阅读说明：本技术 一种甲基苯丙胺富集及快速检测的方法 (Methamphetamine enrichment and rapid detection method ) 是由 狄斌 闫昆 胡驰 藿湘雲 戚立凯 于 2021-07-30 设计创作，主要内容包括：本发明公开了一种甲基苯丙胺富集及快速检测的方法,向富集的甲基苯丙胺样品中加入超分子荧光探针,混匀,测定荧光强度。基于葫芦脲主体Q8与吖啶类染料客体自组装构建了超分子荧光探针,甲基苯丙胺能够与染料客体竞争主体分子,引起荧光变化,实现对甲基苯丙胺的快速检测。该方法巧妙地将富集与检测结合起来,显著地提高了检测方法的灵敏度和专属性。(The invention discloses a method for enriching and rapidly detecting methamphetamine. Based on self-assembly of the cucurbituril host Q8 and the acridine dye guest, the supramolecular fluorescent probe is constructed, methamphetamine can compete with the dye guest for host molecules to cause fluorescence change, and 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.)

1. A method for enriching and rapidly detecting methamphetamine is characterized in that: adding a super-molecular fluorescent probe into the enriched methamphetamine sample, uniformly mixing, measuring the fluorescence intensity, and drawing a standard curve by taking the concentration of the methamphetamine as an abscissa and the fluorescence intensity as an ordinate.

2. The method for enrichment and rapid detection of methamphetamine according to claim 1, wherein: the supramolecular fluorescent probe is obtained by self-assembling a supramolecular cucurbituril main body Q8 and an acridine dye object after mixing.

3. The method for enrichment and rapid detection of methamphetamine according to claim 1, wherein: using Fe₃O₄The @ PDA @ C18 magnetic nano material enriches methamphetamine.

4. The method for enrichment and rapid detection of methamphetamine according to claim 1, wherein: said Fe₃O₄The magnetic nano material of @ PDA @ C18 has Fe in the core₃O₄The magnetic particle comprises a polydopamine layer wrapped on the outer layer, and a hydrophobic group C18 wrapped on the outermost layer.

5. The method for enrichment and rapid detection of methamphetamine according to claim 4, wherein: said Fe₃O₄The preparation method of the magnetic particles comprises the following steps: taking FeCl₃·6H₂Dissolving O and anhydrous sodium acetate in ethylene glycol respectively, mixing uniformly, reacting at high temperature and high pressure, cleaning and drying after the reaction is finished.

6. The method for enrichment and rapid detection of methamphetamine according to claim 3, wherein: said Fe₃O₄The preparation method of the @ PDA @ C18 magnetic nano material comprises the following steps: fe₃O₄Magnetic particles are placed in a weak base Tris buffer solution, then a proper amount of dopamine hydrochloride is added, stirring is carried out, after the reaction is finished, cleaning and drying are carried out for standby application; taking Fe₃O₄@ PDA is put in toluene, added with chloro (dimethyl) octadecylsilane, mechanically stirred at room temperature, and washed and dried in vacuum after the reaction is finished.

7. The method for enrichment and rapid detection of methamphetamine according to claim 2, wherein: the acridine dye object comprises acridine orange, acridine yellow, proflavine and neutral red.

Technical Field

The invention relates to a compound analysis and detection method, in particular to a method for enriching and rapidly detecting methamphetamine.

Background

The methamphetamine hydrochloride is pure white crystal in appearance, occupies a very high proportion in drug abuse, has very strong toxicity, can cause irreversible damage to a nervous system after long-term intake of the methamphetamine hydrochloride, and is easy to generate dependence on the methamphetamine hydrochloride by an eater to cause addiction to the methamphetamine hydrochloride.

At present, sewage epidemiology is one of the well-accepted methods which can objectively monitor and evaluate the regional illegal drug abuse situation at home and abroad. The concentration of drugs and their metabolites in the wastewater is very low and the background matrix environment is complex. At present, the detection usually depends on the determination of drug components in sewage by LC-MS/MS after solid phase extraction. However, the traditional solid phase extraction method has poor selectivity to analytes, complicated operation steps and time and labor waste. And mass spectrometry instruments are expensive and require specialized technicians to be equipped for use.

In conclusion, the prior art lacks a high-sensitivity analysis method of a fluorescent supramolecular sensor capable of rapidly detecting the content of methamphetamine in a sewage sample, so that the problem that the combination of magnetic solid-phase extraction and a supramolecular fluorescent probe for detecting the methamphetamine is urgently needed to be solved is developed.

Disclosure of Invention

The invention aims to provide a fluorescence detection method based on magnetic solid-phase extraction combined with a supramolecular fluorescence probe, which has high sensitivity and good specificity and is used for quickly detecting methamphetamine.

The invention provides a method for enriching and rapidly detecting methamphetamine.

Furthermore, the supramolecular fluorescent probe is obtained by self-assembling a supramolecular cucurbituril main body Q8 and an acridine dye guest after mixing.

Further, Fe is used₃O₄The @ PDA @ C18 magnetic nano material enriches methamphetamine.

Further, said Fe₃O₄The magnetic nano material of @ PDA @ C18 has Fe in the core₃O₄The magnetic particle comprises a polydopamine layer wrapped on the outer layer, and a hydrophobic group C18 wrapped on the outermost layer.

Further, said Fe₃O₄The preparation method of the magnetic particles comprises the following steps: taking FeCl₃·6H₂Dissolving O and anhydrous sodium acetate in ethylene glycol, mixing, reacting at high temperature and high pressure, and cleaningAnd (5) washing and drying.

Further, Fe₃O₄The preparation method of the @ PDA @ C18 magnetic nano material comprises the following steps: fe₃O₄Magnetic particles are placed in a weak base Tris buffer solution, then a proper amount of dopamine hydrochloride is added, stirring is carried out, after the reaction is finished, cleaning and drying are carried out for standby application; taking Fe₃O₄@ PDA is put in toluene, added with chloro (dimethyl) octadecylsilane, mechanically stirred at room temperature, and washed and dried in vacuum after the reaction is finished.

Further, the acridine dye guest comprises acridine orange, acridine yellow, proflavine and neutral red.

First synthesize Fe₃O₄The @ PDA @ C18 magnetic nano material is used for further optimizing the enrichment condition. And then uniformly mixing the supramolecular cucurbituril main body Q8 and the acridine dye guest preferably in a molar concentration ratio of 1: 2, and carrying out self-assembly to obtain the supramolecular fluorescent probe. And (3) uniformly mixing the enriched methamphetamine sample with the supramolecular fluorescent probe, measuring the fluorescence intensity, and drawing a standard curve by taking the concentration of the methamphetamine as a horizontal coordinate and the fluorescence intensity as a vertical coordinate.

The magnetic nano material is Fe₃O₄@ PDA @ C18, has the ability of enriching methamphetamine, and has magnetic Fe in core₃O₄The magnetic particle and the poly dopamine layer wrapped by the outer layer have a large amount of catechol structures and provide rich pi-pi structures. In addition, the outermost layer is coated with hydrophobic group C18 to finally obtain the amphiphilic magnetic nano material Fe₃O₄@[email protected]。

Said preparation of Fe₃O₄A method of magnetic particles, preferably comprising the steps of: taking a proper amount of FeCl₃·6H₂Dissolving O and anhydrous sodium acetate in ethylene glycol respectively, mixing uniformly under stirring, placing in a polytetrafluoroethylene high-pressure reaction kettle, reacting at 200 ℃ for 12h, cleaning after the reaction is finished, and drying for later use.

Said preparation of Fe₃O₄The method of @ PDA @ C18, preferably comprising the steps of: taking a proper amount of Fe₃O₄Magnetic particles are put into weak base Tris buffer solution, and then proper amount of dopa hydrochloride is addedAnd (4) mechanically stirring amine for 12 hours, finishing the reaction, washing and drying for later use. Then taking a proper amount of Fe₃O₄@ PDA is put into toluene, added with chloro (dimethyl) octadecylsilane, mechanically stirred at room temperature for 12h, and after the reaction is finished, cleaned and dried in vacuum for standby.

Preferably the enrichment conditions are as follows: 100mL of sample to be detected with pH of 9.5 is put into a plastic bottle, and Fe is added₃O₄@ PDA @ C18 magnetic material 10mg, the material was dispersed evenly by ultrasound, shaken at room temperature for 15min, then the solution was discarded by magnetic separation, 1500. mu.L volume of elution solution (5% formic acid) was added, transferred to a 1.5mL EP tube, vortexed for 5min, then magnetic separated to obtain the enriched sample solution.

The supramolecular cucurbituril Q8 can cause dye fluorescence quenching after being combined with acridine dye guest, the lowest fluorescence value is achieved when the optimal molar ratio is 1: 2, and after the object to be detected, namely methamphetamine, is added, the guest competes with the dye to be combined with the host Q8, so that the fluorescence value of the dye is recovered.

The structural formulas of the main body cucurbituril Q8 and the acridine dye guest are as follows:

preferably, the volume of the enriched methamphetamine is 350 mu L, the volume of the fluorescent probe is 350 mu L, the mixture is mixed and vortexed for 2-3 seconds, and the mixture is added into a 96-well plate, wherein the volume of the three plates is 200 mu L per well.

Preferably, the fluorescence intensity is a fluorescence signal value at an excitation wavelength of 430nm and an emission wavelength of 520 nm.

Has the advantages that: compared with the prior art, the invention has the following advantages:

the method has high sensitivity, good specificity and simple and convenient method, and can be used for quantitative detection of methamphetamine in a sewage sample.

Drawings

FIG. 1 is a schematic diagram of the principle of magnetic solid phase extraction combined with supramolecular fluorescent probe detection in the present invention;

fig. 2 is a TEM image of a magnetic material: a is Fe₃O₄@ PDA, B is Fe₃O₄@[email protected]；

FIG. 3 is a standard graph established for the process at different methamphetamine concentrations;

FIG. 4 is a fluorescence image of the detection method under other interfering substances.

Detailed Description

The detection scheme of the invention is shown in figure 1, magnetic beads and a sample containing methamphetamine are incubated, after 15min, magnetic separation is carried out, supernatant is discarded, 5% formic acid is added, vortex is carried out for 5min, eluent obtained by magnetic separation is mixed with a supramolecular fluorescent probe, and the fluorescence intensity is measured. The content of the methamphetamine in the sample and the fluorescence intensity are in a linear relation, so that the detection of the methamphetamine in the sewage is realized.

The method for detecting methamphetamine in sewage sample based on magnetic solid phase extraction combined with supramolecular fluorescent probe

1. Magnetic material Fe₃O₄Preparation of @ PDA @ C18

150mL of ethylene glycol was measured, placed in a 500mL round-bottom flask, and 8.1g of FeCl was added₃·6H₂And O, dissolving the mixture by magnetic stirring to obtain a 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. Adding anhydrous sodium acetate solution dropwise to FeCl under stirring₃Obtaining brown yellow turbid liquid in the solution, transferring the solution to a polytetrafluoroethylene high-pressure reaction kettle, and reacting for 12 hours at the temperature of 200 ℃ to prepare Fe with the particle size of about 200-₃O₄And (4) nano ions. Repeatedly washing with pure water and ethanol for three times, and vacuum drying at 60 deg.C for 24 hr. Subsequently taking the prepared Fe₃O₄Placing 800mg of nano particles in 1000mLpH 8.5 10mM Tris buffer solution, performing ultrasonic treatment to uniformly disperse the nano particles, then adding 800mg of dopamine hydrochloride, and mechanically stirring the mixture at room temperature for 12 hours to obtain Fe₃O₄@ PDA magnetic material, repeatedly washing with pure water and ethanol, and vacuum drying at 60 deg.C for 12 h. Then, the obtained Fe₃O₄@ PDA material 360mg, placed in 250mL toluene, ultrasonic assisted dissolution, followed by addition of 480mg chloro (dimethyl) octadecylSilane, mechanically stirring for 12h to obtain Fe₃O₄@ PDA @ C18 magnetic material, repeatedly washed with pure water and ethanol, and vacuum dried at 60 deg.C for 12 h.

TEM of the magnetic material obtained is shown in FIG. 2, Fe prepared by this method₃O₄@ PDA @ C18 size is about 250nm, Fe₃O₄The outer core is wrapped with 30-40nm polydopamine layer. The outermost layer contains irregular primary spherical protrusions and is grafted C18.

2. Establishment of standard curve for detecting methamphetamine by fluorescent probe

Preparing a supramolecular fluorescent probe: a mother liquor of 100. mu.MQ 8 and 1mM proflavin was prepared and stored at 4 ℃. Dissolving a certain volume of mother liquor of Q8 and proflavine in ultrapure water to make the final concentration of 10 mu MQ8 and 20 mu M proflavine, and uniformly mixing for later use.

An enrichment step: 100mL of ultrapure water having a pH of 9.5 was taken out and charged into a plastic bottle, and methamphetamine solution was added in an equal volume so that the final concentrations thereof were 0ng/mL, 10ng/mL, 20ng/mL, 30ng/mL, 40ng/mL, 50ng/mL, 60ng/mL, 70ng/mL and 80 ng/mL. Adding Fe₃O₄@ PDA @ C18 magnetic material 10mg, the material was dispersed evenly by ultrasound, shaken at room temperature for 15min, then the solution was discarded by magnetic separation, 1500. mu.L volume of elution solution (5% formic acid) was added, transferred to a 1.5mL EP tube, vortexed for 5min, then magnetic separated to obtain the enriched sample solution.

A detection step: and (3) adding 350 mu L of the enriched sample solution into 350 mu L of the fluorescent probe solution, uniformly mixing, and repeatedly measuring each concentration for 3 times, wherein the excitation wavelength is 430nm, and the emission wavelength is a fluorescent signal value under 520 nm. The concentration of methamphetamine hydrochloride is used as the abscissa and the fluorescence intensity is used as the ordinate, a standard curve is drawn, and the result is shown in fig. 3, so that a linear equation y of 994890x +76756301 is obtained

3. Specialization inspection

Putting 100mL of ultrapure water with the pH value of 9.5 into a plastic bottle, adding methamphetamine and various other interference substances, wherein the final concentration of the mecamylamine, the cocaine, the heroin, the triazolam and the alprazolam is 40 ng/mL; tryptophan, tyrosine, phenylalanine, glucose, sodium chloride and potassium chloride of 400ng/mL are enriched and detected according to the method, and the interference of the substances on the detection method is examined. The results are shown in FIG. 4, where no significant fluorescence change was observed for interfering substances other than the target.