RP1, MBA probes detect the standard curve of NE at the interface; (C) detecting Raman spectra of NE in a solution by using RP1 and MBA probes; (D) standard curves for detection of NE in solution by RP1, MBA probes.
Fig. 4: (A) 10. Mu.M RP1, MBA probes detect reproducibility of 4nM NE at the interface; (B) 10. Mu.M RP1, MBA probes detect reproducibility of 1. Mu.M NE in solution; (C) 10. Mu.M flexible molecule FB1, MBA probes detect reproducibility of 10nM NE at the interface; (D) 10. Mu.M flexible molecules FB1, MBA detect reproducibility of 1. Mu.M NE in solution; (E) 10 μM RP1, MBA probes detect 4nM NE at the interface and use alkynyl peaks as reproducibility after reference calibration; (F) 10. Mu.M RP1, MBA probes detect 1. Mu.M NE in solution and use alkynyl peaks as reproducibility after calibration as a reference.
Fig. 5: experimental diagrams of the selectivity of RP1 and MBA probes to common neurotransmitters, metal ions, amino acids and bioactive substances contained in organisms; wherein A is neurotransmitter, B is metal ion, C is amino acid, and D is other bioactive substance. The light bars in the figure show the raman intensities of neurotransmitters, metal ions, amino acids and bioactive substances added, and the dark bars show the raman intensities of the same amount of NE added to the group corresponding to each light bar.
Fig. 6: (A, D) a time-dependent profile and histogram of SERS peak intensities for a no drug control; (B, E) a map and histogram of the SERS peak intensity over time for an injection of 1mg/kg drug; (C, F) SERS peak intensity versus time for 2.5mg/kg drug injection and histogram.
Fig. 7: the construction process of the high-reproducibility surface-enhanced Raman spectrum platform is used for detecting norepinephrine in mouse cerebrospinal fluid.
Detailed Description
The invention will be described in further detail with reference to the following specific examples and drawings. The procedures, conditions, experimental methods, etc. for carrying out the present invention are common knowledge and common knowledge in the art, except for the following specific references, and the present invention is not particularly limited.
Example 1: preparation of gold nanoparticles
49mL of ultrapure water and 0.58mL of chloroauric acid solution with a certain concentration are taken, placed in a 100mL round bottom flask, heated and boiled, and then 0.3mL of 1%wt sodium citrate solution is quickly added, followed by heating and refluxing. After cooling to room temperature, the nanoparticle size was characterized by SEM, UV absorption spectrum and particle size distribution, respectively, indicating successful preparation of gold particles with a particle size of 20nm (FIGS. 1A-C). Measuring Raman spectrum of mercaptobenzoic acid and SERS spectrum modified on synthesized gold nano particles respectively by using 4-mercaptobenzoic acid as standard molecule, and calculating the synthesized gold nano particlesThe enhancement factor of the particles is 7.05 x 10 3 (FIG. 1D).
Example 2: modification and detection of probe molecules
RP1 and MBA solutions with total concentration of 10 mu M were respectively reacted with gold nanoparticles prepared in example 1 of the present invention, and SERS spectra and XPS data before and after the reaction were measured (FIG. 2). Raman characteristic peaks (fig. 2B) ascribed to the probe molecules appear, and peaks (161.4 eV) ascribed to Au-S bonds (163.2 eV) and B (fig. 2D-E) appear in XPS data, indicating that the probe molecules are modified on gold particles. Next, SERS spectra and XPS data after the reaction of NE and probe molecules were measured and a peak (400.0 eV) attributed to N was observed (FIG. 2F), indicating that NE and probe molecules interacted and SERS spectra were observed at 868cm, respectively -1 1271cm -1 The symmetrical and asymmetrical stretching vibration peaks of C-O-C belonging to oxazolidine (figure 2C) and the overall peak intensity are obviously increased, which indicates that the distance between nano particles is reduced and the electromagnetic field intensity is increased after NE reacts with probe molecules.
Example 3: in vitro detection of NE by RP1 and MBA probes
Reacting 10 mu MRP1 and MBA with NE with different concentration at normal temperature and normal pressure, recording SERS spectra of NE with different concentration detected on liquid/liquid interface, and then making I 868 /I 2207 And NE concentration, yielding a linear range of 0.6nM to 40nM (FIGS. 3A-B). Reacting 10 mu MRP1 and MBA with NE with different concentration at normal temperature and normal pressure, recording SERS spectra of detecting NE with different concentration in solution, and then making I 868 /I 2207 And NE concentration, yielding a linear range of 0.25 μm-5 μm (FIGS. 3C-D). By comparing the linear relation between the probe molecules and the NE detection at the interface and the non-interface after the reaction, the detection concentration at the interface is reduced by about 3 orders of magnitude, and the detection requirement of the trace concentration can be realized.
Example 4: reproductivity comparison
RP1, MBA and NE are detected on an interface and a non-interface respectively after being reacted, the reproducibility of 50 detection results is compared, the SERS intensity of alkynyl peaks serving as reference peaks/new peaks is respectively used for quantification, and the reproducibility of 50 detection results is compared. After the flexible probe molecules FB1, MBA and NE of the same reactive group react, the flexible probe molecules are detected on an interface and a non-interface respectively, and the reproducibility of 50 detection results is compared.
10. Mu.M RP1, MBA were reacted with 4nM NE and tested at the interface with 3.9% reproducibility using the new peak intensity as a quantification (FIG. 4A) and 2.0% reproducibility using the alkynyl peak as a reference (FIG. 4E); 10. Mu.M RP1, MBA and 1. Mu.M NE were tested on non-interfaces with a reproducibility of 9.0% using the intensity of the new peak as a quantification (FIG. 4B) and 6.9% using the alkynyl peak as a reference (FIG. 4F). 10. Mu.M FB1, MBA were reacted with 10nM NE, with a reproducibility of 8.6% on the interface (FIG. 4C) and 1. Mu.M NE, with a reproducibility of 15.8% on the non-interface (FIG. 4D). The data show that the reproducibility can be remarkably improved by using the interface as a detection platform under the condition of the same probe molecule; under the same detection platform condition, the reproducibility can be further increased by using the rigid probe compared with the flexible molecule; under the condition of the same detection platform and the same probe molecule, alkynyl peaks are used as references, so that the reproducibility can be increased.
Example 5: selectivity experiment
The RP1 and MBA are reacted with common neurotransmitters, metal ions, amino acids, bioactive substances and the like contained in organisms to perform selectivity experiments.
10. Mu.M of RP1, MBA probes were incubated with neurotransmitter (200. Mu.M AA,50nM Ch, DA,5-HT, ACh, EP and GABA) (FIG. 5A) metal ions (50 mM K + 150mM Na + 1mM Ca 2+ And Mg (magnesium) 2+ Cu of 10. Mu.M 2+ ，Zn 2+ ，Fe 3+ ，Al 3+ And Mn of 2+ ) (FIG. 5B), amino acids (10. Mu.M Arg, cys, glu, gly, lys, met, phe, his, leu, ile, ser, thr and Val) (FIG. 5C) and other bioactive substances (20. Mu.M UA,1mM lactic acid, 500nM H) 2 O 2 5mM glucose and 10uM of 5-HIAA) (FIG. 5D). After the reaction, the corresponding SERS intensity is obtained, 4nM NE is added into each group of solution, the corresponding SERS intensity is obtained, and I is drawn according to each group of curves 868 /I 2207 A drawing.
Example 6: detection of NE in cerebrospinal fluid of mice under stress stimulation and administration and drug-free treatment
And detecting cerebrospinal fluid by using SERS spectrum, judging whether the difference of the content of NE in the cerebrospinal fluid of the mice stimulated by the stress corresponding to the drug treatment exists, and further exploring the relation between the drug and the release amount of NE in the brain.
Implanting microdialysis probe (CMA 7 Tub) into the prefrontal cortex of mice at 2μL.min -1 Is infused with cerebrospinal fluid for at least 90 minutes to reach equilibrium, and a constant temperature blanket is used to maintain the temperature of the mice throughout the experiment. Mice were subjected to tail pressure at 90 minutes from the start of the experiment and maintained for 30 minutes to cause acute stress stimulation, and cerebrospinal fluid collected every 30 minutes was reacted with RP1, MBA throughout the experiment and detected by SERS. As can be seen from FIGS. 6A and 6D, 868cm -1 The SERS peak at 120 minutes had a significant increase, indicating that acute stress resulted in a dramatic increase in the release of NE from the cerebrospinal fluid and that the NE concentration remained above the basal concentration for 60 minutes. Next, mice were treated with drug administration, 1mg/kg of tranquilization was injected 30 minutes at the beginning of the experiment, tail pressure was applied to the mice for 30 minutes at 90 minutes, and the data of FIGS. 6B and 6E showed 868cm -1 The SERS peak intensity is lower than that of the non-drug control group, which shows that the drug can reduce the basic concentration of the NE in the cerebrospinal fluid and has the relieving effect on the release of the NE concentration caused by acute stress. We explored the effect of different drug concentrations on NE release, FIGS. 6C and 6F show the changes in NE concentration in cerebrospinal fluid after 2.5mg/kg of tranquilization, 868cm -1 The peak intensity at the site is lower than 1mg/kg, which indicates that the higher the drug concentration, the more obvious the inhibition effect on the release amount of NE. And detecting cerebrospinal fluid by using SERS spectrum, judging whether the difference of the content of NE in the cerebrospinal fluid of the mice stimulated by the stress corresponding to the drug treatment exists, and further exploring the relation between the drug and the release amount of NE in the brain.
The protection of the present invention is not limited to the above embodiments. Variations and advantages that would occur to one skilled in the art are included in the invention without departing from the spirit and scope of the inventive concept, and the scope of the invention is defined by the appended claims.
Claims (14)
1. The construction method of the high-reproducibility surface-enhanced Raman spectrum platform is characterized by comprising the following specific steps of:
step (1), designing and synthesizing a rigid probe molecule RP1 which specifically reacts with norepinephrine;
step (2), synthesizing gold nanoparticles as a Raman enhancement substrate;
step (3), modifying the rigid probe molecule RP1 and the rigid probe molecule MBA obtained in the step (1) on the gold nanoparticle obtained in the step (2) through Au-S bond; wherein, the structures of the rigid probe molecules RP1 and MBA are respectively shown as a formula (A) and a formula (B):
and (4) adding a second organic solvent to form a liquid/liquid interface Raman spectrum detection platform.
2. The method of construction according to claim 1, wherein the method of preparation of rigid probe molecule RP1 comprises the steps of: taking p-iodobenzaldehyde and ethynyl thiophene as raw materials, and reacting in a dry first organic solvent under the catalysis of cuprous iodide and bis (triphenylphosphine) palladium dichloride to generate a rigid probe molecule RP1, wherein the reaction process is shown as a reaction formula (I):
3. the construction method according to claim 2, wherein the molar ratio of p-iodobenzaldehyde to ethynylthiophene is (5-6): (5-6); and/or the total addition of the mixed catalyst of the cuprous iodide and the bis (triphenylphosphine) palladium dichloride is 2-3 times of the total amount of the reaction raw materials; and/or the molar ratio of the cuprous iodide to the bis (triphenylphosphine) palladium dichloride in the catalyst is (1-2): 1-2; and/or the first organic solvent comprises one or more of triethylamine, tetrahydrofuran, acetonitrile and acetone; and/or the dosage volume of the first organic solvent is 20-30mL; and/or, the temperature of the reaction is 10-40 ℃; and/or the reaction time is 12-20h.
4. The construction method according to claim 1, wherein in the step (2), the preparation method of the gold nanoparticles comprises the following specific steps: mixing 8-12mM chloroauric acid solution into ultrapure water according to the volume ratio of 1-1.5%, heating and boiling, adding 0.1-2mL of 0.7-2.5%wt sodium citrate solution, and heating and refluxing to generate gold nanoparticles with the particle size of 15-23nm; the specific particle size of the gold nanoparticles is determined by ultraviolet absorption.
5. The construction method according to claim 1, wherein in the step (3), the specific method for modifying the rigid probe molecule on the gold nanoparticle is as follows: dispersing RP1 and MBA solutions with the total concentration of 1-20 mu M and the same concentration into the gold nanoparticle solution obtained in the step (2), and oscillating for 0.5-8 hours for modification; the volume ratio of the probe solution to the gold nanoparticle solution is (1-10): (20-30).
6. The construction method according to claim 1, wherein in the step (4), the second organic solvent is one or more selected from oleic acid, n-butanol, n-hexane, 1, 2-dichloroethane, and acetone; the volume of the second organic solvent is 100-200 mu L; and (3) adding the second organic solvent, and standing for 1-10 minutes.
7. The high reproducibility surface-enhanced raman spectroscopy platform of any one of claims 1-6.
8. Use of the high reproducibility surface-enhanced raman spectroscopy platform of claim 7 for in vivo/in vitro detection of norepinephrine.
9. A method for detecting norepinephrine in vitro by a surface enhanced raman spectroscopy platform, the method comprising:
step a, jointly modifying RP1 rigid probe molecules shown in a formula (A) and MBA rigid probe molecules shown in a formula (B) on the surfaces of gold nanoparticles to form functionalized gold nanoparticles;
step b, reacting the functionalized gold nanoparticles obtained in the step a with norepinephrine with the content to be detected;
and c, measuring the Raman spectrum of the norepinephrine to be measured, and obtaining the content of the norepinephrine to be measured by measuring the linear relation between the peak intensity of the characteristic Raman spectrum and the norepinephrine in advance.
10. The method of claim 9, wherein in step b, the functionalized gold nanoparticles are reacted with norepinephrine for a time of 0.5 to 10 minutes.
11. The method of claim 9, wherein the linear relationship equation is y = 0.036x +0.81, r 2 ＝0.999。
12. A method for detecting norepinephrine in murine cerebrospinal fluid by surface enhanced raman spectroscopy, the method comprising:
step a, jointly modifying RP1 rigid probe molecules shown in a formula (A) and MBA rigid probe molecules shown in a formula (B) on the surfaces of gold nanoparticles to form functionalized gold nanoparticles;
step b, reacting the functionalized gold nanoparticles obtained in the step a with norepinephrine in cerebrospinal fluid;
and c, measuring the Raman spectrum of the norepinephrine in the cerebrospinal fluid, and obtaining the content of the norepinephrine in the cerebrospinal fluid by measuring the linear relation between the peak intensity of the characteristic Raman spectrum and the norepinephrine in advance.
13. The method of claim 12, wherein in step b, the functionalized gold nanoparticles are reacted with norepinephrine for a time of from 0.5 to 10 minutes.
14. The method of claim 12, wherein the linear relationship equation is y = 0.036x +0.81, r 2 ＝0.999。
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