CN114839177B - Method for detecting effective components of Chinese herbal medicine without labels based on surface-enhanced Raman spectroscopy technology and application - Google Patents
Method for detecting effective components of Chinese herbal medicine without labels based on surface-enhanced Raman spectroscopy technology and application Download PDFInfo
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- G—PHYSICS
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- 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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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The application discloses a method and application for detecting active ingredients of Chinese herbal medicine without labels based on a surface-enhanced Raman spectrum technology, and relates to a method and application for detecting active ingredients of Chinese herbal medicine without labels based on a surface-enhanced Raman spectrum technology. The application aims to solve the problems of complex and long time consumption of the conventional method for identifying the effective components of the Chinese herbal medicine, and the method is used for reducing silver nitrate by sodium borohydride to generate silver sol, adding halide for incubation to prepare a reinforced substrate, mixing with a sample to be detected, adding an aggregating agent, and carrying out SERS detection. The enhanced substrate successfully detects SERS characteristic peaks of the standard substance of the effective components of the Chinese herbal medicine in methanol solution and water solution, simultaneously successfully detects SERS signals of the rhizome and leaf extraction solution of coptis chinensis, identifies and identifies the effective components of the Chinese herbal medicine through the characteristic peaks of the standard substance, detects the SERS signals of berberine in artificial serum, and has good sensitivity and repeatability. The application is applied to the field of traditional Chinese medicine detection.
Description
Technical Field
The application relates to a method for detecting active ingredients of Chinese herbal medicine without labels based on a surface-enhanced Raman spectrum technology and application thereof.
Background
Common identification methods of the effective components of the Chinese herbal medicine are chromatography and mass spectrometry. The chromatography mainly includes thin layer chromatography, gas chromatography and high performance liquid chromatography. At present, different methods for detecting the effective components of the Chinese herbal medicine have the limitation. Wherein, the thin layer chromatography is not suitable for separating biological macromolecules, and the prior preparation plate making is time-consuming and labor-consuming; the gas chromatography has small adaptation range and is only suitable for components with certain vapor pressure and good thermal stability; high performance liquid chromatography takes a long time, and chromatography mass spectrometry pretreatment is complex and takes a long time. The combination of liquid chromatography and high resolution mass spectrometry is a classical method for analysis of active ingredients of traditional Chinese medicine, but the method has high equipment cost, complex sample preparation and requires qualified and trained personnel. Therefore, a rapid and simple detection method is needed to meet the social demands.
Disclosure of Invention
The application aims to solve the problems of complex and long time consumption of the conventional method for identifying the effective components of the Chinese herbal medicine, and provides a method for detecting the effective components of the Chinese herbal medicine without labels based on a surface-enhanced Raman spectrum technology and application thereof.
The application discloses a method for detecting effective components of Chinese herbal medicine without labels based on a surface-enhanced Raman spectrum technology, which comprises the following steps: 1. preparing a reinforced substrate: a. preparing a sodium borohydride solution with the concentration of 0.5-0.9g/L, then preparing a silver nitrate solution with the concentration of 4-14g/L, adding the silver nitrate solution into the sodium borohydride solution, and stirring to obtain silver sol, wherein the volume ratio of the silver nitrate solution to the sodium borohydride solution is 1 (50-90); b. centrifuging the silver sol, removing the supernatant, and incubating the centrifuged product and halide solution at room temperature for 30-180min to obtain a reinforced substrate; the volume ratio of the centrifuged product to the halide solution was 10: (0.5-3);
2. mixing the enhanced substrate with a sample to be detected, adding an aggregating agent, fully mixing, absorbing the solution by a capillary, and performing SERS detection, wherein the wavelength of a SERS detection laser is 633nm, the scanning time is 10-60s, and the laser energy is 20mW; wherein the volume ratio of the reinforcing substrate, the sample to be tested and the aggregating agent is (1-20): (0.5-5): (0.5-3).
The effective components of the Chinese herbal medicine are the mixture of various active medicines, and the Raman signals of the effective components of the Chinese herbal medicine cannot be accurately distinguished by adopting a traditional substrate. The silver nano-particles after incubation can eliminate the influence of borate interference signals, and the generated SERS signals are all derived from the sample. And a 'hot spot' gap suitable for entering of drug molecules is formed, and a clear SERS signal is obtained. And then introducing an aggregating agent, wherein the aggregating agent can be combined with the residual borate on the surface of the silver sol to form a compound, so that the interference of the borate is eliminated again. Meanwhile, the aggregation of silver nano particles can be guided, and the Raman signal of drug molecules is enhanced. The enhanced substrate developed by the application successfully detects SERS characteristic peaks of standard substances of active ingredients (including alkaloids, flavonoids, lignin, other phenolic acids and the like) of Chinese herbal medicines in methanol solution and aqueous solution, and simultaneously successfully detects SERS signals of alcohol extraction (or water extraction) solutions of roots, stems and leaves of coptis, and the active ingredients of the Chinese herbal medicines are identified and identified through the characteristic peaks of the standard substances. The method further detects SERS signals of berberine (coptis active ingredient) in the fetal bovine serum, which proves that the application has better sensitivity and reproducibility.
One of the main obstacles limiting the development of Chinese herbal medicines at present is that the detection of the effective components of the Chinese herbal medicines has no unified standard, the prepared enhancement substrate can generate a SERS signal of a 'hot spot' enhancement sample to be detected based on a Surface Enhanced Raman Spectrum (SERS) technology, the SERS characteristic fingerprint peak of the effective components of the Chinese herbal medicines is successfully detected, and a unified standard method for detecting the effective components of the Chinese herbal medicines by utilizing the surface enhanced Raman spectrum technology is established and can be subsequently used for constructing a SERS fingerprint spectrum library of the effective components of the Chinese herbal medicines. The method can be completed within 5 minutes from sample preparation, detection and spectrogram analysis, and has the advantages of simple and convenient operation and low detection cost.
Drawings
FIG. 1 is a SERS spectrum of the methanol extraction solution and the aqueous extraction solution of berberine of example 1; wherein a is methanol extraction solution, and b is water extraction solution;
FIG. 2 is a SERS spectrum of the methanol extraction solution and the aqueous extraction solution of palmatine of example 1; wherein a is methanol extraction solution, and b is water extraction solution;
FIG. 3 is a SERS spectrum of the methanol extraction solution and the aqueous extraction solution of coptisine in example 1; wherein a is methanol extraction solution, and b is water extraction solution;
FIG. 4 is a SERS spectrum of methyl coptisine methanol extraction solution and aqueous extraction solution of example 1; wherein a is methanol extraction solution, and b is water extraction solution;
FIG. 5 is a SERS spectrum of the methanol extract solution of isorhamnetin and wogonin of example 1; wherein 1 is isorhamnetin and 2 is wogonin;
FIG. 6 is a SERS spectrum of bergenin methanol solution of example 1;
FIG. 7 is a SERS spectrum of methanol extraction solution and water extraction solution of Coptis root in example 2; wherein a is methanol extraction solution, and b is water extraction solution;
FIG. 8 is a SERS spectrum of methanol extraction solution and water extraction solution of rhizoma Coptidis stem in example 2; wherein a is methanol extraction solution, and b is water extraction solution;
FIG. 9 is a SERS spectrum of methanol extraction solution and water extraction solution of Coptis chinensis leaves in example 2; wherein a is methanol extraction solution, and b is water extraction solution;
FIG. 10 is a SERS spectrum of a different system of example 3; wherein 1 is silver sol reduced by sodium borohydride, 2 is silver sol after incubation of iodide ions, and 3 is silver sol after incubation of coptis root alcohol extraction solution and iodide ions; 4 is coptis root alcohol extraction solution and silver sol introduced with calcium ion aggregation agent;
FIG. 11 is a SERS profile of berberine solids of example 4 and its use in fetal bovine serum; wherein c is a solid SERS spectrum of berberine; d is a berberine embryo bovine serum SERS spectrogram;
FIG. 12 is a SERS spectrum of berberine at different concentrations in fetal bovine serum according to example 4.
Detailed Description
The first embodiment is as follows: the method for detecting the effective components of the Chinese herbal medicine without labels based on the surface-enhanced Raman spectrum technology in the embodiment comprises the following steps: 1. preparing a reinforced substrate: a. preparing a sodium borohydride solution with the concentration of 0.5-0.9g/L, then preparing a silver nitrate solution with the concentration of 4-14g/L, adding the silver nitrate solution into the sodium borohydride solution, and stirring to obtain silver sol, wherein the volume ratio of the silver nitrate solution to the sodium borohydride solution is 1 (50-90); b. centrifuging the silver sol, removing the supernatant, and incubating the centrifuged product and halide solution at room temperature for 30-180min to obtain a reinforced substrate; the volume ratio of the centrifuged product to the halide solution was 10: (0.5-3);
2. mixing the enhanced substrate with a sample to be detected, adding an aggregating agent, fully mixing, absorbing the solution by a capillary, and performing SERS detection, wherein the wavelength of a SERS detection laser is 633nm, the scanning time is 10-60s, and the laser energy is 20mW; wherein the volume ratio of the reinforcing substrate, the sample to be tested and the aggregating agent is (1-20): (0.5-5): (0.5-3).
The second embodiment is as follows: the first difference between this embodiment and the specific embodiment is that: in the first step, the silver nitrate solution is directly poured or dripped into the sodium borohydride solution, and if the silver nitrate solution is dripped into the sodium borohydride solution, the dripping speed is 8-12 drops/second. The other is the same as in the first embodiment.
And a third specific embodiment: this embodiment differs from the first or second embodiment in that: the stirring in the first step is stirring at 500 rpm for 40min. The other is the same as the first or second embodiment.
The specific embodiment IV is as follows: this embodiment differs from one of the first to third embodiments in that: the centrifugation of the silver sol in step one was carried out at 8500 rpm for 35min. The other is the same as in one of the first to third embodiments.
Fifth embodiment: this embodiment differs from one to four embodiments in that: the concentration of the halide solution in the first step is 1mM, wherein the halide is sodium chloride, potassium chloride, sodium bromide, potassium bromide, sodium iodide or potassium iodide. The others are the same as in one to one fourth embodiments.
Specific embodiment six: this embodiment differs from one of the first to fifth embodiments in that: the aggregating agent in the second step is 5-15mM calcium chloride solution or magnesium chloride solution. The others are the same as in one of the first to fifth embodiments.
Seventh embodiment: the method for detecting the effective components of the Chinese herbal medicine without labels based on the surface-enhanced Raman spectrum technology is applied to detecting the effective components of the Chinese herbal medicine without labels.
Eighth embodiment: the present embodiment is different from the seventh embodiment in that: the method for detecting the effective components of the Chinese herbal medicine comprises the following steps: comparing the SERS characteristic peak of the Chinese herbal medicine extraction solution with the SERS characteristic peak of the Chinese herbal medicine active ingredient standard, and identifying the active ingredients of the Chinese herbal medicine extraction solution. The other is the same as in the seventh embodiment.
Detailed description nine: this embodiment differs from the seventh or eighth embodiment in that: the Chinese herbal medicine is rhizoma Coptidis, and the effective components are alkaloids, flavonoids, lignin or phenolic acids. The others are the same as in the seventh or eighth embodiment.
Detailed description ten: this embodiment differs from one of the seventh to ninth embodiments in that: the alkaloids are berberine, palmatine, coptisine or methyl coptisine; the flavonoids are wogonin or isorhamnetin; lignin is bergenin; the phenolic acid is ferulic acid or chlorogenic acid. The others are the same as in one of the seventh to ninth embodiments.
Eleventh embodiment: the method for detecting the effective components of the Chinese herbal medicine based on the surface-enhanced Raman spectroscopy technology without labels is applied to detecting the effective components of the Chinese herbal medicine in the fetal calf serum.
Twelve specific embodiments: this embodiment differs from embodiment eleven in that: the method for detecting the effective components of the Chinese herbal medicine in the fetal calf serum comprises the following steps: detecting the SERS signal of the fetal calf serum containing the active ingredients of the Chinese herbal medicines, comparing the SERS signal with the SERS signal of the pure product of the active ingredients, and identifying and determining the active ingredients in the fetal calf serum through characteristic peaks. The other is the same as in embodiment eleven.
Thirteen specific embodiments: this embodiment differs from the eleventh or twelfth embodiment in that: the Chinese herbal medicine is rhizoma Coptidis, and the effective components are alkaloids, flavonoids, lignin or phenolic acids. The others are the same as in the eleventh or twelfth embodiment.
The beneficial effects of the application are verified by the following tests:
example 1,
A method for detecting the effective components of Chinese herbal medicine without labels based on surface-enhanced Raman spectroscopy technology comprises the following steps:
1. preparing a reinforced substrate: a. 0.25g of sodium borohydride is weighed and dissolved in 350mL of ionized water, and the mixture is stirred in a three-necked flask at a rotating speed of 1650 revolutions per minute for 8 minutes to obtain sodium borohydride solution; 0.045g of silver nitrate is dissolved in 5mL of deionized water to obtain a silver nitrate solution, and then the silver nitrate solution is poured into a sodium borohydride solution and stirred (500 revolutions per minute) in a three-neck flask for 40 minutes to obtain silver sol; b. centrifuging silver sol at 8500 rpm for 35min, removing supernatant, and incubating 10 μl of the centrifugate with 1 μl of 1mM potassium iodide solution at room temperature for 60min to obtain enhanced substrate;
2. mixing the reinforced substrate with a sample to be detected, adding a calcium chloride solution, fully mixing, and performing SERS detection under the detection conditions: the laser wavelength is 633nm, the scanning time is 35s, and the laser energy is 20mW.
The sample to be tested is rhizoma coptidis active ingredient standard substance solution, and the preparation of the rhizoma coptidis active ingredient standard substance solution is carried out: accurately weighing rhizoma Coptidis active ingredient standard substance 5 mg-5 ml, and metering with methanol (or deionized water) to scale mark to obtain 1mg/ml alcohol (or water) solution.
Standard substance: alkaloids (berberine, palmatine, coptisine and methylcoptisine), flavonoids (wogonin and isorhamnetin), lignans (bergenin) and other phenolic acids (ferulic acid and chlorogenic acid).
Sample to be measured:
1. Mu.L of an alcoholic solution (or aqueous solution) of berberine, palmatine and coptisine, respectively, was mixed with 10. Mu.L of the reinforcing substrate, then 1. Mu.L of 10mM calcium chloride solution was added thereto, and the mixture was thoroughly mixed by shaking and was detected by capillary suction.
1. Mu.L of methyl coptisine in alcohol was mixed with 15. Mu.L of the reinforcing substrate, then 1. Mu.L of 10mM calcium chloride solution was added, and the mixture was thoroughly mixed by shaking and detected by capillary suction.
1. Mu.L of aqueous solution of methylcoptisine was mixed with 10. Mu.L of the reinforcing substrate, then 1. Mu.L of 10mM calcium chloride solution was added, and the mixture was thoroughly mixed by shaking and detected by capillary suction.
1. Mu.L of wogonin and isorhamnetin in an alcoholic solution were mixed with 2. Mu.L of the reinforcing substrate, respectively, and then 1. Mu.L of 10mM calcium chloride solution was added thereto, and the mixture was thoroughly mixed by shaking and detected by capillary suction.
mu.L of bergenin in an alcoholic solution was mixed with 1. Mu.L of the reinforcing substrate, then 1. Mu.L of 10mM calcium chloride solution was added, and the mixture was thoroughly mixed by shaking and detected by capillary suction.
The detection results are shown in fig. 1-6, and berberine (characteristic peaks: 727, 1207,1494 and 1630cm -1 ) Coptisine (characteristic peak: 713 773, 1286 and 1468cm -1 ) Palmatine (characteristic peak: 507 734, 1390 and 1424m -1 ) Methyl coptisine (characteristic peak: 1032 1289, 1332 and 1492m -1 ) Wogonin (characteristic peak: 616 1003, 1243 and 1393m -1 ) Isorhamnetin (characteristic peak: 1272 1364, 1499 and 1586m -1 ) Chlorogenic acid (characteristic peak: 972 1262, 1369 and 1615m -1 ) Ferulic acid (characteristic peak: 626 1231 and 1480m -1 ) And bergenin (characteristic peaks: 1028 1348 and 1457m -1 ) Is a characteristic peak of (2). The SERS characteristic peaks of the active ingredient standard substances of alkaloids (berberine, palmatine, coptisine, methyl coptisine and the like), flavonoids (wogonin, isorhamnetin and the like), lignin (bergenin and the like) and other phenolic acids (ferulic acid, chlorogenic acid and the like) in the Chinese herbal medicine coptis in methanol solution and aqueous solution are successfully detected.
Example 2
A method for detecting the effective components of Chinese herbal medicine without labels based on surface-enhanced Raman spectroscopy technology comprises the following steps:
1. preparing a reinforced substrate: a. 0.25g of sodium borohydride is weighed and dissolved in 350mL of ionized water, and the mixture is stirred in a three-necked flask at a rotating speed of 1650 revolutions per minute for 8 minutes to obtain sodium borohydride solution; 0.045g of silver nitrate is dissolved in 5mL of deionized water to obtain a silver nitrate solution, and then the silver nitrate solution is poured into a sodium borohydride solution and stirred (500 revolutions per minute) in a three-neck flask for 40 minutes to obtain silver sol; b. centrifuging silver sol for 35min under the condition of 8500 r/min, removing supernatant, and then taking 10 mu L of centrifugal product and 1 mu L of sodium chloride solution with the concentration of 1mM to incubate for 60min at room temperature to obtain a reinforced substrate;
2. mixing the reinforced substrate with a sample to be detected, adding a calcium chloride solution, fully mixing, and performing SERS detection under the detection conditions: the laser wavelength is 633nm, the scanning time is 35s, and the laser energy is 20mW.
Sample to be measured:
mu.L of the root alcoholic solution was mixed with 10. Mu.L of the reinforcing substrate, then 1. Mu.L of 10mM calcium chloride solution was added, and the mixture was thoroughly mixed by shaking and detected by capillary suction.
1. Mu.L of the stem alcoholic solution was mixed with 15. Mu.L of the reinforcing substrate, then 1. Mu.L of 10mM calcium chloride solution was added, and the mixture was thoroughly mixed by shaking and detected by capillary suction.
mu.L of the leaf alcoholic solution was mixed with 15. Mu.L of the reinforcing substrate, then 1. Mu.L of 10mM calcium chloride solution was added, and the mixture was thoroughly mixed by shaking and detected by capillary suction.
mu.L of the root aqueous solution was mixed with 8. Mu.L of the reinforcing substrate, then 1. Mu.L of 10mM calcium chloride solution was added, and the mixture was thoroughly mixed by shaking and detected by capillary suction.
mu.L of the stem aqueous solution was mixed with 8. Mu.L of the reinforcing substrate, then 1. Mu.L of 10mM calcium chloride solution was added, and the mixture was thoroughly mixed by shaking and detected by capillary suction.
mu.L of the leaf aqueous solution was mixed with 10. Mu.L of the reinforcing substrate, then 1. Mu.L of 10mM calcium chloride solution was added, and the mixture was thoroughly mixed by shaking and detected by capillary suction.
The above alcohol solution is a nail alcohol extraction solution.
The SERS spectrograms of the ethanol extraction and water extraction solutions of the roots, stems and leaves of coptis are shown in figures 7-9, compared with the SERS characteristic peaks of the standard product of the embodiment 1, the SERS characteristic peak positions and the SERS characteristic peak intensities of the ethanol extraction (or water extraction) solutions of the stems and leaves of coptis are obviously different, and the effective components in the solution of the sample to be detected can be simply and rapidly identified. According to the difference in peak position and shared peak intensity of characteristic peaks of active ingredients in the root extract of FIG. 7 (728, 1291, 1494 and 1623cm -1 ) It can be recognized that the extract mainly contains alkaloids such as berberine and coptisine and derivatives thereof. According to the peak position and shared peak intensity differences of the characteristic peaks of the active ingredients in the stem extract of FIG. 8 (728, 1289, 1492, 1574 and 1623cm -1 ) It can be recognized that the extract mainly contains alkaloids such as berberine and coptisine and derivatives thereof. According to FIG. 9, the peak position and the peak intensity difference of the characteristic peaks of the active ingredients in the leaf extract (604, 1194, 1257 and 1514 cm) -1 ) Can identify that the extracting solution mainly contains flavonoid substancesSuch as wogonin.
EXAMPLE 3,
SERS detection of the alcohol extract solution of Coptis root of example 2 with different silver sols
Sodium borohydride reduced silver sol (ag@bo): 0.25g of sodium borohydride is weighed and dissolved in 350mL of ionized water, and the mixture is stirred in a three-necked flask at a rotating speed of 1650 revolutions per minute for 8 minutes to obtain sodium borohydride solution; 0.045g of silver nitrate is dissolved in 5mL of deionized water to obtain a silver nitrate solution, and then the silver nitrate solution is poured into a sodium borohydride solution and stirred (500 revolutions per minute) in a three-neck flask for 40 minutes to obtain silver sol;
silver sol after incubation: centrifuging silver sol at 8500 rpm for 35min, removing supernatant, and incubating 10 μl of the centrifugate with 1 μl of 1mM potassium iodide solution at room temperature for 60min to obtain enhanced substrate;
introducing a calcium ion aggregating agent: 10. Mu.L of the reinforcing substrate was mixed with 1. Mu.L of the sample to be tested, and then 1. Mu.L of 10mM calcium chloride solution was added thereto for thorough mixing.
SERS detection conditions: the laser wavelength is 633nm, the scanning time is 35s, and the laser energy is 20mW.
The SERS detection is carried out after different silver sols, coptis root alcohol extraction solutions and different silver sols are mixed respectively, and the result is shown in fig. 10, and the SERS signal (curve 1 in the figure) of stronger borate can be observed by utilizing the silver sol (Ag@BO) reduced by sodium borohydride in the traditional method, so that the obtained signal can not be distinguished from a sample or impurities by utilizing the Ag@BO enhanced substrate. It is noted that the active ingredients of the herbal medicine are themselves a mixture of various active drugs, which means that ag@bo cannot be used to accurately distinguish the raman signal of the active ingredients in the herbal medicine. In the embodiment, iodide ions are introduced to modify Ag@BO, the obtained sol system (Ag@I) almost has no Raman signal (curve 2 in the figure), and a weak but clear SERS signal (curve 3 in the figure) can be observed when the sol system is added into the coptis root alcohol extraction solution. The silver nano-particles after incubation are explained to eliminate the influence of borate interference signals, and the generated SERS signals are all derived from the sample. And a 'hot spot' gap suitable for entering of drug molecules is formed, and a clear SERS signal is obtained. After the calcium ion aggregating agent is introduced, calcium ions can be combined with the residual borate on the surface of the silver sol to form a compound, and the interference of the borate is eliminated again. Meanwhile, calcium ions can guide silver nano particles to aggregate, so that the Raman signal of the drug molecules is enhanced (curve 4 in the figure).
EXAMPLE 4,
A method for rapidly detecting effective components of Chinese herbal medicine without labels based on a surface-enhanced Raman spectrum technology comprises the following steps: 1. preparing a reinforced substrate: a. 0.25g of sodium borohydride is weighed and dissolved in 350mL of ionized water, and the mixture is stirred in a three-necked flask at a rotating speed of 1650 revolutions per minute for 8 minutes to obtain sodium borohydride solution; 0.045g of silver nitrate is dissolved in 5mL of deionized water to obtain a silver nitrate solution, and then the silver nitrate solution is poured into a sodium borohydride solution and stirred (500 revolutions per minute) in a three-neck flask for 40 minutes to obtain silver sol; b. centrifuging silver sol at 8500 rpm for 35min, removing supernatant, and incubating 10 μl of the centrifugate with 1 μl of 1mM potassium iodide solution at room temperature for 60min to obtain enhanced substrate;
2. mixing 10 mu L of the enhanced substrate with 1 mu L of a sample to be detected, adding 1 mu L of 10mM calcium chloride solution, fully mixing, and carrying out SERS detection under the detection conditions: the laser wavelength is 633nm, the scanning time is 35s, and the laser energy is 20mW.
Sample to be measured: berberine is dissolved in fetal calf serum to prepare mixed solutions with different concentrations; 100nM, 1. Mu.M, 10. Mu.M, 100. Mu.M, 1mM, respectively.
SERS detection of berberine solid and berberine in fetal bovine serum with the enhanced substrate of this example is shown in FIG. 11, in which the berberine solid is at 732, 1203, 1277, 1398, 1517 and 1624cm -1 Distinct SERS characteristic peaks appear, the Raman peak signal of berberine in the fetal bovine serum is obviously enhanced, but partial peaks are slightly offset. The shift in peak position is due to the high polarity of serum, resulting in interconversion of berberine isomers in solution and destruction of intramolecular hydrogen bonds. 531 and 728cm -1 The peak at which is a distorted isoquinoline ring of 728cm -1 The peak at which is significantly enhanced over the peak of the solid. 1273-1630cm -1 Information within rangeThe number peaks are mainly due to vibrations of the aromatic ring. Through 728, 1273-1630cm -1 The characteristic peaks of (2) can identify and determine berberine in serum. In FIG. 12, the SERS spectra of berberine fetal bovine serum were detected at different concentrations, and characteristic peak signals were clearly observed even at low concentrations of 100 nM. Therefore, the embodiment successfully detects the signal of the effective component (berberine) of coptis in the fetal bovine serum and determines the lowest concentration of the berberine.
Claims (6)
1. A method for detecting effective components of Chinese herbal medicine without labels based on a surface-enhanced Raman spectrum technology is characterized by comprising the following steps: 1. preparing a reinforced substrate: a. 0.25g of sodium borohydride is weighed and dissolved in 350mL ion water, and the mixture is stirred in a three-neck flask for 8 minutes at a rotating speed of 1650 revolutions per minute to obtain sodium borohydride solution; dissolving 0.045g of silver nitrate in 5mL of deionized water to obtain a silver nitrate solution, and then pouring the silver nitrate solution into a sodium borohydride solution and stirring in a three-neck flask for 40 minutes to obtain silver sol; b. centrifuging silver sol at 8500 rpm for 35min, removing supernatant, and incubating the centrifugated product and halide solution at room temperature for 30-180min to obtain enhanced substrate; the volume ratio of the centrifuged product to the halide solution was 10: (0.5-3);
mixing the reinforced substrate with a sample to be detected, adding an aggregating agent, fully mixing, sucking the solution by a capillary, and performing SERS detection, wherein the wavelength of a SERS detection laser is 633-nm, the scanning time is 10-60s, and the laser energy is 20mW; wherein the volume ratio of the reinforcing substrate, the sample to be tested and the aggregating agent is (1-20): (0.5-5): (0.5-3); wherein the aggregating agent is 5-15mM calcium chloride solution or magnesium chloride solution, and the sample to be tested is alcohol extract solution, water extract solution of Chinese herbal medicine or fetal calf serum containing effective components of Chinese herbal medicine;
the method for detecting the effective components of the Chinese herbal medicine based on the surface-enhanced Raman spectrum technology without labels is applied to detecting the effective components of the Chinese herbal medicine or applied to detecting the effective components of the Chinese herbal medicine in the fetal bovine serum.
2. The method for detecting the active ingredients of the Chinese herbal medicine without labels based on the surface-enhanced Raman spectroscopy technology according to claim 1, wherein the concentration of the halide solution in the first step is 1mM, and the halide is sodium chloride, potassium chloride, sodium bromide, potassium bromide, sodium iodide or potassium iodide.
3. The method for detecting the effective components of the Chinese herbal medicine based on the surface-enhanced Raman spectroscopy without labels according to claim 1, wherein the Chinese herbal medicine is coptis chinensis, and the effective components are alkaloids, flavonoids, lignin or phenolic acids.
4. The method for detecting the effective components of the Chinese herbal medicine without labels based on the surface-enhanced Raman spectroscopy technology according to claim 3, wherein the alkaloids are berberine, palmatine, coptisine or methylcoptisine; the flavonoids are wogonin or isorhamnetin; lignin is bergenin; the phenolic acid is ferulic acid or chlorogenic acid.
5. The method for detecting the effective components of the Chinese herbal medicine without labels based on the surface-enhanced Raman spectroscopy technology as claimed in claim 1, wherein the method for detecting the effective components of the Chinese herbal medicine comprises the following steps: comparing the detected SERS characteristic peak with the SERS characteristic peak of the traditional Chinese medicine active ingredient standard substance, and identifying the active ingredients of the Chinese herbal medicine.
6. The method for detecting the effective components of the Chinese herbal medicine based on the surface-enhanced Raman spectroscopy without labels according to claim 1, wherein the method for detecting the effective components of the Chinese herbal medicine in the fetal bovine serum comprises the following steps: detecting the SERS signal of the fetal calf serum containing the active ingredients of the Chinese herbal medicines, comparing the SERS signal with the SERS signal of the pure product of the active ingredients, and identifying and determining the active ingredients in the fetal calf serum through characteristic peaks.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101339132A (en) * | 2008-08-27 | 2009-01-07 | 福建师范大学 | Method for detecting traditional Chinese medicine soup ingredient using surface reinforced Raman spectrum |
| CN101811193A (en) * | 2010-04-06 | 2010-08-25 | 浙江大学 | Method for preparing silver nano sheet self-assembling material |
| CN102398041A (en) * | 2010-09-15 | 2012-04-04 | 吉林师范大学 | Method for assembling silver nanoparticle film by using gas-liquid interface |
| CN108579696A (en) * | 2018-03-12 | 2018-09-28 | 中国人民解放军第二军医大学 | A kind of theophylline molecular engram material and its preparation method and application of silver nano-grain doping |
| CN113447469A (en) * | 2021-06-16 | 2021-09-28 | 浙江工业大学 | Method for detecting heavy metal in traditional Chinese medicine based on Raman spectrum combined with molecular probe |
| CN113640275A (en) * | 2021-08-02 | 2021-11-12 | 中国科学院南海海洋研究所 | Organic matter Raman detection method based on surface enhanced Raman spectrum |
| CN113702350A (en) * | 2021-07-13 | 2021-11-26 | 哈尔滨医科大学 | Novel coronavirus detection method and kit based on surface enhanced Raman spectroscopy |
-
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- 2022-04-25 CN CN202210440615.0A patent/CN114839177B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101339132A (en) * | 2008-08-27 | 2009-01-07 | 福建师范大学 | Method for detecting traditional Chinese medicine soup ingredient using surface reinforced Raman spectrum |
| CN101811193A (en) * | 2010-04-06 | 2010-08-25 | 浙江大学 | Method for preparing silver nano sheet self-assembling material |
| CN102398041A (en) * | 2010-09-15 | 2012-04-04 | 吉林师范大学 | Method for assembling silver nanoparticle film by using gas-liquid interface |
| CN108579696A (en) * | 2018-03-12 | 2018-09-28 | 中国人民解放军第二军医大学 | A kind of theophylline molecular engram material and its preparation method and application of silver nano-grain doping |
| CN113447469A (en) * | 2021-06-16 | 2021-09-28 | 浙江工业大学 | Method for detecting heavy metal in traditional Chinese medicine based on Raman spectrum combined with molecular probe |
| CN113702350A (en) * | 2021-07-13 | 2021-11-26 | 哈尔滨医科大学 | Novel coronavirus detection method and kit based on surface enhanced Raman spectroscopy |
| CN113640275A (en) * | 2021-08-02 | 2021-11-12 | 中国科学院南海海洋研究所 | Organic matter Raman detection method based on surface enhanced Raman spectrum |
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