CN113588616B - Methamphetamine enrichment and rapid detection method - Google Patents

Methamphetamine enrichment and rapid detection method Download PDF

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CN113588616B
CN113588616B CN202110868837.8A CN202110868837A CN113588616B CN 113588616 B CN113588616 B CN 113588616B CN 202110868837 A CN202110868837 A CN 202110868837A CN 113588616 B CN113588616 B CN 113588616B
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methamphetamine
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CN113588616A (en
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狄斌
闫昆
胡驰
藿湘雲
戚立凯
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China Pharmaceutical University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
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    • 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
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N21/643—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" non-biological material
    • 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
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6432—Quenching

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Abstract

The invention discloses a method for enriching and rapidly detecting methamphetamine, which comprises the steps of adding a supermolecule fluorescent probe into an enriched methamphetamine sample, uniformly mixing, and measuring the fluorescence intensity. Supermolecule fluorescent probes are constructed based on self-assembly of cucurbituril host Q8 and acridine dye objects, and methamphetamine can compete with the dye objects for host molecules to cause fluorescence change, so that rapid detection of the methamphetamine is realized. The method skillfully combines enrichment and detection, and obviously improves the sensitivity and specificity of the detection method.

Description

Methamphetamine enrichment and rapid detection method
Technical Field
The invention relates to a compound analysis and detection method, in particular to a methamphetamine enrichment and rapid detection method.
Background
The chemical name of the methamphetamine is methamphetamine, the methamphetamine is pure white crystal, the methamphetamine has very high proportion in drug abuse, the methamphetamine has very strong toxicity, irreversible damage can be caused to a nervous system after long-term intake of the methamphetamine, and a user can easily rely on the methamphetamine to cause addiction to the methamphetamine.
At present, sewage epidemiology is one of the most accepted methods at home and abroad for objectively monitoring and evaluating illegal drug abuse conditions in areas. Sewage poisoning products and metabolites thereof have very low concentration and complex background matrix environment. At present, the detection often depends on the determination of drug components in sewage by utilizing LC-MS/MS after solid phase extraction. However, the traditional solid phase extraction method has poor selectivity to the analyte, and the operation steps are complicated, and are time-consuming and labor-consuming. And mass spectrometry instrumentation is expensive, requiring the equipment to be equipped with specialized technicians.
In summary, the present art lacks an analysis method of a fluorescent supramolecular sensor capable of rapidly detecting the content of methamphetamine in a sewage sample with high sensitivity, so that developing a combination of magnetic solid phase extraction and a supramolecular fluorescent probe for detecting methamphetamine is an urgent problem to be solved.
Disclosure of Invention
The invention aims to provide a fluorescent detection method with high sensitivity and good specificity based on magnetic solid phase extraction combined with a supermolecule fluorescent probe, which is used for rapidly detecting methamphetamine.
The invention provides a method for enriching and rapidly detecting methamphetamine, which comprises the steps of adding a supermolecule fluorescent probe into an enriched methamphetamine sample, uniformly mixing, measuring the fluorescence intensity, taking the methamphetamine concentration as an abscissa and the fluorescence intensity as an ordinate, and drawing a standard curve.
Further, the supermolecule fluorescent probe is obtained by self-assembly after mixing a supermolecule cucurbituril host Q8 and an acridine dye object.
Further, fe is adopted 3 O 4 The magnetic nanomaterial @ PDA @ C18 enriches the methamphetamine.
Further, the Fe 3 O 4 The core of the @ PDA @ C18 magnetic nano material is Fe 3 O 4 The magnetic particles are coated with a polydopamine layer on the outer layer and a hydrophobic group C18 on the outermost layer.
Further, the Fe 3 O 4 The preparation method of the magnetic particles comprises the following steps: feCl is taken 3 ·6H 2 O and anhydrous sodium acetate are respectively dissolved in glycol, evenly mixed, reacted at high temperature and high pressure, washed and dried after the reaction is finished.
Further, fe 3 O 4 The preparation method of the @ PDA@C18 magnetic nanomaterial comprises the following steps: fe (Fe) 3 O 4 Placing magnetic particles in weak base Tris buffer solution, then adding a proper amount of dopamine hydrochloride, stirring, finishing the reaction, cleaning and drying for later use; taking Fe 3 O 4 Putting PDA into toluene, adding chloro (dimethyl) octadecylsilane, mechanically stirring at room temperature, cleaning after the reaction is finished, and drying in vacuum.
Further, the acridine dye guest comprises acridine orange, acridine yellow, proflavine and neutral red.
First of all Fe is synthesized 3 O 4 The enrichment condition is further optimized by the @ PDA @ C18 magnetic nano material. And then uniformly mixing the supermolecular cucurbituril host Q8 and the acridine dye guest preferably according to the molar concentration ratio of 1:2, and self-assembling to obtain the supermolecular fluorescent probe. And uniformly mixing the enriched methamphetamine sample with a supermolecule fluorescent probe, measuring the fluorescence intensity, and drawing a standard curve by taking the methamphetamine concentration as an abscissa and the fluorescence intensity as an ordinate.
The magnetic nano material is Fe 3 O 4 PDA@C18 with methamphetamine enrichment capability, and magnetic Fe in the core 3 O 4 The magnetic particles, the polydopamine layer coated on the outer layer has a great number of catechol structures, and provides rich pi-pi structures. In addition, the outermost layer is wrapped with a hydrophobic group C18, and finally the amphiphilic magnetic nano material Fe is obtained 3 O 4 @PDA@C18。
Said preparation of Fe 3 O 4 A method of magnetic particles, the preferred steps comprising: taking a proper amount of FeCl 3 ·6H 2 O and anhydrous sodium acetate are respectively dissolved in glycol, stirred and mixed uniformly, placed in a polytetrafluoroethylene high-pressure reaction kettle for reaction for 12 hours at 200 ℃, and cleaned and dried for standby after the reaction is finished.
Said preparation of Fe 3 O 4 The method @ PDA @ C18, preferably comprises the steps of: taking a proper amount of Fe 3 O 4 And (3) placing the magnetic particles in a weak base Tris buffer solution, then adding a proper amount of dopamine hydrochloride, mechanically stirring for 12 hours, and after the reaction is finished, cleaning and drying for later use. Then taking a proper amount of Fe 3 O 4 Putting PDA into toluene, adding chloro (dimethyl) octadecylsilane, mechanically stirring at room temperature for 12h, and cleaning and vacuum drying for later use.
Preferably the enrichment conditions are as follows: taking 100mL of a sample to be tested with pH of 9.5, putting the sample into a plastic bottle, and adding Fe 3 O 4 10mg of magnetic material @ PDA @ C18, shaking at room temperature for 15min with ultrasound to disperse the material uniformly, then magnetically separating the solution, adding 1500. Mu.L volume of elution solution (5% formic acid), transferring to a 1.5mL EP tube, vortexing for 5min, and magnetically separating to obtain an enriched sample solution.
The supramolecular cucurbituril Q8 can cause fluorescence quenching of the dye after being combined with an acridine dye object, the fluorescence reaches the minimum value when the optimal molar ratio is 1:2, and after methamphetamine which is an object to be detected is added, the dye fluorescence value is recovered because the object competes with the dye for combining with the host Q8.
The host cucurbituril Q8 and the acridine dye guest have the following structural formula:
Figure 1
preferably, the volume of methamphetamine after enrichment is 350. Mu.L, the fluorescent probe is 350. Mu.L, mixed, vortexed for 2-3 seconds, added to a 96-well plate, three parallel, and 200. Mu.L per well.
Preferably, the fluorescence intensity is a fluorescence signal value with an excitation wavelength of 430nm and an emission wavelength of 520 nm.
The beneficial effects are that: compared with the prior art, the invention has the following advantages:
the method has high sensitivity, good specificity and simple method, and can be used for quantitative detection of methamphetamine in sewage samples.
Drawings
FIG. 1 is a schematic diagram of the detection principle of the magnetic solid phase extraction combined with supermolecule fluorescent probe in the invention;
fig. 2 is a TEM image of a magnetic material: a is Fe 3 O 4 @PDA, B is Fe 3 O 4 @PDA@C18；
FIG. 3 is a graph of the standard curve established for the method at different methamphetamine concentrations;
FIG. 4 is a fluorescent chart of the detection method under other interfering substances.
Detailed Description
The detection schematic diagram of the invention is shown in figure 1, magnetic beads and a sample containing methamphetamine are incubated, the supernatant is removed after 15min of magnetic separation, 5% formic acid is added, vortex is carried out for 5min, eluent obtained by magnetic separation is mixed with a supermolecule fluorescent probe, and the fluorescence intensity is measured. The content of methamphetamine in the sample is in linear relation with the fluorescence intensity, so that the methamphetamine in the sewage is detected.
Method for detecting methamphetamine in sewage sample based on magnetic solid phase extraction combined with supermolecule fluorescent probe in embodiment
1. Magnetic material Fe 3 O 4 Preparation of @ PDA @ C18
150mL of ethylene glycol was measured and placed in a 500mL round bottom flask, and 8.1g of ethylene glycol was addedFeCl 3 ·6H 2 O, magnetic stirring to dissolve the solution, and obtaining yellow clear solution. Another 150mL of ethylene glycol was measured and placed in a 250mL round bottom flask, 21.6g of anhydrous sodium acetate was added and dissolved by magnetic stirring to give a clear and transparent solution. Dropwise adding anhydrous sodium acetate solution into FeCl under stirring 3 Obtaining a brown turbid liquid in the solution, transferring the turbid liquid into a polytetrafluoroethylene high-pressure reaction kettle, and reacting for 12 hours at 200 ℃ to obtain Fe with the particle size of about 200-300nm 3 O 4 Nano ions. Repeatedly washing with pure water and ethanol for three times, and vacuum drying at 60deg.C for 24 hr. The Fe prepared is then taken 3 O 4 The nanometer particle 800mg is placed in 1000ml of 10mM Tris buffer solution with pH of 8.5, ultrasonic treatment is carried out to ensure that the nanometer particle is evenly dispersed, then 800mg of dopamine hydrochloride is added, and the mixture is mechanically stirred for 12 hours at room temperature, thus obtaining Fe 3 O 4 PDA magnetic material is repeatedly washed by pure water and ethanol, and vacuum dried for 12 hours at 60 ℃. Then, the prepared Fe 3 O 4 360mg of @ PDA material is placed in 250mL of toluene, ultrasonic dissolution is carried out, 480mg of chloro (dimethyl) octadecylsilane is added, and mechanical stirring is carried out for 12 hours, thus obtaining Fe 3 O 4 The magnetic material @ PDA @ C18 was repeatedly washed with pure water and ethanol and dried in vacuo at 60℃for 12h.
The TEM of the obtained magnetic material is shown in FIG. 2, and Fe prepared by the method 3 O 4 The dimension of @ PDA @ C18 is about 250nm, fe 3 O 4 The outer core is wrapped with 30-40nm polydopamine layer. The outermost layer contains irregular protoglobular protrusions, which are grafted C18.
2. Establishment of standard curve for detecting methamphetamine by fluorescent probe
Preparing a supermolecule fluorescent probe: stock solutions of 100. Mu.MQ 8 and 1mM proflavine were prepared and stored at 4 ℃. A certain volume of Q8 and profen mother solution is dissolved in ultrapure water to make the final concentration of the profen 10 mu MQ8 and the profen 20 mu M, and the profen is uniformly mixed for standby.
Enrichment: 100mL of ultrapure water at pH 9.5 was placed in a plastic bottle, and the same volume of methamphetamine solution was added to give final concentrations of 0ng/mL, 10ng/mL, 20ng/mL, 30ng/mL, 40ng/mL, 50ng/mL, 60ng/mL, 70ng/mL, 80ng/mL. Adding Fe 3 O 4 @PDA@C18 magnetic material 10mg, ultrasound to make the material disperse uniformly, shaking at room temperature for 15min, then magnetic separation to discard the solution, adding 1500. Mu.L volume of elution solution (5% formic acid), transferring to 1.5mL EP tube, vortexing for 5min, then magnetic separation to obtain enriched sample solution.
The detection step comprises: taking 350 mu L of fluorescent probe solution, adding 350 mu L of enriched sample solution, uniformly mixing, wherein the excitation wavelength is 430nm, the emission wavelength is a fluorescent signal value under 520nm, and repeatedly measuring each concentration for 3 times. The standard curve is drawn by taking the concentration of methamphetamine hydrochloride as the abscissa and the fluorescence intensity as the ordinate, and the result is shown in figure 3, thus obtaining the linear equation y=994890x+76756301
3. Investigation of specificity
Taking 100mL of ultrapure water with the pH of 9.5, adding methamphetamine and various other interfering substances into a plastic bottle, and enabling the final concentration of the mecalcanone, the cocaine, the heroin, the triazolam and the alprazolam to be 40ng/mL; tryptophan, tyrosine, phenylalanine, glucose, sodium chloride and potassium chloride with concentration of 400ng/mL are enriched and detected according to the method, and the interference condition of the substances on the detection method is examined. As a result, as shown in FIG. 4, there was no significant change in fluorescence of the interfering substances except for the target.

Claims (4)

1. A method for enriching and rapidly detecting methamphetamine is characterized by comprising the following steps: fe is added to 3 O 4 Incubating @ PDA @ C18 magnetic nanomaterial with a sample containing methamphetamine, magnetically separating and discarding the supernatant after 15min, adding 5% formic acid, vortexing for 5min, mixing the eluent obtained by magnetic separation with a supermolecule fluorescent probe, measuring fluorescence intensity, taking methamphetamine concentration as an abscissa and fluorescence intensity as an ordinate, drawing a standard curve, mixing a supermolecule cucurbituril host Q8 with an acridine dye object, self-assembling to obtain the supermolecule fluorescent probe, and adopting Fe 3 O 4 Enriching methamphetamine by using @ PDA @ C18 magnetic nano material, wherein Fe is as follows 3 O 4 The core of the @ PDA @ C18 magnetic nano material is Fe 3 O 4 The magnetic particles are coated with a polydopamine layer on the outer layer and a hydrophobic group C18 on the outermost layer; the saidFe 3 O 4 The preparation method of the magnetic particles comprises the following steps: feCl is taken 3 ·6H 2 O and anhydrous sodium acetate are respectively dissolved in glycol, evenly mixed, reacted at high temperature and high pressure, washed and dried after the reaction is finished; the Fe is 3 O 4 The preparation method of the @ PDA@C18 magnetic nanomaterial comprises the following steps: fe (Fe) 3 O 4 Placing magnetic particles in weak base Tris buffer solution, then adding a proper amount of dopamine hydrochloride, stirring, finishing the reaction, cleaning and drying for later use; taking Fe 3 O 4 Putting PDA into toluene, adding chloro (dimethyl) octadecylsilane, mechanically stirring at room temperature, cleaning after the reaction is finished, and drying in vacuum.
2. The method for enriching and rapidly detecting methamphetamine according to claim 1, wherein the method comprises the steps of: the Fe is 3 O 4 The preparation method of the magnetic particles specifically comprises the following steps: 150 g mL of ethylene glycol was measured and placed in a 500 g mL round bottom flask, and 8.1g g of FeCl was added 3 ·6H 2 O, magnetically stirring to dissolve the solution, and obtaining yellow clear solution; 150mL of ethylene glycol is additionally measured and placed in a 250mL round-bottom flask, 21.6g anhydrous sodium acetate is added, and magnetic stirring is carried out to dissolve the ethylene glycol, so as to obtain a clear and transparent solution; dropwise adding anhydrous sodium acetate solution into FeCl under stirring 3 Transferring the brown yellow turbid liquid into a polytetrafluoroethylene high-pressure reaction kettle, and reacting at 200 ℃ for 12h to obtain Fe with the particle size of 200-300nm 3 O 4 Magnetic particles.
3. The method for enriching and rapidly detecting methamphetamine according to claim 1, wherein the method comprises the steps of: the Fe is 3 O 4 The preparation method of the @ PDA @ C18 magnetic nano material specifically comprises the following steps: taking Fe 3 O 4 Magnetic particles 800 and mg are placed in 1000mL 10mM Tris buffer solution with pH of 8.5, ultrasonic treatment is carried out to ensure that the magnetic particles are uniformly dispersed, then 800mg dopamine hydrochloride is added, and the mixture is mechanically stirred at room temperature for 12h to obtain Fe 3 O 4 Cleaning magnetic material @ PDA with pure water and ethanol repeatedly, and vacuum drying at 60deg.C for 12 h; then, the prepared Fe 3 O 4 @ PDA material 360mg, disposed250 In the mL of toluene, ultrasonic dissolution is carried out, 480mg chlorine (dimethyl) octadecylsilane is added, and mechanical stirring is carried out for 12h, thus obtaining Fe 3 O 4 Pda@c18 magnetic nanomaterial.
4. The method for enriching and rapidly detecting methamphetamine according to claim 1, wherein the method comprises the steps of: the acridine dye object comprises acridine orange, acridine yellow, proflavine and neutral red.
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