CN112945933A - Method for rapidly detecting triphenylmethane chemicals in living biological tissues in situ - Google Patents
Method for rapidly detecting triphenylmethane chemicals in living biological tissues in situ Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 34
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
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- 239000011259 mixed solution Substances 0.000 claims description 6
- 229910052697 platinum Inorganic materials 0.000 claims description 6
- HKZLPVFGJNLROG-UHFFFAOYSA-M silver monochloride Chemical compound [Cl-].[Ag+] HKZLPVFGJNLROG-UHFFFAOYSA-M 0.000 claims description 6
- -1 triphenylmethane chemical compounds Chemical class 0.000 claims description 6
- 238000002484 cyclic voltammetry Methods 0.000 claims description 5
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Inorganic materials [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 claims description 5
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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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- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
The invention relates to a method for rapidly detecting triphenylmethane chemicals in a living biological tissue in situ, which is characterized in that a gold-silver double-function substrate is inserted into a hollow cavity of a stainless steel hollow tube needle, the stainless steel hollow tube needle with the substrate is pricked into the living biological tissue, and the triphenylmethane chemicals in the living biological tissue are extracted; and taking out the stainless steel hollow tube needle with the substrate, exciting and irradiating the gold-silver bifunctional substrate by using a Raman spectrometer, carrying out Raman spectrum detection, and analyzing and detecting according to Raman characteristic peaks. The detection method can quickly and efficiently enrich the triphenylmethane chemicals in the biological tissue, carries out online detection, does not need to carry out pretreatment on the biological tissue, does not need to collect blood, realizes the integration of extraction and detection, does not need complicated sample pretreatment, and is suitable for on-site quick sampling detection.
Description
The technical field is as follows:
the invention relates to a method for rapidly detecting triphenylmethane chemicals in living biological tissues in situ, belonging to the technical field of analytical chemistry.
Background art:
with the rapid development of national economy, the living standard of urban and rural residents is continuously improved, and the dietary structure of people is deeply changed. The aquatic products have the advantages of low fat, low cholesterol, high protein, rich nutrition, delicious taste and the like, and are one of the foods favored by consumers.
Compared with other meat products, microorganisms are easier to reproduce in aquatic products, various pathogenic bacteria, viruses and parasites can be easily found in intestinal tracts, skins and muscles of the aquatic products, farmers can use pesticides, bactericides and the like in the breeding process in order to prevent and treat diseases of aquatic animals or pollution of the aquatic animals by the microorganisms, and the common bactericides in the breeding process are triphenylmethane chemicals, are dyes and bactericides, can cause cancers and are non-edible substances which are prohibited to be added in foods in China at the Ming Dynasty. The illegal or improper use of these drugs can cause quality safety problems such as drug residues. Therefore, the research on the rapid detection technology for aquatic product safety is accelerated, the aquatic product quality is improved, and a healthy aquatic product quality supervision system is established, so that the method has important practical significance and profound strategic significance for improving the competitiveness of aquatic products in the global market in China.
Triphenylmethane chemicals mainly comprise malachite green, leucomalachite green, and crystal violet; at present, the detection of the triphenylmethane chemical residues is mainly gas chromatography and high performance liquid chromatography, the detection of the triphenylmethane chemical in aquatic products is usually carried out by extracting, enriching or concentrating, and then carrying out chromatographic separation and detection by an electron capture, hydrogen flame, ultraviolet or other detectors. Along with the enhancement of the awareness of food safety, pollution-free green health foods are increasingly popular. Therefore, development of a detection method for rapidly detecting a triphenylmethane chemical is required.
Surface Enhanced Raman Spectroscopy (SERS) is a fast, non-destructive method of detection that can yield molecular features, is not sensitive to water, is simple to pre-treat samples, and has received wide attention in environmental monitoring, food safety, and medical and health applications. The Solid Phase Microextraction (SPME) is a sample pretreatment technology which does not need a solvent and is simple to operate, and is suitable for field sampling in a complex system. The solid phase micro-extraction and the surface enhanced Raman spectroscopy are combined, so that the integration of extraction and detection can be realized, and the detection efficiency is improved. In the SERS detection technology, the selection and preparation of an SERS active substrate become the key for obtaining a high-quality SERS signal.
The existing analysis method can realize the detection of the substance to be detected in the meat tissue extract of the aquatic products. Such as: and (3) measuring malachite green in the meat tissue of the aquatic product by using the surface enhanced Raman spectroscopy of the N-isopropylacrylamide @ gold substrate. And (3) measuring the purine content in the meat tissue of the aquatic product based on the surface enhanced Raman spectroscopy of the gold @ silver substrate. In the case of living organisms, the method is generally performed by collecting blood and then using a liquid chromatography method. The liquid chromatography has the advantage of high accuracy, but the pretreatment of the sample is more complicated and the time consumption is long when the liquid chromatography is used for measurement.
Because the environment in an organism is complex, the to-be-detected object needs to be quickly and efficiently enriched, and the damage to the organism is small, the current detection means is only suitable for detection in a water body or a non-living organism, and the realization of quick in-situ detection in the living organism is still a challenge at present.
The invention content is as follows:
aiming at the defects of the prior art, the invention provides a method for rapidly detecting triphenylmethane chemicals in living biological tissues in situ.
The method can realize convenient and rapid sampling and Raman detection of the triphenylmethane chemical in the living biological tissue, and is very suitable for rapid field analysis of the living body. The gold-silver bifunctional substrate has good uniformity, high stability and stronger Raman enhancement factor.
The invention is realized by the following technical scheme:
a method for rapidly detecting triphenylmethane chemicals in living biological tissues in situ comprises the following steps:
1) preparing a gold-silver bifunctional substrate by using an electrochemical method;
2) inserting the gold-silver double-function substrate into a hollow cavity of a stainless steel hollow tube needle, pricking the stainless steel hollow tube needle with the substrate into a living biological tissue, and extracting triphenylmethane chemicals in the living biological tissue;
3) and taking out the stainless steel hollow tube needle with the substrate, exciting and irradiating the gold-silver bifunctional substrate by using a Raman spectrometer, carrying out Raman spectrum detection, and analyzing and detecting according to Raman characteristic peaks.
Preferably, the gold-silver bifunctional substrate in step 1) is prepared as follows:
a. preparation of porous silver wire
Ultrasonically cleaning a silver wire with the diameter of 0.15mm for 5-10min by using acetone, ethanol and ultrapure water respectively, taking the cleaned silver wire as a working electrode, a silver/silver chloride electrode as a reference electrode, a platinum electrode as a counter electrode to form an electrode system, placing the electrode system in 0.1mol/L hydrochloric acid solution for cyclic voltammetry scanning, etching the silver wire, taking out the silver wire, and washing the silver wire for 3 times by using the ultrapure water to obtain a porous silver wire;
b. preparing a gold-silver bifunctional substrate:
taking porous silver extraction wire as a working electrode, a silver/silver chloride electrode as a reference electrode, a platinum electrode as a counter electrode to form a three-electrode system, and placing the three-electrode system in KNO3With HAuCl4The mixed solution is deposited for 350-450s by using constant voltage, and is washed for 3 times by using ultrapure water after being taken out, so that the gold-silver dual-function substrate is obtained.
Preferably, in step a, the voltage is-0.2V during cyclic voltammetry scanning, the scanning is carried out for 15 circles, and the scanning speed is 10 mV/s.
Preferably, according to the invention, in step b, KNO3With HAuCl4KNO in the mixed solution of3Has a concentration of 0.1mol/L, HAuCl4The concentration of (A) is 1mmol/L, and the constant voltage is-0.4V.
Preferably, in step 2), the stainless steel hollow tube needle with the substrate is inserted into the living body biological tissue by using the tip of the stainless steel hollow tube needle, and then the hollow tube jacket is retracted backwards, so that the gold-silver double-function substrate in the tube is exposed and contacted with the living body biological tissue.
According to the invention, in the step 2), the stainless steel hollow tube needle has an outer diameter of 0.54-0.75mm, an inner diameter of 0.25-0.45mm, and a length less than that of the gold-silver bifunctional substrate, and preferably, the length is 3 cm.
Most preferably, the stainless steel hollow tube needle has an outer diameter of 0.64mm and an inner diameter of 0.33 mm.
Preferably, in step 2) according to the present invention, the triphenylmethane chemical is malachite green, leucomalachite green or crystal violet.
Preferably, according to the invention, in step 2), the extraction time is 8 to 12 min.
Preferably, in step 3), the wavelength of the laser irradiation is 785nm, the laser power is 300mW, and the integration time is 1 s.
Preferably, in step 3), the raman spectrometer is a QE65000 raman spectrometer.
The detection method can detect the triphenylmethane chemicals in aquatic products and can also detect the triphenylmethane chemicals remaining in fruits and vegetables.
The invention has the technical characteristics and advantages that:
1. the detection method can quickly and efficiently enrich the triphenylmethane chemicals in the living body, carries out online detection, does not need to carry out pretreatment on living tissues and collect blood, realizes the integration of extraction and detection, does not need complex sample pretreatment, and is suitable for on-site quick sampling detection.
2. The gold-silver double-function substrate in the detection method has high specificity to the triphenylmethane chemicals in the living body, can quickly and efficiently enrich the triphenylmethane chemicals from the living body organism, and is not influenced by the complex environment in the organism; meanwhile, the preparation method of the substrate is simple, the time consumption is short, the morphology size can be adjusted through the potential and the time, the prepared substrate is pure, and the background signal is low; the method has the advantages of multiple substrate hot spots, high sensitivity, large specific surface area, high extraction efficiency and contribution to adsorption of molecules of an object to be detected; the gold nanoparticles protect silver from being oxidized, and meanwhile, the gold nanoparticles also have the function of enhancing signals.
3. The detection method adopts the stainless steel hollow tube needle to insert the gold-silver bifunctional substrate into the living tissue, can protect the gold-silver bifunctional substrate from being damaged when entering the biological tissue, and has small damage to the living tissue.
4. The detection method can detect the in vivo extraction for 10min, has high extraction efficiency, and is beneficial to rapid and continuous monitoring of the in vivo.
Drawings
FIG. 1 is a schematic diagram of the rapid in situ detection of triphenylmethane chemical compounds in living biological tissues according to the present invention.
Fig. 2 is an SEM image of the gold-silver bifunctional substrate prepared in example 1.
FIG. 3 is a graph showing the linear relationship between the concentrations of the malachite green standard substances and the characteristic peak intensities in experimental example 1.
FIG. 4 is a graph showing the linear relationship between the concentrations of the leuco malachite green standard and the intensities of the characteristic peaks in experimental example 1.
FIG. 5 is a Raman signal spectrum collected from fish of different concentrations in Experimental example 2.
FIG. 6 is a Raman signal spectrum of fish collected at different extraction times in Experimental example 2.
FIG. 7 shows a bubble number of 10 in Experimental example 3-6And (3) Raman signals collected in the fish body in the MG solution of M at intervals.
FIG. 8 shows a bubble number of 10 in Experimental example 3-6The concentration of leucomalachite green corresponding to Raman signals collected at intervals in the body of the fish in the MG solution of M.
FIG. 9 shows a bubble number of 10 in Experimental example 3-5And (3) transferring the fish in the MG solution of M for 10h into clear water, and timing, wherein Raman signals are collected in the fish body at intervals.
FIG. 10 shows a bubble number 10 in Experimental example 3-4And (3) starting timing after 1h of fish in the MG solution of M is transferred into clear water, and collecting Raman signals in the fish body at intervals.
FIG. 11 shows a bubble number of 10 in Experimental example 3-6And (3) collecting Raman signals in vivo at intervals of a fish in CV solution of M.
FIG. 12 shows a bubble number of 10 in Experimental example 3-5And (3) transferring the fish in the CV solution of M into clear water, and then starting timing, wherein Raman signals are collected in the fish body at intervals.
FIG. 13 is a graph of the effect of different voltages on the Raman intensity of a gold-silver bifunctional substrate.
Fig. 14 is a graph of the effect of different deposition times on the raman intensity of a gold-silver bifunctional substrate.
The specific implementation mode is as follows:
in order to make the technical problems, technical solutions and advantages of the present invention more apparent, the following detailed description is given with reference to the accompanying drawings and specific examples, but not limited thereto, and the present invention is not described in detail and is in accordance with the conventional techniques in the art.
Example 1:
preparing a gold-silver bifunctional substrate:
a. preparing the porous silver extraction wire: cutting a silver wire with the diameter of 0.15mm to be 6cm long, respectively cleaning the silver wire by using acetone, ethanol and ultrapure water, taking the cleaned silver wire as a working electrode, taking a silver/silver chloride electrode as a reference electrode, taking a platinum electrode as a counter electrode to form a three-electrode system, placing the three-electrode system in 0.1mol/L hydrochloric acid solution prepared from ultrapure water and concentrated hydrochloric acid, and cleaning the prepared porous silver wire by adopting a cyclic voltammetry, wherein the voltage range is-0.2V-0.2V, the sweeping speed is 10mV/s, and the cycle is 15 circles, so that the prepared porous silver wire is cleaned for 3 times by using the ultrapure water.
b. Preparing a gold-silver bifunctional substrate: containing 0.1mol/L KNO3With 1mmol/L HAuCl4The mixed solution is used as electrolyte, the porous silver extraction wire is used as a working electrode, the silver/silver chloride electrode is used as a reference electrode, the platinum electrode is used as a counter electrode to form a three-electrode system, a constant potential deposition method is utilized, the potential is minus 0.4V, 350-550s are deposited, the mixed solution is taken out and washed for 5min, and the gold-silver dual-function substrate is obtained.
An SEM image of the gold-silver bifunctional substrate is shown in fig. 2. In the preparation process of the gold-silver bifunctional substrate, the voltage and the deposition time affect the substrate Raman signal, and the influence of different voltages and different deposition times on the Raman intensity of the gold-silver bifunctional substrate is shown in FIGS. 13 and 14, as can be seen from FIGS. 13 and 14, the voltage is-0.4V, and the obtained Raman signal of the gold-silver bifunctional substrate is strongest when the deposition time is 500 s.
Example 2:
a method for rapidly detecting triphenylmethane chemicals in living biological tissues in situ comprises the following steps:
1) inserting the gold-silver double-function substrate into a hollow cavity of a stainless steel hollow tube needle, pricking the stainless steel hollow tube needle with the substrate into a fish body by using a sharp head of the stainless steel hollow tube needle, then retreating the outer sleeve of the hollow tube backwards to enable the gold-silver double-function substrate in the tube to be exposed and contacted with the fish, and extracting triphenylmethane chemicals in the fish for 10 min;
2) and taking out the stainless steel hollow tube needle with the substrate, and detecting by using a portable Raman spectrometer, wherein the laser wavelength is 785nm, the laser intensity is 150mW, and the integration time is 1s to obtain a Raman spectrum.
Experimental example 1:
1) preparing malachite green standard substance solutions with different concentrations, immersing the gold-silver bifunctional substrate into the malachite green standard substance solution for solid-phase microextraction, and then performing Raman detection for 3 times in parallel. With the gradual increase of the concentration of malachite green in the malachite green standard solution, 1174cm in the Raman spectrogram-1The peak intensity is gradually enhanced, the intensity at the peak is selected to be combined with a linear curve to realize the quantitative detection of the malachite green, and the linear relation between the concentration of the malachite green standard substance and the characteristic peak intensity is shown in figure 3.
2) Preparing leuco malachite green standard substance solutions with different concentrations, immersing the gold-silver dual-function substrate into the leuco malachite green standard substance solution for solid-phase microextraction, and then performing Raman detection for 3 times in parallel. With the gradual increase of the concentration of the leucomalachite green in the leucomalachite green standard solution, 1375cm in the Raman spectrogram-1The intensity of the nearby peak is gradually enhanced, the peak intensity and a combination linear curve are selected to realize the quantitative detection of the leucomalachite green, and the linear relation between the concentration of the leucomalachite green standard substance and the intensity of the characteristic peak is shown in figure 4.
Experimental example 2:
1. respectively in blank solution, 10-6M、10-5M and 10-4M in MG solution of malachite green soaked fish. After 4 days, the raman signal in the fish in the blank solution was collected and detected by the method of example 2. 10-6M、10-5M and 10-4After death of the fish in M in Malachite Green MG solution, the detection was performed by the method of example 2. The results of the detection are shown in FIG. 5. As can be seen from the view in figure 5,fish soaked in the blank solution had no apparent raman peaks. 10-6M、10-5M and 10-4The in vivo signal of fish soaked in MG solution of M increases with increasing MG solution concentration after death.
2. At 10-5Soaking the fish in the MG solution of M for 5h, then pricking the fish body with a stainless steel hollow tube needle tip, retreating the hollow tube sleeve backwards to expose the gold-silver double-function substrate in the tube to be contacted with the fish body tissue, carrying out solid phase micro-extraction by using the gold-silver double-function substrate, changing the extraction time (respectively 5min, 10min, 15min, 20min and 25min), taking out the gold-silver double-function substrate, and carrying out detection by using a portable Raman spectrometer, wherein the laser wavelength is 785nm, the laser intensity is 150mW, and the integration time is 1s to obtain a Raman spectrogram. The results of the detection are shown in FIG. 6. As can be seen from fig. 6, as the extraction time is extended, the intensity of the collected signal increases, but the rate of increase decreases. The difference between the signal acquired by extraction for 30min and the signal acquired by extraction for 10min is smaller. The signal intensity and the extraction efficiency are comprehensively considered, and the extraction time is optimal within 10 min.
Experimental example 3:
1. soaking fish in the water 10-6M in MG solution, fish were tested at intervals using the method of example 2. The raman signal collected is shown in fig. 7. The collected signal was compared with the signal of the standard solution of Experimental example 1, and leucomalachite green was collected from the fish. From 10-6The MG solution concentration of M is lower and the survival time of the fish is longer. At 10-6Signals were detected by soaking M in MG solution for 23 h. As the soaking time increased, a gradual increase in the signal in the fish was detected. In the solution with lower MG concentration, the leucomalachite green signals in the fish body at different time can be continuously and rapidly detected, and the concentration of the leucomalachite green in the fish body can be estimated according to the linear relation between the Raman signals and the concentration of the standard solution. The corresponding leucomalachite green concentrations in fish bodies for different soaking times are shown in fig. 8.
2. Soaking fish in the water 10-5M in MG solution for 10h, then transferred to clear water to start timing, and the detection is carried out by the method of example 2 at intervals. 10-5M of MThe G solution has high concentration, the survival time of the fish is short, and the fish is firstly soaked in 10 percent-5M in MG solution for 10h, then transferred to clear water, and the metabolism in the fish body is studied. The raman signal collected is shown in fig. 9. And comparing the collected signal with a standard solution signal, wherein the collected signal in the fish body is leucomalachite green. The signal in the fish decreases as the time the fish leave the MG solution increases. After 32h, a significant signal was still detected, but by 56h the signal was not significant. In the solution with higher MG concentration, after the fish is separated from the MG solution environment, the leucomalachite green signals in the fish body at different time can be continuously and rapidly detected, and the metabolic process of the fish can be researched.
3. Soaking fish in the water 10-4M in MG solution for 1h, then transferred to clear water to start timing, and the detection is carried out by the method of example 2 at intervals. 10-4The MG solution concentration of M is high, the survival time of the fish is short, and the fish is firstly soaked in 10-4And (4) carrying out 1h on the MG solution of M, then transferring to clear water, and researching the metabolism condition in the fish body. The raman signal collected is shown in fig. 10. The collected signals are compared with the signals of the standard solution, the signals collected in the fish body are the signals of malachite green when the fish is just put into the clear water, and the signals of the malachite green collected in the fish body after 6 hours are hidden. Therefore, the MG solution is presumed to have high concentration, so that malachite green signals can be collected in the fish body firstly, and are metabolized along with the fish body after being placed in clear water, and the collected malachite green signals are leucomalachite green signals. After 6h the signal in the fish decreased as the fish left the MG solution for longer. The signal can still be detected at 116 h. In the solution with higher MG concentration, after the fish is separated from the MG solution environment, the leucomalachite green signals in the fish body at different time can be continuously and rapidly detected, and the metabolic process of the fish can be researched.
4. Soaking fish in the water 10-6In the CV solution of M, the detection was performed by the method of example 2 at regular intervals. The fish is always soaked in 10-6M in CV solution, the assay was performed by the method of example 2 at intervals. The raman signal collected is shown in fig. 11. 10-6The CV solution concentration of M is lower and the survival time of the fish is longer. At 10-6Signals were detected by soaking M in MG solution for 24 h. With the soaking time being prolongedLong, the signal in the fish body is gradually increased. In a solution with lower CV concentration, the leuco crystal violet signal in the fish body at different time can be continuously and rapidly detected.
5. Soaking fish in the water 10-5M in CV solution for 3h, then transferred to clear water to start timing, and the detection is carried out by the method of example 2 at intervals. 10-5The CV solution of M has a higher concentration and the fish has a shorter survival time, and the fish is firstly soaked in 10-5And (3) allowing the solution of M in CV to stand for 3h, and then transferring the solution to clear water to study the metabolism condition in the fish body. The raman signal collected is shown in fig. 12. The signal in the fish decreases as the time the fish leave the CV solution increases. The signal still detected after 72 h. In the solution with higher CV concentration, after the fish is separated from the CV solution environment, the leuco crystal violet signals in the fish body at different times can be continuously and rapidly detected, and the metabolic process of the fish can be researched.
Claims (10)
1. A method for rapidly detecting triphenylmethane chemicals in living biological tissues in situ comprises the following steps:
1) preparing a gold-silver bifunctional substrate by using an electrochemical method;
2) inserting the gold-silver double-function substrate into a hollow cavity of a stainless steel hollow tube needle, pricking the stainless steel hollow tube needle with the substrate into a living biological tissue, and extracting triphenylmethane chemicals in the living biological tissue;
3) and taking out the stainless steel hollow tube needle with the substrate, exciting and irradiating the gold-silver bifunctional substrate by using a Raman spectrometer, carrying out Raman spectrum detection, and analyzing and detecting according to Raman characteristic peaks.
2. The method for rapid in-situ detection of triphenylmethane chemicals in living biological tissues according to claim 1, wherein the gold-silver bifunctional substrate in step 1) is prepared by the following method:
a. preparation of porous silver wire
Ultrasonically cleaning a silver wire with the diameter of 0.15mm for 5-10min by using acetone, ethanol and ultrapure water respectively, taking the cleaned silver wire as a working electrode, a silver/silver chloride electrode as a reference electrode, a platinum electrode as a counter electrode to form an electrode system, placing the electrode system in 0.1mol/L hydrochloric acid solution for cyclic voltammetry scanning, etching the silver wire, taking out the silver wire, and washing the silver wire for 3 times by using the ultrapure water to obtain a porous silver wire;
b. preparing a gold-silver bifunctional substrate:
taking porous silver extraction wire as a working electrode, a silver/silver chloride electrode as a reference electrode, a platinum electrode as a counter electrode to form a three-electrode system, and placing the three-electrode system in KNO3With HAuCl4The mixed solution is deposited for 350-450s by using constant voltage, and is washed for 3 times by using ultrapure water after being taken out, so that the gold-silver dual-function substrate is obtained.
3. The method for rapid in situ detection of triphenylmethane chemical compounds in living biological tissues according to claim 2, wherein in the step a, the voltage is-0.2V during cyclic voltammetry scanning, the scanning is performed for 15 cycles, and the scanning speed is 10 mV/s.
4. The method for rapid in situ detection of triphenylmethane chemical compounds in living biological tissues as claimed in claim 2, wherein in step b, KNO3With HAuCl4KNO in the mixed solution of3Has a concentration of 0.1mol/L, HAuCl4The concentration of (A) is 1mmol/L, and the constant voltage is-0.4V.
5. The method for rapid in situ detection of triphenylmethane chemical compounds in living body biological tissues as claimed in claim 1, wherein in the step 2), the stainless steel hollow tube needle with the substrate is inserted into the living body biological tissues by using the sharp tip of the stainless steel hollow tube needle, and then the hollow tube sheath is retracted backwards, so that the gold-silver double-function substrate in the tube is exposed and contacted with the living body biological tissues.
6. The method for rapid in situ detection of triphenylmethane chemical compounds in living biological tissues as claimed in claim 1, wherein the stainless steel hollow tube needle has an outer diameter of 0.54-0.75mm and an inner diameter of 0.25-0.45mm, and a length less than that of the gold-silver bifunctional substrate, preferably, a length of 3 cm.
7. The method of claim 1, wherein the stainless steel hollow tube needle has an outer diameter of 0.64mm and an inner diameter of 0.33 mm.
8. The method for rapid in situ detection of triphenylmethane chemicals in living biological tissues as claimed in claim 1, wherein in step 2), the triphenylmethane chemicals are malachite green, leucomalachite green or crystal violet, and the extraction time is 8-12 min.
9. The method for rapid in-situ detection of triphenylmethane chemical compounds in living biological tissues as claimed in claim 1, wherein in step 3), the laser irradiation wavelength is 785nm, the laser power is 300mW, and the integration time is 1 s.
10. The method for rapid in situ detection of triphenylmethane chemicals in living biological tissues as claimed in claim 1, wherein in step 3), the Raman spectrometer is QE65000 Raman spectrometer.
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