CN113447469A - Method for detecting heavy metal in traditional Chinese medicine based on Raman spectrum combined with molecular probe - Google Patents
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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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Abstract
The invention provides a method for detecting traditional Chinese medicine heavy metal based on Raman spectrum combined molecular probe, which utilizes a chemical reduction method to prepare silver nanoparticles as a substrate material of a surface enhanced Raman spectrum technology, takes a 4, 4-bipyridine probe (Dpy) as a beacon molecule, and indirectly detects mercury ions in a traditional Chinese medicine system by detecting the Raman response difference value of the Dpy based on the principle that the Dpy molecule can be specifically combined with the mercury ions; the method for detecting the heavy metal ions has stronger selectivity and higher sensitivity, and can realize the trace detection of the heavy metals of the traditional Chinese medicine samples on site by adopting the portable Raman spectrometer.
Description
Technical Field
The invention belongs to the field of detection of heavy metals in traditional Chinese medicines, and particularly relates to a method for detecting heavy metals in traditional Chinese medicines based on Raman spectrum combined with a molecular probe. In particular to a method for detecting heavy metal ions in a traditional Chinese medicine injection based on a Surface Enhanced Raman Spectroscopy (SERS) combined with specific probe molecules.
Background
Most heavy metal ions such as lead, cadmium, arsenic, mercury, copper and the like can cause serious environmental pollution and even threaten human health and safety once entering a biological chain without being treated. The mercury ions are the most toxic liquid heavy metal ions, and even if the mercury ions invade the human body in a very low amount, the mercury ions can cause the pathological changes of the lung, the kidney, the liver and the nervous system. At present, the Chinese medicine industry in China develops rapidly, various Chinese medicine preparations enter the market, but the Chinese medicine preparation and Chinese medicine decoction pieces are shown in flight inspection and medicine sampling inspection results of various medicine monitoring departments, are a serious disaster area, and the people in the industry generally think that the exceeding of heavy metals, the exceeding of pesticides and the like become pain points of the industry development and are the road barrows with the largest Chinese medicine product export.
At present, a plurality of methods for detecting heavy metal ions in traditional Chinese medicinal materials comprise an inductively coupled plasma mass spectrometry technology, an atomic absorption spectrometry technology, a chromatography method, an electrochemical method, a fluorescence spectrometry method and the like. Although these techniques are well-developed for detecting heavy metals, most of these methods have the disadvantages of complicated pretreatment operation, toxic reagents, expensive detection instrument, and unsuitability for on-site analysis. The current problem of heavy metal residue is becoming more and more significant, and in order to further ensure the safety of medicinal materials, a method for rapidly detecting heavy metal residue on site needs to be established.
The Raman spectrum and the infrared spectrum are the same as the molecular vibration spectrum and can reflect the characteristic structure of molecules. The Surface-enhanced Raman spectroscopy (SERS) has the advantages of convenient use, high detection speed, no damage, simple sample pretreatment, capability of on-site trace substance detection and the like, and is widely applied to the fields of environmental protection, national defense safety, biomedicine and the like. The SERS detection generally requires gold, silver or other nanomaterials as a substrate to enhance the raman signal of a target molecule, but since heavy metal ions do not have raman response and cannot be directly detected by raman spectroscopy, a probe molecule with a raman signal is required to indirectly detect heavy metal ions in a traditional Chinese medicine. The probes which are usually required not only have good Raman activity, but also can specifically bind with heavy metal ions. However, the traditional Chinese medicine components are very complex and contain active ingredients such as various sugars, amino acids, alkaloids and the like, and the substances bring great fluorescence interference to the detection of heavy metal ions.
At present, no relevant report is found on a method for detecting heavy metal ions in traditional Chinese medicines by combining surface enhanced Raman spectroscopy with a specific molecular probe technology. The invention provides a new idea for safety evaluation of traditional Chinese medicine as a novel detection method for heavy metal ions in traditional Chinese medicine.
Disclosure of Invention
The invention provides a method for detecting heavy metal ions in a traditional Chinese medicine injection based on surface-enhanced Raman spectroscopy combined with a 4, 4-bipyridine (Dpy) probe molecular technology, which improves the sensitivity and reliability of heavy metal ion detection by optimizing the enhancement effect of a Raman substrate and can realize the rapid detection of heavy metals in traditional Chinese medicines on site by using a portable Raman spectrometer.
The method utilizes a chemical reduction method to prepare silver nanoparticles as a substrate material of a Surface Enhanced Raman Spectroscopy (SERS) technology, takes a 4, 4-bipyridine probe (Dpy) as a beacon molecule, and indirectly detects mercury ions in a traditional Chinese medicine system by detecting the Raman response difference value of the Dpy based on the principle that the Dpy molecule can be specifically combined with the mercury ions. The method for detecting the heavy metal ions has stronger selectivity and higher sensitivity, and can realize the trace detection of the heavy metals of the traditional Chinese medicine samples on site by adopting the portable Raman spectrometer.
The technical scheme of the invention is as follows:
a method for detecting heavy metals in traditional Chinese medicine based on Raman spectrum combined with molecular probes comprises the following steps:
(1) preparation of silver nanoparticles (Ag NPs)
Mixing silver nitrate and ultrapure water, heating to reflux (90-100 ℃), adding 1 wt% trisodium citrate solution (the ultrapure water is used as a solvent), stirring for 20min under a reflux state, stopping heating, continuing stirring, and cooling to room temperature (20-30 ℃) to obtain a silver nanoparticle solution;
the mass ratio of the silver nitrate to the ultrapure water to the 1 wt% trisodium citrate solution is 0.018: 100: 2;
the obtained silver nanoparticles are gray green, do not need to be separated from the solution, and are stored in a brown thin-necked bottle at the temperature of 4 ℃ in a dark place;
(2) establishment of a Standard Curve
In Hg (NO)3)2Preparing a series of standard working solutions of 50-1000 ng/mL by using ultrapure water as a solvent as a standard substance;
mixing the silver nanoparticle solution prepared in the step (1) and 10-4M4, 4-bipyridine solution (ultrapure water is used as a solvent), 0.1M NaCl solution (ultrapure water is used as a solvent), and standard working solution in a volume ratio of 1: 1: 0.75: 1, sequentially adding the materials into a cuvette, uniformly mixing the materials at room temperature, and carrying out surface enhanced Raman testing;
with 4, 4-bipyridine at 1296cm-1Taking the change of the signal as a quantitative basis, and recording the Raman response value before adding the mercury ions as I0The Raman response value after adding the mercury ions is recorded as I, the concentration of the mercury ions is taken as the abscissa, I0-I establishing a standard curve for the ordinate;
all samples are detected by a portable Raman spectrometer and are provided with a semiconductor laser (laser wavelength 785nm) with the power of 300mW and a Peltier refrigeration CCD detector at the temperature of-70 ℃; the spectrum scanning range is 250-2350 cm-1The acquisition time of each spectrum is set to 10s, 3 times of accumulation are carried out, and all experiments are carried out in parallel for 3 times; cosmic ray interference is removed by using a self-contained algorithm, an average value is calculated by comparing three experimental data, and then an SERS spectrum is drawn by using Origin2019b software; recording Dpy at 1296cm-1The signal change of (3) as a quantitative basis; record Dpy (1296 cm)-1) And establishing a standard curve for the Raman intensity difference before and after the reaction and the mercury ions with different concentrations to realize the detection of the heavy metal ions in the sample.
(3) Detection of actual samples
According to the method of the step (2), silver nanoparticle solution 10-4M4, 4-bipyridine solution, 0.1M NaCl solution and a sample to be detected are mixed according to the volume ratio of 1: 1: 0.75: 1, sequentially adding the materials into a cuvette, uniformly mixing the materials at room temperature, and carrying out surface enhanced Raman testing;
4, 4-bipyridine at 1296cm-1Substituting the signal change value into the standard curve established in the step (2), and calculating to obtainObtaining the concentration of mercury ions in the sample;
the sample to be tested, such as qingkailing injection, is analyzed without any pretreatment.
The principle of the method of the invention is (taking the detection of mercury ions in the Chinese medicinal injection qingkailing as an example):
silver nanoparticle materials (Ag NPs) are used as an enhancement substrate, a 4, 4-bipyridine (Dpy) probe is used as an inducer for specifically recognizing mercury ions, the Dpy is combined with the Ag NPs through Ag-N bonds, and a coagulant NaCl is used for inducing the aggregation of the Ag NPs, so that the Raman signal of a probe molecule Dpy is significantly enhanced. Since specific binding of Dpy-Hg is stronger than that of Dpy-Ag NPs, Hg is present in the system at this time2+So that the Dpy molecule is peeled off from the surface of the Ag NPs, the Raman intensity of the Ag NPs is reduced, and the rapid and sensitive research of indirectly detecting the heavy metal ion residue based on the change of Raman spectrum signals is realized.
The enhanced substrate silver nanoparticle is prepared by reducing silver nitrate by trisodium citrate, and compared with other common Raman substrates, the enhanced substrate silver nanoparticle has the advantages of simple synthesis process, good stability and stronger Raman signal enhancement effect. The preparation process is optimized, the optimal reaction time is 20min, and the dosage of trisodium citrate is 2 mL.
The Dpy probe molecule has specificity for detecting mercury ions, and N on the pyridine ring of the Dpy probe molecule can be matched with Hg2+Chelation occurs, Dpy molecules are forced to peel off from the surface of the silver nanoparticles, and the content of mercury ions is indirectly detected through the reduction value of a Raman signal. Finally selecting the Dpy concentration to be 10 by investigating the influence of the Dpy concentration, the NaCl concentration and the adding sequence of the Dpy concentration on the SERS signal intensity- 4M, NaCl concentration was 0.1M, the order of addition was AgNPs-Dpy-NaCl.
Compared with the prior art, the method for detecting the heavy metal in the traditional Chinese medicine based on the Raman spectrum and the molecular probe has the following advantages:
(1) the method has high sensitivity and high detection speed, and the detection time of a single sample only needs 30 s.
(2) Compared with the traditional heavy metal detection and analysis method, the method has the advantages of no need of sample pretreatment, no need of expensive instruments, no need of consumption of chemical reagents, no pollution to the environment and avoidance of waste of a large amount of resources and cost.
(3) The portable Raman spectrometer used by the invention has convenient and flexible use and can realize on-site rapid detection.
(4) The detection sensitivity of the method provided by the invention can reach 0.05mg/L at least, and is far lower than the detection limit of mercury ions 0.2mg/kg specified by pharmacopoeia.
Drawings
FIG. 1 is a schematic diagram of the principle of detecting mercury ions by combining Raman spectroscopy with a probe.
FIG. 2 is a representation of the UV and electron microscopy of silver nanoparticles prepared according to the present invention.
FIG. 3 shows the reaction time condition optimization for preparing silver nanoparticles according to the present invention.
Fig. 4 shows the dosage optimization of the reducing agent for preparing silver nanoparticles according to the present invention.
FIG. 5 is an optimized graph of the concentration of the probe of the present invention.
FIG. 6 is a graph of the optimization of the setting accelerator concentration according to the invention.
FIG. 7 is a SERS spectrum for detecting mercury ions according to the present invention.
FIG. 8 is a diagram illustrating the selective examination of the method according to the present invention.
FIG. 9 is a stability test chart of the method of the present invention.
Detailed Description
The invention will be further described in the following by means of specific embodiments with reference to the attached drawings, to which, however, the scope of protection of the invention is not limited.
Example 1
1. Synthesis and characterization of silver nanoparticles
(1) Synthesis and characterization of silver nanoparticles
Firstly, 0.018g of silver nitrate is weighed into a 250mL three-neck flask, then 100mL of ultrapure water is added, heating reflux is carried out under a constant temperature magnetic stirrer, and 2mL of 1% trisodium citrate prepared in advance is quickly added when the solution is boiled. Keeping the solution boiling, heating for 20min, stopping heating, stirring for 30min, cooling to room temperature to obtain grayish green Ag NPs, and storing in brown bottle at 4 deg.C in dark place. The characterization is shown in FIG. 2.
(2) Principle for detecting mercury ions by using silver nanoparticles modified by probe Dpy
The experimental principle of the invention is as follows: two pyridine structures on a 4, 4-bipyridine (Dpy) molecule can be adsorbed on the surface of Ag NPs in the form of Ag-N bonds, so that a strong SERS signal is presented under the action of Raman laser. However, when Hg is present in the system2+When Dpy will preferentially react with Hg2+The complexation, i.e., Dpy, will be stripped from the surface of the Ag NPs, resulting in a reduction in the SERS signal. Thus, the change in SERS signal of Dpy can be used to indirectly detect Hg in a sample solution2+Content, and further realize Hg2+And (4) carrying out quantitative detection. Fig. 1 shows the detection principle.
(3) Silver nanoparticle synthesis condition optimization
The invention optimizes several main conditions in the experiment. First, the effect of reducing agent dosage and synthesis time on SERS signal intensity was investigated. Secondly, the probe Dpy concentration is investigated, and finally the influence of the NaCl concentration and the addition sequence thereof on the SERS signal intensity is studied. The optimization results are shown in fig. 3 to 6.
2. Preparation of the solution
Preparation of a standard solution: accurately suck 0.1mL of 1000 mug/mL Hg2+The standard solution was diluted with ultrapure water in a 10mL volumetric flask and brought to a constant volume of 10mL to give 10. mu.g/mL Hg2+And (4) stock solution. Taking a proper amount of mercury ion stock solution, and diluting the mercury ion stock solution into a standard working solution of 50-1000 ng/mL step by using ultrapure water for later use.
3. Detection of mercury ions by surface enhanced Raman spectroscopy
Respectively taking 200 μ L of Ag NPs, 200 μ L of Dpy, 150 μ L of NaCl and 200 μ L of Hg with different concentrations2+The solutions were sequentially added to a 3mL cuvette, thoroughly mixed at room temperature and subjected to surface enhanced raman testing. Recording Dpy at 1296cm-1The change in signal is used as a basis for quantification. All samples were taken with a portable raman spectrometer equipped with a semiconductor laser at 300mW and a Peltier refrigerated CCD detector at-70 ℃. The spectrum scanning range is 250-2350 cm-1The acquisition time for each spectrum was set to 10s, 3 times cumulatively, and all experiments were performed in parallel 3 times.Cosmic ray interference is removed by a self-contained algorithm, an average value is obtained by comparing three experimental data, and then an SERS spectrum is drawn by Origin2019b software.
4. Method Selective investigation
To evaluate the invention on Hg2+The specificity of detection is to 4 different heavy metal ions (Cu) under the same detection condition2+、Cd2+、Pb2+、As2+) Detection was performed. The metal ion concentrations were all 10 ppm. Using SERS intensity (I/I)0) A comparison is made wherein I0And I is the SERS intensity of the blank sample and the SERS intensity after the heavy metal is added respectively. As shown in FIG. 8, the ratio of these interfering ions detected alone was significantly higher than that of Hg detected alone2+. The results show that Hg is removed2+In addition, the influence of other four heavy metal ions on SERS intensity is small, which shows that the method has little influence on Hg2+Has higher selectivity in detection.
5. Method stability survey
The reproducibility and stability of the substrate are critical to restrict the practical application, in order to prove the enhanced reproducibility and stability of the synthesized Ag NPs to SERS signals, the reproducibility and stability experiments are respectively carried out on the Ag NPs prepared in the same batch and different batches, and FIG. 9(A) shows that the same batch of ten Raman tests are carried out at 1296cm-1Change in raman peak intensity, RSD 3.8%, fig. 9(B) shows Ag NPs versus Dpy (10 days, 15 days, 20 days, 25 days) in storage of Ag NPs for 1 day, 5 days, 10 days, 15 days, 20 days, 25 days-4M) raman signal enhances the change in stability. Therefore, the prepared Ag NPs have better reproducibility.
6. Analysis of actual samples
In order to prove that the invention provides a method for detecting Hg in traditional Chinese medicine based on SERS Raman spectrum combined with functionalized molecular probe Dpy2+The Raman analysis of the injection of qingkailing was carried out under optimum conditions using the standard addition method by adding Hg at concentrations of 200, 500, 600 and 1000ppb2+The recovery rate of the sample with the standard qingkailing is in the range of 95.22-100.07%, and the experimental result shows that the Dpy functionalized Ag NPs have the actual Hg sample2+Potential for detection.
TABLE 1 analysis of actual samples
The above-described embodiments are not limited to the scope of the present invention, and all modifications and variations based on the basic idea of the present invention are included in the scope of the present invention.
Claims (5)
1. A method for detecting heavy metal in traditional Chinese medicine based on Raman spectrum combined with molecular probe is characterized by comprising the following steps:
(1) preparation of silver nanoparticles
Mixing silver nitrate and ultrapure water, heating to reflux, then adding 1 wt% trisodium citrate solution, stirring for 20min in a reflux state, stopping heating, continuing stirring, and cooling to room temperature to obtain a silver nanoparticle solution;
(2) establishment of a Standard Curve
In Hg (NO)3)2Preparing a series of standard working solutions of 50-1000 ng/mL by using ultrapure water as a solvent as a standard substance;
mixing the silver nanoparticle solution prepared in the step (1) and 10-4Sequentially adding the M4, 4-bipyridyl solution, the 0.1M NaCl solution and the standard working solution into a cuvette, uniformly mixing at room temperature, and performing surface enhanced Raman testing;
with 4, 4-bipyridine at 1296cm-1Taking the change of the signal as a quantitative basis, and recording the Raman response value before adding the mercury ions as I0The Raman response value after adding the mercury ions is recorded as I, the concentration of the mercury ions is taken as the abscissa, I0-I establishing a standard curve for the ordinate;
(3) detection of actual samples
According to the method of the step (2), silver nanoparticle solution 10-4Sequentially adding the M4, 4-bipyridyl solution, the 0.1M NaCl solution and a sample to be tested into a cuvette, uniformly mixing at room temperature, and then carrying out surface enhanced Raman testing;
4, 4-bipyridine at 1296cm-1Substituting the signal change value into the standard curve established in the step (2), and calculating to obtain the concentration of the mercury ions in the sample.
2. The method for detecting the heavy metal in the traditional Chinese medicine based on the Raman spectrum combined with the molecular probe as claimed in claim 1, wherein in the step (1), the mass ratio of the silver nitrate to the ultrapure water to the 1 wt% trisodium citrate solution is 0.018: 100: 2.
3. the method for detecting the heavy metal in the traditional Chinese medicine based on the Raman spectrum combined with the molecular probe as claimed in claim 1, wherein in the step (2), the silver nanoparticle solution is 10-4The volume ratio of the M4, 4-bipyridyl solution to the 0.1M NaCl solution to the standard working solution is 1: 1: 0.75: 1.
4. the method for detecting the heavy metal in the traditional Chinese medicine based on the Raman spectrum combined with the molecular probe as claimed in claim 1, wherein in the step (3), the sample to be detected is qingkailing injection.
5. The method for detecting the heavy metal in the traditional Chinese medicine based on the Raman spectrum combined with the molecular probe as claimed in claim 1, wherein in the step (3), the silver nanoparticle solution is 10-4The volume ratio of the M4, 4-bipyridyl solution to the 0.1M NaCl solution to the sample to be detected is 1: 1: 0.75: 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN202110666390.6A CN113447469A (en) | 2021-06-16 | 2021-06-16 | Method for detecting heavy metal in traditional Chinese medicine based on Raman spectrum combined with molecular probe |
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| CN111812069A (en) * | 2020-06-30 | 2020-10-23 | 上海应用技术大学 | Method for rapidly detecting zinc ions in traditional Chinese medicine on site |
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| CN115855928A (en) * | 2023-02-27 | 2023-03-28 | 合肥工业大学 | Mercury ion detection method and kit based on nucleic acid macroarray and bifunctional molecules |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111812069A (en) * | 2020-06-30 | 2020-10-23 | 上海应用技术大学 | Method for rapidly detecting zinc ions in traditional Chinese medicine on site |
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| CN114839177A (en) * | 2022-04-25 | 2022-08-02 | 哈尔滨医科大学 | Method for label-free detection of effective components of Chinese herbal medicine based on surface enhanced Raman spectroscopy and application |
| CN114839177B (en) * | 2022-04-25 | 2023-10-10 | 哈尔滨医科大学 | Method for detecting effective components of Chinese herbal medicine without labels based on surface-enhanced Raman spectroscopy technology and application |
| CN115855928A (en) * | 2023-02-27 | 2023-03-28 | 合肥工业大学 | Mercury ion detection method and kit based on nucleic acid macroarray and bifunctional molecules |
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