CN116297389B - Drug detection method capable of causing driving operation disability in biological sample at accident site - Google Patents
Drug detection method capable of causing driving operation disability in biological sample at accident site Download PDFInfo
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- G—PHYSICS
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- 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
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Abstract
The invention discloses a method for detecting a medicine which can cause driving operation disability in an accident scene biological sample, comprising the following steps: step one, preparing nano gold sol; step two, extracting a biological sample of an accident scene to prepare a sample extracting solution; step three, mixing the sample extracting solution with the nano gold sol and an agglomerating agent to prepare a sample solution to be detected; and step four, detecting whether a medicine which can cause driving operation disability exists in the driver body by using a portable Raman spectrum. The detection method provided by the invention based on the novel SERS detection technology improves the specificity, sensitivity and detection speed of the on-site biological sample detection of accidents, reduces the detection cost, ensures that the detection method of the medicine which can cause the incapacitation of driving operation is quicker and more accurate, is convenient for the rapid detection of biological samples in the field, burst, site and process, can effectively implement supervision and inspection, and ensures the safety and rapid tracking of accident clues.
Description
Technical Field
The present invention relates to a biological sample detection method. More particularly, the present invention relates to a method for detecting a drug in a biological sample at an accident site, which can cause disability in driving operations.
Background
Because driving operators are weak links in a man-machine-environment system, the development of accident medicine investigation has important significance for preventing and reducing errors of human factors and improving safety. The drug detection is a normally open item in accident medicine investigation, because the drug detection positive prompts drivers that a disease condition requiring medication exists before life. Over-the-counter medications are some of the common medications detected in fatal accident medical surveys because of their ready availability, some of which can impair handling ability and cause serious accidents.
The analysis of the important physical evidence at the first time of the accident scene has important significance for judging the accident cause, and the rapid detection method for harmful substances in biological samples such as serum of personnel at the accident scene, for example diphenhydramine and diazepam harmful substances is relatively absent, so that the investigation of related accident clues and physical evidence samples can not be rapidly carried out on the scene. The traditional detection method has the following technical defects of liquid chromatography, colloidal gold immunochromatography, other chemical color development technologies and the like: 1. the liquid chromatography and other methods have high detection accuracy and reliable results, but are complex to operate, high in cost, long in detection period and not suitable for the rapid detection of illegal additives; 2. the colloidal gold immunochromatography technology is simple to operate and high in sensitivity, but is high in single detection cost and poor in stability, can be only used for samples such as urine and the like, and has high limitation; 3. other technical means such as chemical color development technology have low requirements on experimental operators and easy judgment of results, but have simple principle, high detection limit and high false positive rate, and are easy to generate false negative and false positive.
Disclosure of Invention
It is an object of the present invention to solve at least the above problems and to provide at least the advantages to be described later.
The invention also aims to provide a drug detection method capable of causing driving operation disability in the biological sample of the accident scene, which fills the blank of the rapid detection technology of the drug capable of causing driving operation disability in the biological sample of the accident scene (such as air force flight, motor vehicle driving and the like) in wild, burst, complex environment and the like, and overcomes the defects of high detection cost, long detection time and low detection accuracy of the existing laboratory detection technology.
To achieve these objects and other advantages and in accordance with the purpose of the invention, there is provided a method for detecting a drug that can cause disablement of driving operations in a biological sample at an accident site, comprising the steps of:
step one, preparing nano gold sol;
step two, extracting a biological sample of an accident scene to prepare a sample extracting solution;
step three, mixing the sample extracting solution with the nano gold sol and the agglomerating agent according to the volume ratio of 1:3:1 to obtain a sample solution to be detected;
and fourthly, carrying out laser Raman spectrum scanning on the sample liquid to be detected by using a portable Raman spectrometer, collecting a Raman spectrum chart, analyzing characteristic peaks, and judging whether a medicine which can cause driving operation disability exists in the biological sample.
Preferably, in the method for detecting a drug capable of disabling driving operation in the biological sample at the accident site, the step one, the preparation of the nano gold sol specifically includes: placing chloroauric acid aqueous solution with the mass fraction of 0.01% into a round bottom flask, heating to boil under stirring, adding sodium citrate solution with the mass fraction of 1% into the chloroauric acid aqueous solution, stirring and heating until the liquid in the flask turns into mauve, stopping heating, cooling to room temperature, centrifuging at the rotating speed of 10000 revolutions per minute, removing supernatant, adding equal volume of deionized water for resuspension, and storing at normal temperature in a dark place for standby; wherein the volume ratio of chloroauric acid aqueous solution to sodium citrate solution is 100:1.
Preferably, in the method for detecting the medicine which can cause driving operation disability in the biological sample at the accident site, the agglomerating agent is 1mol/L sodium chloride aqueous solution.
In the method for detecting the medicine which can cause driving operation disability in the accident scene biological sample, the accident scene biological sample is extracted to prepare a sample extracting solution, and the sample extracting solution specifically comprises the following components: adding a medium-polarity solvent into a biological sample, carrying out vortex oscillation for 10 seconds at the rotation speed of 2800 rpm, then centrifuging for 10 seconds at the rotation speed of 10000 rpm, removing lower serum, adding an extractant into supernatant, carrying out vortex oscillation for 10 seconds at the rotation speed of 2800 rpm, then centrifuging for 10 seconds at the rotation speed of 10000 rpm, and collecting the supernatant, namely a sample extract.
Preferably, in the method for detecting the medicine which can cause the driving operation disability in the accident scene biological sample, the medicine which can cause the driving operation disability is diphenhydramine, wherein the medium-polarity solvent is ethyl acetate, and the extracting agent is 1mol/L sulfuric acid aqueous solution; observation of Raman Spectroscopy 990cm -1 And 833cm -1 And if the position of the peak is a characteristic peak with the signal to noise ratio greater than 10, the biological sample contains diphenhydramine.
Preferably, in the method for detecting the medicine which can cause the driving operation disability in the accident scene biological sample, the medicine which can cause the driving operation disability is diazepam, wherein the medium-polarity solvent is cyclohexane, and the extracting agent is nitric acid aqueous solution with the concentration of 1 mol/L; observation of Raman Spectroscopy 1141cm -1 And 687cm -1 And if the position shows a characteristic peak with the signal to noise ratio larger than 10, the biological sample contains diazepam.
Preferably, in the method for detecting the medicine which can cause driving operation disability in the biological sample at the accident site, the excitation wavelength of the portable Raman spectrometer is 785nm, and the spectrum scanning range is 300-2800cm -1 The laser power was 200mW and the integral scan time was 5 seconds. The invention at least comprises the following beneficial effects:
the invention fills the blank of the rapid detection technology of the medicines which can cause driving operation disability in biological samples such as serum in the field, burst and complex environment of accident sites, and overcomes the defects of high detection cost, long detection time and low detection accuracy of the existing laboratory detection technology; the invention provides a rapid detection method for a drug which can cause driving operation disability in biological samples such as serum based on a surface enhanced Raman spectroscopy, and the method has the advantages of high sensitivity, high detection speed, low detection cost and the like. The invention develops a detection method based on a new SERS detection technology, improves the specificity, sensitivity and detection speed of the detection of the medicines which can cause driving operation disability in biological samples such as accident site serum and the like, reduces the detection cost, ensures that the detection method of the medicines which can cause driving operation disability is quicker and more accurate, is convenient for the rapid detection of the biological samples such as wild, burst, site, serum and the like in the process, can effectively implement supervision and inspection, and ensures safe and rapid tracking of accident clues.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.
Drawings
FIG. 1 is a Raman spectrum corresponding to diphenhydramine with different concentrations according to the present invention;
fig. 2 is a graph of raman spectra corresponding to different concentrations of diazepam according to the present invention.
Detailed Description
The present invention is described in further detail below with reference to the drawings and examples to enable those skilled in the art to practice the invention by referring to the description.
It will be understood that terms, such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.
The experimental methods described in the following embodiments are conventional methods unless otherwise indicated, and the reagents and materials are commercially available.
In the description of the present invention, the terms "transverse", "longitudinal", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc. indicate orientations or positional relationships based on the orientations or positional relationships shown in the drawings, are merely for convenience in describing the present invention and simplifying the description, and do not indicate or imply that the devices or elements referred to must have a specific orientation, be configured and operated in a specific orientation, and thus are not to be construed as limiting the present invention.
The invention provides a drug detection method capable of causing driving operation disability in an accident scene biological sample, which comprises the following steps of:
step one, preparing nano gold sol;
step two, extracting a biological sample of an accident scene to prepare a sample extracting solution;
step three, mixing the sample extracting solution with the nano gold sol and the agglomerating agent according to the volume ratio of 1:3:1 to obtain a sample solution to be detected; sequentially adding 300 microliters of nano gold sol and 100 microliters of agglomerating agent into 100 microliters of sample extracting solution;
and fourthly, carrying out laser Raman spectrum scanning on the sample liquid to be detected by using a portable Raman spectrometer, collecting a Raman spectrum chart, analyzing characteristic peaks, and judging whether a medicine which can cause driving operation disability exists in the biological sample.
In the technical scheme, the invention provides the method for detecting the medicine which can cause the driving operation disability in the biological sample at the accident site, and the method for rapidly detecting the medicine which can cause the driving operation disability in the biological sample such as serum and the like based on the surface enhanced Raman spectroscopy. The biological sample of the accident site related by the invention is serum.
In another technical scheme, the method for detecting the medicine which can cause driving operation disability in the biological sample at the accident site comprises the following steps: placing chloroauric acid aqueous solution with the mass fraction of 0.01% into a round bottom flask, heating to boil under stirring, adding sodium citrate solution with the mass fraction of 1% into the chloroauric acid aqueous solution, stirring and heating until the liquid in the flask turns into mauve, stopping heating, cooling to room temperature, centrifuging at the rotating speed of 10000 revolutions per minute, removing supernatant, adding equal volume of deionized water for resuspension, and storing at normal temperature in a dark place for standby; wherein the volume ratio of chloroauric acid aqueous solution to sodium citrate solution is 100:1.
The technical scheme can also comprise the following technical details so as to better realize the technical effects:
the nano gold sol is modified, and specifically comprises the following steps:
step a, preparing an agarose water solution with the mass concentration of 5g/L, heating to 80 ℃, dispersing microcrystalline graphene oxide in the agarose water solution, stirring at constant temperature for 40min, immediately refrigerating at 5 ℃ for 30min, and obtaining graphene oxide hydrogel; the mass ratio of the microcrystalline graphene oxide to agarose is 1.5:1;
b, placing graphene oxide hydrogel in deionized water with 10 times of volume, standing for 36 hours, replacing the deionized water every 9 hours, placing the graphene oxide hydrogel in acetone with 5 times of volume, standing for 48 hours, and replacing the acetone every 8 hours to obtain replaced graphene oxide hydrogel; freezing the replaced graphene oxide hydrogel under the condition of-50, and taking out the graphene oxide hydrogel after 24 hours to obtain graphene oxide aerogel;
c, dispersing the nano gold sol in methanol to obtain a methanol solution of the nano gold sol; immersing graphene oxide aerogel in a methanol solution of nano gold sol, removing the methanol solvent after 3 hours, and drying and crushing through supercritical ethanol to obtain the nano gold sol; the mass ratio of the graphene oxide aerogel to the nano gold sol is 1:4;
further, graphene oxide in the above technical scheme is modified, specifically: adding graphene oxide and 3-mercaptopropyl triethoxysilane into ethanol according to the ratio of 25g to 1L, fully and uniformly stirring, heating to 50 ℃, stirring at constant temperature for reaction for 4 hours, washing, and separating to obtain modified graphene oxide.
In the technical scheme, chloroauric acid and sodium citrate are used as raw materials to prepare the nano gold sol, and graphene oxide is used for modifying the nano gold sol, and the nano gold sol is specifically: firstly, preparing microcrystalline graphene oxide into hydrogel, sequentially replacing the graphene oxide hydrogel with water and acetone, and then freezing the replaced graphene oxide to prepare graphene oxide aerogel, so that the porosity on the outer surface of the graphene oxide is greatly improved; then dipping the aerogel of the graphene oxide in a methanol solution of the nano gold sol, so that the nano gold sol can be adsorbed in a pore canal of the aerogel of the graphene oxide, and further the modified nano gold sol with obviously enhanced stretched surface spectral activity is obtained;
the graphene oxide has pi-pi double bond and electrostatic force performance, the detection target can be locked by combining the nano gold sol with the graphene oxide, the graphene oxide enhances a Raman scanning signal by utilizing a chemically enhanced action mechanism, and the effect of doubly enhancing the Raman scanning signal of the analyte is achieved by combining an electromagnetic enhancement mechanism of the nano gold sol; before the graphene oxide is combined with the nano gold sol, the graphene oxide is sequentially subjected to hydrogel and aerogel treatment, so that the pore canal of the graphene oxide is enlarged, the surface of the graphene oxide becomes rough and porous, more nano gold sol can be adsorbed, and the Raman signal can be further enhanced.
In another technical scheme, in the method for detecting the medicine which can cause driving operation disability in the biological sample at the accident site, the agglomerating agent is 1mol/L sodium chloride aqueous solution; the aqueous solution of sodium chloride is stored at normal temperature and in a dark place for standby. The sodium chloride aqueous solution is used as an agglomerating agent, can pertinently promote the adsorption and agglomeration of the medicament and the nano gold sol which can cause the driving operation disability, and generates more hot spots so as to generate stronger Raman spectrum signals.
In another technical scheme, the method for detecting the medicine which can cause driving operation disability in the biological sample of the accident scene comprises the following steps of extracting the biological sample of the accident scene to prepare a sample extracting solution, wherein the sample extracting solution specifically comprises the following steps: adding a medium-polarity solvent into a biological sample, carrying out vortex oscillation for 10 seconds at the rotation speed of 2800 rpm, then centrifuging for 10 seconds at the rotation speed of 10000 rpm, removing lower serum, adding an extractant into supernatant, carrying out vortex oscillation for 10 seconds at the rotation speed of 2800 rpm, then centrifuging for 10 seconds at the rotation speed of 10000 rpm, and collecting the supernatant, namely a sample extract; if the biological sample is solid, pulverizing the solid sample into powder in advance, wherein the dosage ratio of the biological sample, the medium-polarity solvent and the extractant is 1g to 2mL to 0.5mL; the biological sample is liquid, and the volume ratio of the biological sample, the medium-polarity solvent and the extracting agent is 1:1:0.5.
According to the technical scheme, the serum biological sample is firstly subjected to medium-polarity solvent and centrifugal treatment to extract the medicine which can cause driving operation disability in the sample, then the extracting agent is subjected to back extraction under the centrifugal treatment, and then the medicine is mixed with nano gold or nano silver sol and an agglomerating agent for dissolution, and a portable Raman spectrometer is used for detection, so that the detection limit is lower.
In another technical scheme, in the method for detecting the medicine which can cause driving operation disability in the accident scene biological sample, the medicine which can cause driving operation disability is diphenhydramine, wherein the medium-polarity solvent is ethyl acetate, and the extracting agent is 1mol/L sulfuric acid aqueous solution; observation of Raman Spectroscopy 990cm -1 And 833cm -1 And if the position of the peak is a characteristic peak with the signal to noise ratio greater than 10, the biological sample contains diphenhydramine.
Diphenhydramine has the structure of
Diphenhydramine is mainly toxic to inhibit and then excite the central nervous system, and finally produces failure inhibition, and the severity depends on the dosage. Should be immediately sent to hospital for emergency treatment once mistaking or overdosing of the product is found. Manifested by anorexia, nausea, emesis, constipation or diarrhea, thirst, frequent urination or dysuria, hematuria, hearing impairment, blurred vision, movement disorder, superficial respiration, tachycardia, fever and substernal pain; convulsions, coma, heart inhibition and respiratory paralysis may occur in severe cases.
The surface enhanced Raman spectrum of diphenhydramine is shown in figure 1, when the Raman spectrum is 990, 833cm -1 When raman characteristic peaks appear at equal positions, the samples contain diphenhydramine, and as can be seen from fig. 1, the characteristic peaks are enhanced with the increase of the diphenhydramine content in the samples (the peak height is increased); otherwise, the sample is considered to contain no diphenhydramine or less than the detection limit of the method.
In another technical scheme, the biological sample of the accident scene can causeThe medicine detection method for driving operation incapacitation can lead the medicine for driving operation incapacitation to be diazepam, wherein the medium-polarity solvent is cyclohexane, and the extracting agent is nitric acid aqueous solution with the concentration of 1 mol/L; observation of Raman Spectroscopy 1141cm -1 And 687cm -1 And if the position shows a characteristic peak with the signal to noise ratio larger than 10, the biological sample contains diazepam.
Diazepam, also known as diazepam, is an organic compound of the formula C 16 H 13 ClN 2 O, which is classified as a second type of psychotropic drug management.
Clinically used for treatment: (1) anxiety disorders, and various functional neurological disorders. (2) Especially has excellent curative effect on anxiety insomnia. (3) Epilepsy: can be used with other antiepileptic drugs for treating epileptic seizure and seizure, and can be administered by intravenous injection for controlling status epilepticus. (4) Convulsions caused by various reasons, such as eclampsia, tetanus, infantile hyperpyrexia convulsion, etc. (5) Muscle spasms caused by cerebrovascular accidents, spinal cord injury, central myotonia, lumbar muscle strain, endoscopy and the like. (6) Other: migraine, tension headache, singultus, reflex muscle spasm caused by inflammation, panic, alcohol withdrawal syndrome, and can be used for treating familial, senile and essential tremor, and for administration before anesthesia.
On day 10 and 27 of 2017, the world health organization international cancer research institute published a list of carcinogens for preliminary collation reference, diazepam in the 3-class carcinogen list.
The surface enhanced Raman spectrum of diazepam is shown in the following figure 2, when the Raman spectrum is 687cm and 1141cm -1 When raman characteristic peaks appear at equal positions, the characteristic peaks are enhanced with the increase of the content of the diazepam in the sample (the peak height is increased) as the diazepam is contained in the sample as shown in fig. 1; otherwise, the sample is considered to contain no diazepam or less than the detection limit of the method.
In another technical scheme, the method for detecting the medicine in the biological sample at the accident scene, which can cause driving operation disability, has the excitation wavelength of 785nm and the spectral scanning range of 300-2800cm -1 The laser power was 200mW and the integral scan time was 5 seconds.
Example 1 ]
The invention provides a drug detection method capable of causing driving operation disability in an accident scene biological sample, which comprises the following steps of:
step one, preparing nano gold sol, which specifically comprises the following steps: placing chloroauric acid aqueous solution with the mass fraction of 0.01% into a round bottom flask, heating to boil under stirring, adding sodium citrate solution with the mass fraction of 1% into the chloroauric acid aqueous solution, stirring and heating until the liquid in the flask turns into mauve, stopping heating, cooling to room temperature, centrifuging at the rotating speed of 10000 revolutions per minute, removing supernatant, adding equal volume of deionized water for resuspension, and storing at normal temperature in a dark place for standby; wherein, the volume ratio of chloroauric acid aqueous solution to sodium citrate solution is 100:1;
step two, extracting biological samples (serum) of the accident scene to prepare sample extracting solution, which specifically comprises the following steps: adding a medium-polarity solvent ethyl acetate into a biological sample, carrying out vortex oscillation for 10 seconds at the rotation speed of 2800 rpm, then centrifuging for 10 seconds at the rotation speed of 10000 rpm, removing lower serum, adding an extractant into supernatant fluid, carrying out vortex oscillation for 10 seconds at the rotation speed of 2800 rpm, then centrifuging for 10 seconds at the rotation speed of 10000 rpm, and collecting the supernatant fluid, namely a sample extracting solution; the biological sample is solid, the solid sample is crushed into powder in advance, and the powdery biological sample is mixed with the medium-polarity solvent and the extracting agent according to the dosage ratio of 1g to 2mL to 0.5mL.
Step three, mixing the sample extracting solution with the nano gold sol and the agglomerating agent according to the volume ratio of 1:3:1 to obtain a sample solution to be detected; the method comprises the steps of carrying out a first treatment on the surface of the The agglomerating agent is 1mol/L sodium chloride aqueous solution;
step four, carrying out laser Raman spectrum scanning (the excitation wavelength of the portable Raman spectrometer is 785nm, and the spectrum scanning range is 300-2800 cm) on the sample liquid to be detected by using the portable Raman spectrometer -1 Laser power 200mW, integral scanning time 5 seconds), collecting a Raman spectrum, analyzing characteristic peaks, and judging whether a medicine which can cause driving operation disability exists in a biological sample or not: observation of Raman Spectroscopy 990cm -1 And 833cm -1 Whether the position is large in signal-to-noise ratioThe appearance of the characteristic peak at 10 indicates that diphenhydramine is contained in the biological sample.
In the embodiment, the detection limit of the diphenhydramine is 1mg/kg, so that the quick, accurate and micro-trace measurement of the diphenhydramine in the serum biological sample of the accident site is realized.
Example 2 ]
The invention provides a drug detection method capable of causing driving operation disability in an accident scene biological sample, which comprises the following steps of:
step one, preparing nano gold sol, which specifically comprises the following steps: placing chloroauric acid aqueous solution with the mass fraction of 0.01% into a round bottom flask, heating to boil under stirring, adding sodium citrate solution with the mass fraction of 1% into the chloroauric acid aqueous solution, stirring and heating until the liquid in the flask turns into mauve, stopping heating, cooling to room temperature, centrifuging at the rotating speed of 10000 revolutions per minute, removing supernatant, adding equal volume of deionized water for resuspension, and storing at normal temperature in a dark place for standby; wherein, the volume ratio of chloroauric acid aqueous solution to sodium citrate solution is 100:1;
the nano gold sol is modified, and specifically comprises the following steps:
step a, preparing an agarose water solution with the mass concentration of 5g/L, heating to 80 ℃, dispersing microcrystalline graphene oxide in the agarose water solution, stirring at constant temperature for 40min, immediately refrigerating at 5 ℃ for 30min, and obtaining graphene oxide hydrogel; the mass ratio of the microcrystalline graphene oxide to agarose is 1.5:1;
b, placing graphene oxide hydrogel in deionized water with 10 times of volume, standing for 36 hours, replacing the deionized water every 9 hours, placing the graphene oxide hydrogel in acetone with 5 times of volume, standing for 48 hours, and replacing the acetone every 8 hours to obtain replaced graphene oxide hydrogel; freezing the replaced graphene oxide hydrogel under the condition of-50, and taking out the graphene oxide hydrogel after 24 hours to obtain graphene oxide aerogel;
c, dispersing the nano gold sol in methanol to obtain a methanol solution of the nano gold sol; immersing graphene oxide aerogel in a methanol solution of nano gold sol, removing the methanol solvent after 3 hours, and drying and crushing through supercritical ethanol to obtain the nano gold sol; the mass ratio of the graphene oxide aerogel to the nano gold sol is 1:4;
step two, extracting biological samples (serum) of the accident scene to prepare sample extracting solution, which specifically comprises the following steps: adding a medium-polarity solvent ethyl acetate into a biological sample, carrying out vortex oscillation for 10 seconds at the rotation speed of 2800 rpm, then centrifuging for 10 seconds at the rotation speed of 10000 rpm, removing lower serum, adding an extractant into supernatant fluid, carrying out vortex oscillation for 10 seconds at the rotation speed of 2800 rpm, then centrifuging for 10 seconds at the rotation speed of 10000 rpm, and collecting the supernatant fluid, namely a sample extracting solution; the biological sample is liquid, and the volume ratio of the biological sample, the medium-polarity solvent and the extractant is 1mL to 0.5mL.
Step three, mixing the sample extracting solution with the nano gold sol and the agglomerating agent according to the volume ratio of 1:3:1 to obtain a sample solution to be detected; the method comprises the steps of carrying out a first treatment on the surface of the The agglomerating agent is 1mol/L sodium chloride aqueous solution;
step four, carrying out laser Raman spectrum scanning (the excitation wavelength of the portable Raman spectrometer is 785nm, and the spectrum scanning range is 300-2800 cm) on the sample liquid to be detected by using the portable Raman spectrometer -1 Laser power 200mW, integral scanning time 5 seconds), collecting a Raman spectrum, analyzing characteristic peaks, and judging whether a medicine which can cause driving operation disability exists in a biological sample or not: observation of Raman Spectroscopy 990cm -1 And 833cm -1 And if the position of the peak is a characteristic peak with the signal to noise ratio greater than 10, the biological sample contains diphenhydramine.
In the embodiment, the detection limit of the diphenhydramine is 0.2mg/kg, so that the quick, accurate and micro-trace measurement of the diphenhydramine in the serum biological sample at the accident site is realized.
Example 3 ]
The invention provides a drug detection method capable of causing driving operation disability in an accident scene biological sample, which comprises the following steps of:
step one, preparing nano gold sol, which specifically comprises the following steps: placing chloroauric acid aqueous solution with the mass fraction of 0.01% into a round bottom flask, heating to boil under stirring, adding sodium citrate solution with the mass fraction of 1% into the chloroauric acid aqueous solution, stirring and heating until the liquid in the flask turns into mauve, stopping heating, cooling to room temperature, centrifuging at the rotating speed of 10000 revolutions per minute, removing supernatant, adding equal volume of deionized water for resuspension, and storing at normal temperature in a dark place for standby; wherein, the volume ratio of chloroauric acid aqueous solution to sodium citrate solution is 100:1;
the nano gold sol is modified, and specifically comprises the following steps:
step a, preparing an agarose water solution with the mass concentration of 5g/L, heating to 80 ℃, dispersing microcrystalline graphene oxide in the agarose water solution, stirring at constant temperature for 40min, immediately refrigerating at 5 ℃ for 30min, and obtaining graphene oxide hydrogel; the mass ratio of the microcrystalline graphene oxide to agarose is 1.5:1;
b, placing graphene oxide hydrogel in deionized water with 10 times of volume, standing for 36 hours, replacing the deionized water every 9 hours, placing the graphene oxide hydrogel in acetone with 5 times of volume, standing for 48 hours, and replacing the acetone every 8 hours to obtain replaced graphene oxide hydrogel; freezing the replaced graphene oxide hydrogel under the condition of-50, and taking out the graphene oxide hydrogel after 24 hours to obtain graphene oxide aerogel;
c, dispersing the nano gold sol in methanol to obtain a methanol solution of the nano gold sol; immersing graphene oxide aerogel in a methanol solution of nano gold sol, removing the methanol solvent after 3 hours, and drying and crushing through supercritical ethanol to obtain the nano gold sol; the mass ratio of the graphene oxide aerogel to the nano gold sol is 1:4;
further, graphene oxide in the above technical scheme is modified, specifically: adding graphene oxide and 3-mercaptopropyl triethoxysilane into ethanol according to the ratio of 25g to 1L, fully and uniformly stirring, heating to 50 ℃, stirring at constant temperature for reacting for 4 hours, washing, and separating to obtain modified graphene oxide;
step two, extracting biological samples (serum) of the accident scene to prepare sample extracting solution, which specifically comprises the following steps: adding a medium-polarity solvent ethyl acetate into a biological sample, carrying out vortex oscillation for 10 seconds at the rotation speed of 2800 rpm, then centrifuging for 10 seconds at the rotation speed of 10000 rpm, removing lower serum, adding an extractant into clear liquid, carrying out vortex oscillation for 10 seconds at the rotation speed of 2800 rpm, then centrifuging for 10 seconds at the rotation speed of 10000 rpm, and collecting lower clear liquid, namely sample extract; the biological sample is liquid, and the volume ratio of the biological sample, the medium-polarity solvent and the extractant is 1mL to 0.5mL.
Step three, mixing the sample extracting solution with the nano gold sol and the agglomerating agent according to the volume ratio of 1:3:1 to obtain a sample solution to be detected; the method comprises the steps of carrying out a first treatment on the surface of the The agglomerating agent is 1mol/L sodium chloride aqueous solution;
step four, carrying out laser Raman spectrum scanning (the excitation wavelength of the portable Raman spectrometer is 785nm, and the spectrum scanning range is 300-2800 cm) on the sample liquid to be detected by using the portable Raman spectrometer -1 Laser power 200mW, integral scanning time 5 seconds), collecting a Raman spectrum, analyzing characteristic peaks, and judging whether a medicine which can cause driving operation disability exists in a biological sample or not: observation of Raman Spectroscopy 990cm -1 And 833cm -1 And if the position of the peak is a characteristic peak with the signal to noise ratio greater than 10, the biological sample contains diphenhydramine.
In the embodiment, the detection limit of the diphenhydramine is 0.05mg/kg, so that the quick, accurate and micro-trace measurement of the diphenhydramine in the serum biological sample at the accident site is realized.
Example 4 ]
The invention provides a drug detection method capable of causing driving operation disability in an accident scene biological sample, which comprises the following steps of:
step one, preparing nano gold sol, which specifically comprises the following steps: placing chloroauric acid aqueous solution with the mass fraction of 0.01% into a round bottom flask, heating to boil under stirring, adding sodium citrate solution with the mass fraction of 1% into the chloroauric acid aqueous solution, stirring and heating until the liquid in the flask turns into mauve, stopping heating, cooling to room temperature, centrifuging at the rotating speed of 10000 revolutions per minute, removing supernatant, adding equal volume of deionized water for resuspension, and storing at normal temperature in a dark place for standby; wherein, the volume ratio of chloroauric acid aqueous solution to sodium citrate solution is 100:1;
step two, extracting biological samples (serum) of the accident scene to prepare sample extracting solution, which specifically comprises the following steps: adding a medium-polarity solvent cyclohexane into a biological sample, carrying out vortex oscillation for 10 seconds at the rotation speed of 2800 rpm, centrifuging for 10 seconds at 10000 rpm, removing lower serum, adding 1mol/L nitric acid aqueous solution as an extracting agent into supernatant fluid, carrying out vortex oscillation for 10 seconds at the rotation speed of 2800 rpm, centrifuging for 10 seconds at 10000 rpm, and collecting the supernatant fluid, namely a sample extracting solution; the biological sample is solid, the solid sample is crushed into powder in advance, and the powdery biological sample is mixed with the medium-polarity solvent and the extracting agent according to the dosage ratio of 1g to 2mL to 0.5mL.
Step three, mixing the sample extracting solution with the nano gold sol and the agglomerating agent according to the volume ratio of 1:3:1 to obtain a sample solution to be detected; the method comprises the steps of carrying out a first treatment on the surface of the The agglomerating agent is 1mol/L sodium chloride aqueous solution;
step four, carrying out laser Raman spectrum scanning (the excitation wavelength of the portable Raman spectrometer is 785nm, and the spectrum scanning range is 300-2800 cm) on the sample liquid to be detected by using the portable Raman spectrometer -1 Laser power 200mW, integral scanning time 5 seconds), collecting a Raman spectrum, analyzing characteristic peaks, and judging whether a medicine which can cause driving operation disability exists in a biological sample or not: observation of Raman Spectroscopy 1141cm -1 And 687cm -1 And if the position shows a characteristic peak with the signal to noise ratio larger than 10, the biological sample contains diazepam.
In the embodiment, the detection limit of the diazepam is 1mg/kg, so that the quick, accurate and micro-trace measurement of the diazepam in the serum biological sample of the accident site is realized.
Example 5 ]
The invention provides a drug detection method capable of causing driving operation disability in an accident scene biological sample, which comprises the following steps of:
step one, preparing nano gold sol, which specifically comprises the following steps: placing chloroauric acid aqueous solution with the mass fraction of 0.01% into a round bottom flask, heating to boil under stirring, adding sodium citrate solution with the mass fraction of 1% into the chloroauric acid aqueous solution, stirring and heating until the liquid in the flask turns into mauve, stopping heating, cooling to room temperature, centrifuging at the rotating speed of 10000 revolutions per minute, removing supernatant, adding equal volume of deionized water for resuspension, and storing at normal temperature in a dark place for standby; wherein, the volume ratio of chloroauric acid aqueous solution to sodium citrate solution is 100:1;
the nano gold sol is modified, and specifically comprises the following steps:
step a, preparing an agarose water solution with the mass concentration of 5g/L, heating to 80 ℃, dispersing microcrystalline graphene oxide in the agarose water solution, stirring at constant temperature for 40min, immediately refrigerating at 5 ℃ for 30min, and obtaining graphene oxide hydrogel; the mass ratio of the microcrystalline graphene oxide to agarose is 1.5:1;
b, placing graphene oxide hydrogel in deionized water with 10 times of volume, standing for 36 hours, replacing the deionized water every 9 hours, placing the graphene oxide hydrogel in acetone with 5 times of volume, standing for 48 hours, and replacing the acetone every 8 hours to obtain replaced graphene oxide hydrogel; freezing the replaced graphene oxide hydrogel under the condition of-50, and taking out the graphene oxide hydrogel after 24 hours to obtain graphene oxide aerogel;
c, dispersing the nano gold sol in methanol to obtain a methanol solution of the nano gold sol; immersing graphene oxide aerogel in a methanol solution of nano gold sol, removing the methanol solvent after 3 hours, and drying and crushing through supercritical ethanol to obtain the nano gold sol; the mass ratio of the graphene oxide aerogel to the nano gold sol is 1:4;
step two, extracting biological samples (serum) of the accident scene to prepare sample extracting solution, which specifically comprises the following steps: adding a medium-polarity solvent cyclohexane into a biological sample, carrying out vortex oscillation for 10 seconds at the rotation speed of 2800 rpm, centrifuging for 10 seconds at the rotation speed of 10000 rpm, removing lower serum, adding 1mol/L nitric acid aqueous solution as an extracting agent into supernatant, carrying out vortex oscillation for 10 seconds at the rotation speed of 2800 rpm, centrifuging for 10 seconds at the rotation speed of 10000 rpm, and collecting the supernatant, namely a sample extracting solution; the biological sample is liquid, and the volume ratio of the biological sample, the medium-polarity solvent and the extractant is 1mL to 0.5mL.
Step three, mixing the sample extracting solution with the nano gold sol and the agglomerating agent according to the volume ratio of 1:3:1 to obtain a sample solution to be detected; the method comprises the steps of carrying out a first treatment on the surface of the The agglomerating agent is 1mol/L sodium chloride aqueous solution;
step four, carrying out laser Raman spectrum scanning (the excitation wavelength of the portable Raman spectrometer is 785nm, and the spectrum scanning range is 300-2800 cm) on the sample liquid to be detected by using the portable Raman spectrometer -1 Laser power 200mW, integral scanning time 5 seconds), collecting a Raman spectrum, analyzing characteristic peaks, and judging whether a medicine which can cause driving operation disability exists in a biological sample or not: observation of Raman Spectroscopy 1141cm -1 And 687cm -1 And if the position shows a characteristic peak with the signal to noise ratio larger than 10, the biological sample contains diazepam.
In the embodiment, the detection limit of the diazepam is 0.2mg/kg, so that the quick, accurate and micro-trace measurement of the diazepam in the serum biological sample of the accident site is realized.
Example 6 ]
The invention provides a drug detection method capable of causing driving operation disability in an accident scene biological sample, which comprises the following steps of:
step one, preparing nano gold sol, which specifically comprises the following steps: placing chloroauric acid aqueous solution with the mass fraction of 0.01% into a round bottom flask, heating to boil under stirring, adding sodium citrate solution with the mass fraction of 1% into the chloroauric acid aqueous solution, stirring and heating until the liquid in the flask turns into mauve, stopping heating, cooling to room temperature, centrifuging at the rotating speed of 10000 revolutions per minute, removing supernatant, adding equal volume of deionized water for resuspension, and storing at normal temperature in a dark place for standby; wherein, the volume ratio of chloroauric acid aqueous solution to sodium citrate solution is 100:1;
the nano gold sol is modified, and specifically comprises the following steps:
step a, preparing an agarose water solution with the mass concentration of 5g/L, heating to 80 ℃, dispersing microcrystalline graphene oxide in the agarose water solution, stirring at constant temperature for 40min, immediately refrigerating at 5 ℃ for 30min, and obtaining graphene oxide hydrogel; the mass ratio of the microcrystalline graphene oxide to agarose is 1.5:1;
b, placing graphene oxide hydrogel in deionized water with 10 times of volume, standing for 36 hours, replacing the deionized water every 9 hours, placing the graphene oxide hydrogel in acetone with 5 times of volume, standing for 48 hours, and replacing the acetone every 8 hours to obtain replaced graphene oxide hydrogel; freezing the replaced graphene oxide hydrogel under the condition of-50, and taking out the graphene oxide hydrogel after 24 hours to obtain graphene oxide aerogel;
c, dispersing the nano gold sol in methanol to obtain a methanol solution of the nano gold sol; immersing graphene oxide aerogel in a methanol solution of nano gold sol, removing the methanol solvent after 3 hours, and drying and crushing through supercritical ethanol to obtain the nano gold sol; the mass ratio of the graphene oxide aerogel to the nano gold sol is 1:4;
further, graphene oxide in the above technical scheme is modified, specifically: adding graphene oxide and 3-mercaptopropyl triethoxysilane into ethanol according to the ratio of 25g to 1L, fully and uniformly stirring, heating to 50 ℃, stirring at constant temperature for reacting for 4 hours, washing, and separating to obtain modified graphene oxide;
step two, extracting biological samples (serum) of the accident scene to prepare sample extracting solution, which specifically comprises the following steps: adding a medium-polarity solvent cyclohexane into a biological sample, carrying out vortex oscillation for 10 seconds at the rotation speed of 2800 rpm, centrifuging for 10 seconds at the rotation speed of 10000 rpm, removing lower serum, adding 1mol/L nitric acid aqueous solution as an extracting agent into supernatant, carrying out vortex oscillation for 10 seconds at the rotation speed of 2800 rpm, centrifuging for 10 seconds at the rotation speed of 10000 rpm, and collecting the supernatant, namely a sample extracting solution; the biological sample is liquid, and the volume ratio of the biological sample, the medium-polarity solvent and the extractant is 1mL to 0.5mL.
Step three, mixing the sample extracting solution with the nano gold sol and the agglomerating agent according to the volume ratio of 1:3:1 to obtain a sample solution to be detected; the method comprises the steps of carrying out a first treatment on the surface of the The agglomerating agent is 1mol/L sodium chloride aqueous solution;
step four, carrying out laser Raman spectrum scanning (the excitation wavelength of the portable Raman spectrometer is 785nm, and the spectrum scanning range is 300-2800 cm) on the sample liquid to be detected by using the portable Raman spectrometer -1 Laser power 200mW, integral scanning time 5 seconds), collecting a Raman spectrum, analyzing characteristic peaks, and judging whether a medicine which can cause driving operation disability exists in a biological sample or not: observation of Raman Spectroscopy 1141cm -1 And 687cm -1 And if the position shows a characteristic peak with the signal to noise ratio larger than 10, the biological sample contains diazepam.
In the embodiment, the detection limit of the diazepam is 0.05mg/kg, so that the quick, accurate and micro-trace measurement of the diazepam in the serum biological sample of the accident site is realized.
The number of equipment and the scale of processing described herein are intended to simplify the description of the present invention. Applications, modifications and variations of the present invention will be readily apparent to those skilled in the art.
Although embodiments of the present invention have been disclosed above, it is not limited to the details and embodiments shown and described, it is well suited to various fields of use for which the invention would be readily apparent to those skilled in the art, and accordingly, the invention is not limited to the specific details and illustrations shown and described herein, without departing from the general concepts defined in the claims and their equivalents.
Claims (8)
1. The method for detecting the medicine which can cause driving operation disability in the biological sample at the accident site is characterized by comprising the following steps of:
step one, preparing nano gold sol;
step two, extracting a biological sample of an accident scene to prepare a sample extracting solution;
step three, mixing the sample extracting solution with the nano gold sol and the agglomerating agent according to the volume ratio of 1:3:1 to obtain a sample solution to be detected;
step four, carrying out laser Raman spectrum scanning on the sample liquid to be detected by using a portable Raman spectrometer, collecting a Raman spectrum chart, analyzing characteristic peaks, and judging whether a medicine which can cause driving operation disability exists in a biological sample; the medicine is diphenhydramine or diazepam;
wherein the nano gold sol is modified, and specifically comprises the following steps:
step a, preparing an agarose water solution with the mass concentration of 5g/L, heating to 80 ℃, dispersing microcrystalline graphene oxide in the agarose water solution, stirring at constant temperature for 40min, immediately refrigerating at 5 ℃ for 30min, and obtaining graphene oxide hydrogel; the mass ratio of the microcrystalline graphene oxide to agarose is 1.5:1;
b, placing graphene oxide hydrogel in deionized water with 10 times of volume, standing for 36 hours, replacing the deionized water every 9 hours, placing the graphene oxide hydrogel in acetone with 5 times of volume, standing for 48 hours, and replacing the acetone every 8 hours to obtain replaced graphene oxide hydrogel; freezing the replaced graphene oxide hydrogel at the temperature of-50 ℃, and taking out the graphene oxide hydrogel after 24 hours to obtain graphene oxide aerogel;
c, dispersing the nano gold sol in methanol to obtain a methanol solution of the nano gold sol; immersing graphene oxide aerogel in a methanol solution of nano gold sol, removing the methanol solvent after 3 hours, and drying and crushing through supercritical ethanol to obtain the nano gold sol; the mass ratio of the graphene oxide aerogel to the nano gold sol is 1:4.
2. The method for detecting a drug capable of disabling driving operations in a biological sample at an accident site according to claim 1, wherein the preparing of the nano gold sol comprises the following steps: placing chloroauric acid aqueous solution with the mass fraction of 0.01% into a round bottom flask, heating to boil under stirring, adding sodium citrate solution with the mass fraction of 1% into the chloroauric acid aqueous solution, stirring and heating until the liquid in the flask turns into mauve, stopping heating, cooling to room temperature, centrifuging at the rotating speed of 10000 revolutions per minute, removing supernatant, adding equal volume of deionized water for resuspension, and storing at normal temperature in a dark place for standby; wherein the volume ratio of chloroauric acid aqueous solution to sodium citrate solution is 100:1.
3. The method for detecting a drug which can cause driving operation disability in a biological sample at an accident site according to claim 1, wherein the agglomerating agent is 1mol/L sodium chloride aqueous solution.
4. The method for detecting a drug capable of causing driving disability in a biological sample at an accident site according to claim 1, wherein the second step is to extract the biological sample at the accident site to obtain a sample extract, specifically: adding a medium-polarity solvent into a biological sample, carrying out vortex oscillation for 10 seconds at the rotation speed of 2800 rpm, then centrifuging for 10 seconds at the rotation speed of 10000 rpm, removing lower serum, adding an extractant into supernatant, carrying out vortex oscillation for 10 seconds at the rotation speed of 2800 rpm, then centrifuging for 10 seconds at the rotation speed of 10000 rpm, and collecting the supernatant, namely a sample extract.
5. The method for detecting a drug capable of disabling driving operations in a biological sample at an accident site according to claim 4, wherein the drug capable of disabling driving operations is diphenhydramine, wherein the medium-polarity solvent is ethyl acetate, and the extractant is 1mol/L sulfuric acid aqueous solution; observation of Raman Spectroscopy 990cm -1 And 833cm -1 And if the position of the peak is a characteristic peak with the signal to noise ratio greater than 10, the biological sample contains diphenhydramine.
6. The method for detecting a drug capable of disabling driving operations in a biological sample at an accident site according to claim 4, wherein the drug capable of disabling driving operations is diazepam, wherein the medium-polarity solvent is cyclohexane, and the extractant is 1mol/L nitric acid aqueous solution; observation of Raman Spectroscopy 1141cm -1 And 687cm -1 And if the position shows a characteristic peak with the signal to noise ratio larger than 10, the biological sample contains diazepam.
7. A medicament for disabling driving operations in a biological sample at an accident site according to any one of claims 5 to 6The object detection method is characterized in that the excitation wavelength of the portable Raman spectrometer is 785nm, and the spectrum scanning range is 300-2800cm -1 The laser power was 200mW and the integral scan time was 5 seconds.
8. The method for detecting a drug capable of disabling driving operations in a biological sample at an accident site according to claim 1, wherein the microcrystalline graphene oxide is modified, specifically: adding microcrystalline graphene oxide and 3-mercaptopropyl triethoxysilane into ethanol according to the ratio of 25g to 1L, fully and uniformly stirring, heating to 50 ℃, stirring at constant temperature for reaction for 4 hours, washing, and separating to obtain modified graphene oxide.
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