Detection method and kit for rapid detection of quaternary ammonium oxime reactivators in biological samples

Abstract

The present invention relates to a detection method and a kit for rapid detection of quaternary ammonium oxime reactivators in biological samples, and specifically to a method for detecting quaternary ammonium oxime antitoxic agents, which comprises: 1) using a surface enhanced Raman spectrometer to detect the signal of the quaternary ammonium oxime antitoxic agent in the sample to be tested; 2) based on the signal obtained by the detection, determining the type and / or content of the quaternary ammonium oxime antitoxic agent in the sample to be tested. The detection method has many advantages such as simple operation, fast detection speed, high sensitivity, etc., and can be used for rapid detection of quaternary ammonium oxime rescue drugs for nerve agent poisoning in biological samples.

Description

Technical Field

[0001] The present invention relates to the field of analytical chemistry, and in particular to a detection method and a kit for rapid detection of quaternary ammonium salt oxime reactivators in biological samples. Background Art

[0002] Quaternary ammonium oximes (QAO) reactivators are a very important class of drugs for the prevention and treatment of organophosphorus poisoning. Organophosphorus poisons can react with the serine hydroxyl group at the active site of acetylcholinesterase (AChE) to form a stable PO covalent bond (OP-AChE), which irreversibly inhibits AChE and causes poisoning. The oxime hydroxyl group in the reactivator can attack the phosphorus atom, destroy the PO covalent bond, restore the serine hydroxyl group at the active site of acetylcholinesterase, and restore the activity of the enzyme. This class of compounds has a significant detoxification effect on the peripheral nervous system, but the strong charge of the quaternary ammonium salt structure makes it difficult for them to pass through the blood-brain barrier (BBB) ​​to exert a detoxification effect in the central nervous system. Encapsulating the reactivator through dosage form modification can improve its ability to pass through the blood-brain barrier, thereby enhancing the reactivation effect on the central nervous system. At present, the analytical methods for such compounds in biological samples are mostly high performance liquid chromatography (HPLC), liquid chromatography-tandem mass spectrometry (LC-MS / MS), capillary electrophoresis (CE), etc. These detection methods are highly sensitive and reproducible, but require complex sample pretreatment processes, long analysis time and more professional operators. In addition, since these compounds have strong polarity and poor chromatographic retention behavior, special chromatographic columns or ion pair reagents are required to improve their separation effect; and oxime compounds are easily degraded in biological samples. Methods with complex sample pretreatment steps, slow detection rate and poor sensitivity can no longer meet the needs of qualitative and quantitative detection. In order to effectively evaluate the ability of various quaternary ammonium oxime reactivators to pass through the BBB after their own and dosage form modification, and to quickly and accurately detect the concentration and time-effect / dose-effect relationship of antitoxic drugs in biological samples, and to provide important technical support for responding to NAs poisoning, it is still necessary to explore rapid and highly sensitive detection methods that can be applied to quaternary ammonium oxime reactivators in biological samples. Summary of the invention

[0003] After experimental exploration, the inventors obtained a quaternary ammonium oxime anti-toxin detection method and detection kit based on surface enhanced Raman spectroscopy. The inventors found that the detection method or detection kit of the present invention has many advantages such as simple operation, fast detection speed, and high sensitivity, and can be used for the rapid detection of quaternary ammonium oxime rescue drugs for nerve agent poisoning in biological samples.

[0004] To this end, in a first aspect of the present invention, the present invention provides a method for detecting a quaternary ammonium salt oxime antitoxic agent, comprising:

[0005] 1) Using a surface enhanced Raman spectrometer, the signal of the quaternary ammonium salt oxime antitoxic agent in the sample to be tested is detected;

[0006] 2) Based on the signal obtained by the detection, determine the type and / or content of the quaternary ammonium salt oxime antitoxic agent in the sample to be tested.

[0007] Surface-Enhanced Raman Spectroscopy (SERS) is a fingerprint spectroscopy technology with the advantages of high sensitivity, real-time non-destructive analysis, no need for complex sample pre-treatment, simple operation and fast detection speed. Directly applying SERS to the detection of quaternary ammonium salt oxime reactivators in biological matrices has very low sensitivity, and various biomacromolecules in biological samples, such as blood cells, proteins, amino acids, etc., will seriously inhibit the SERS detection signal, resulting in no detection signal or extremely low sensitivity. In view of this difficult problem, the present invention constructs a "stacked" Au-I - -QAO specific sensing system, using positively charged QAO compounds and coated iodine anions (I - ) nanogold, not only effectively removes the interference of proteins, amino acids and other compounds adsorbed on the surface of nanogold, but also attracts the target analytes to the hot spot of the nanosol, thereby effectively simplifying the sample pretreatment process, significantly improving the detection sensitivity and analysis speed of QAO compounds in biological samples, and avoiding the shortcomings of poor parallelism in the analysis of biological samples due to self-degradation; it realizes the qualitative and quantitative analysis of oxime reactivators in samples such as blood and brain extracts, providing a relatively strong technical support for evaluating the blood-brain barrier penetration ability of such compounds.

[0008] To this end, in some embodiments, the surface enhanced Raman spectroscopy enhancement substrate is selected from gold nanosol, silver nanosol, gold magnetic nanosol or other forms of nano detection substrates.

[0009] In some embodiments, the surface enhanced Raman spectroscopy enhancement substrate is pinhole shell-isolated gold nanoparticles (pinSHINs).

[0010] In some embodiments, the surface enhanced Raman spectroscopy enhancing agent is selected from KF, KI, NaCl, MgSO4, Na2CO3, Na2SO4, NaBr, NaHCO3, and Na2SO3.

[0011] In some embodiments, the surface enhanced Raman spectroscopy enhancing agent is KI.

[0012] In some embodiments, the concentration of the enhancing reagent is 50-200mmol / L (such as 50, 70, 90, 100, 110, 130, 150, 170, 190 or 200mmol / L), preferably 50-150mmol / L, and more preferably 100mmol / L, wherein the concentration of the enhancing reagent refers to the final concentration of the enhancing reagent after the enhancing reagent is mixed with the enhancing substrate and the sample to be tested.

[0013] In some embodiments, step 1) of the method comprises:

[0014] 1-1) Centrifuging the enhanced substrate and discarding the supernatant;

[0015] 1-2) mixing the enhancing agent with the product obtained in step 1-1); preferably, slowly adding the enhancing agent to the bottom of the product obtained in step 1-1);

[0016] 1-3) mixing the sample to be tested with the product obtained in step 1-2);

[0017] 1-4) Using a surface enhanced Raman spectrometer, the product obtained in step 1-3) is detected to obtain the signal of the quaternary ammonium salt oxime antitoxic agent in the sample to be tested.

[0018] In some embodiments, the laser power of the surface enhanced Raman spectroscopy is 0-300 mW, preferably 60-180 mW, and more preferably 60-120 mW.

[0019] In some embodiments, the detection time of the detection is 8-12 s, preferably 10 s.

[0020] In some embodiments, the detection wavelength of the detection is 785 nm.

[0021] In some embodiments, the quantitative peak detected is 1638 cm -1 Peak height at the Raman shift.

[0022] In some embodiments, the surface enhanced Raman spectrometer is a portable or handheld Raman spectrometer.

[0023] In some embodiments, the sample to be tested is selected from a blood sample or a tissue sample (such as a brain tissue sample).

[0024] In some embodiments, the blood sample is pre-centrifuged to remove red blood cells to obtain a plasma sample.

[0025] In some embodiments, the tissue sample is pre-treated by precipitation to remove protein and / or fat tissue.

[0026] In some embodiments, the precipitant used in the precipitation treatment is selected from trifluoroacetic acid, formic acid, acetonitrile, sodium chloride, zinc sulfate, acetic acid, ethanol, acetonitrile, preferably sodium chloride.

[0027] In some embodiments, the concentration of the precipitant (preferably sodium chloride) is 0.5-1.5 mol / L, preferably 1 mol / L, wherein the concentration of the precipitant refers to the final concentration of the precipitant after the precipitant is mixed with the tissue sample.

[0028] In some embodiments, the quaternary ammonium salt oxime antitoxic agent is selected from TMB-4 (bis-phosphodiester, bis-4,4′-pyridinecarboxaldehyde oxime-N,N′dibromotrimethyl salt), HI-6 (amidopralidoxime), Obi (bisphosphodiester), MMB-4 (1,1′-methylenebis[4-[(hydroxyimino)methyl]-pyridine dibromide), PAM (pralidoxime, pyridinecarboxaldehyde oxime iodomethane salt).

[0029] In some embodiments, the porous shell-isolated gold nanoparticles include a core and a shell, the shell encapsulates the core, the core is AuNPs, and the shell is a SiO2 shell.

[0030] In some embodiments, the particle size of the AuNPs is 50-60 nm, preferably 55 nm.

[0031] In some embodiments, the thickness of the SiO2 shell layer is 1-2 nm.

[0032] In some embodiments, the porous shell-isolated gold nanoparticles are prepared by the following method:

[0033] 1) first mixing AuNPs with 3-aminopropyltrimethoxysilane;

[0034] 2) performing a second mixing of the first mixed product and Na2SiO3 to obtain the porous shell-isolated gold nanoparticles.

[0035] In some embodiments, the 3-aminopropyltrimethoxysilane is provided in a form dissolved in water.

[0036] In some embodiments, in the aqueous solution of 3-aminopropyltrimethoxysilane, the concentration of 3-aminopropyltrimethoxysilane is 0.5-1.5 mM, preferably 1 mM.

[0037] In some embodiments, the Na2SiO3 is provided in a form dissolved in water.

[0038] In some embodiments, in the Na2SiO3 aqueous solution, the concentration (w / w) of Na2SiO3 is 0.5%-0.6%, preferably 0.54%.

[0039] In some embodiments, the pH of the Na2SiO3 aqueous solution is greater than 11.

[0040] In some embodiments, the first mixing time is 10-20 min, preferably 15 min.

[0041] In some embodiments, the second mixing time is 2-5 min, preferably 3 min.

[0042] In some embodiments, the AuNPs (gold nanoparticles) are prepared by Frens' sodium citrate reduction of tetrachloroauric acid method.

[0043] In some embodiments, the AuNPs are prepared by the following method:

[0044] 1) mixing tetrachloroauric acid trihydrate with water to obtain a mother solution;

[0045] 2) taking part of the mother liquor and diluting it with water;

[0046] 3) heating the diluted mother liquor to boiling, adding trisodium citrate aqueous solution thereto after condensation and reflux, and continuing heating for 30-50 min (such as 40 min) to obtain the AuNPs.

[0047] In some embodiments, the concentration of tetrachloroauric acid trihydrate in the mother solution is 0.5-1.5 mg / mL, preferably 1 mg / mL.

[0048] In some embodiments, when a portion of the mother liquor is diluted with water, the dilution factor is 8-12 times, preferably 10 times.

[0049] In some embodiments, in the trisodium citrate aqueous solution, the concentration (w / w) of trisodium citrate is 0.5-1.5%, preferably 1%.

[0050] In some embodiments, the volume ratio of the diluted mother liquor to the trisodium citrate aqueous solution is 100:0.8-0.9, preferably 100:0.85.

[0051] In some embodiments, determining the type of quaternary ammonium salt oxime antitoxic agent in the sample to be tested is performed by the following steps:

[0052] 1) Using a surface enhanced Raman spectrometer, the signal of the quaternary ammonium salt oxime antitoxic agent in the sample to be tested is detected;

[0053] 2-1-A) comparing the detected signal with the characteristic fingerprint of each quaternary ammonium salt oxime antitoxic agent;

[0054] 2-2-A) Based on the comparison results, determine the type of quaternary ammonium oxime antitoxic agent in the sample to be tested.

[0055] In some embodiments, determining the content of quaternary ammonium salt oxime antitoxic agent in the sample to be tested is carried out by the following steps:

[0056] 1) Using a surface enhanced Raman spectrometer, the signal of the quaternary ammonium salt oxime antitoxic agent in the sample to be tested is detected;

[0057] 2-1-B) using a surface enhanced Raman spectrometer to detect signals of quaternary ammonium salt oxime antitoxic agents at different preset concentrations (e.g., multiple dilutions);

[0058] 2-2-B) based on different preset concentrations of the quaternary ammonium oxime anti-toxic agent and the signal intensities of the different preset concentrations of the quaternary ammonium oxime anti-toxic agent obtained by detection, a signal intensity-concentration working curve is drawn;

[0059] 2-3-B) Based on the signal of the quaternary ammonium salt oxime anti-toxic agent in the sample to be tested obtained by detection and the signal intensity-concentration working curve drawn, the content of the quaternary ammonium salt oxime anti-toxic agent in the sample to be tested is determined.

[0060] In some embodiments, in step 2-1-B), when detecting the signals of quaternary ammonium oxime antitoxic agents of different preset concentrations (e.g., multiple dilutions), the enhanced substrate of each concentration is prepared by the following method: a first preset volume (e.g., 1 mL) of the enhanced substrate is placed in an EP tube for centrifugation and the supernatant is discarded, and the enhanced substrate remaining at the bottom is mixed evenly, and then a second preset volume (e.g., 10 μL) of the enhanced substrate is taken from the enhanced substrate remaining at the bottom and dispensed into an EP tube for standby use.

[0061] In the second aspect of the present invention, the present invention provides a quaternary ammonium salt oxime anti-toxic agent detection kit based on surface enhanced Raman spectroscopy, which comprises:

[0062] (1) a reinforcing substrate, wherein the reinforcing substrate is as described above;

[0063] (2) an enhancing agent, wherein the enhancing agent is as described above;

[0064] Optionally, the kit further comprises: (3) a reagent for pre-treating the sample to be tested.

[0065] In some embodiments, the reinforcing substrate is a porous shell-isolated gold nanoparticle;

[0066] In some embodiments, the porous shell layer isolates the gold nanoparticles as described above.

[0067] In some embodiments, the sample pre-treatment reagent is a precipitant.

[0068] In some embodiments, the precipitating agent is as described above. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 . Transmission electron microscopy characterization images of AuNPs (a) and Pin SHINs (b);

[0070] Figure 2 SERS spectra of TMB-4 aqueous solution (100 ppb) with different inorganic salts added (a), and the SERS spectra of 1638 cm -1 The influence of peak intensity (b);

[0071] Figure 3 SERS detection spectra of TMB-4 aqueous solution (100 ppb) with KI added at concentrations of 0, 20, 30, 50, 100, 200, 300, 400, and 500 mmol / L (a); Effect of KI concentration on TMB-4 1638 cm -1 The influence of SERS peak intensity at (b);

[0072] Figure 4 .SERS detection spectra of five quaternary ammonium oxime reactivators PAM (10μg / ml), HI-6, MMB4, TMB4, Obi (1μg / ml) in aqueous solution;

[0073] Figure 5a .SERS detection spectra of TMB-4 at different concentrations;

[0074] Figure 5b .1638cm -1 Working curve of SERS peak intensity-TMB-4 concentration (0.5-20ng / ml);

[0075] Figure 5c .SERS detection spectra of Obi at different concentrations;

[0076] Figure 5d .1638cm -1 SERS peak intensity-Obi concentration (0.5-20ng / ml) working curve;

[0077] Figure 5e .SERS detection spectra of HI-6 at different concentrations;

[0078] Figure 5f .1638cm -1SERS peak intensity-HI-6 concentration (0.5-20ng / ml) working curve;

[0079] Figure 5g .SERS detection spectra of MMB-4 at different concentrations;

[0080] Figure 5h .1638cm -1 Working curve of SERS peak intensity-MMB-4 concentration (0.5-20ng / ml);

[0081] Figure 6 . AuNPs and pinSHINs as enhanced substrates, SERS detection spectra of HI-6 plasma sample (50ng / ml);

[0082] Figure 7 . Use pinSHINs to detect the effect of different storage time on HI-6 plasma samples (1 mg / ml) at 1638 cm -1 The impact of peak intensity changes;

[0083] Figure 8 . SERS detection spectra of HI-6 plasma samples at different blood collection time points (a); time-intensity curve of HI-6 plasma samples (b);

[0084] Fig. 9 .When the sample to be tested is a brain homogenate with a HI-6 concentration of 200 ppb, SERS detection diagram (a) of brain homogenate tissue treated with different protein precipitants, wherein direct detection means directly detecting the brain homogenate tissue without adding a precipitant, centrifugal detection means taking the supernatant for detection after centrifugal treatment of the brain homogenate tissue without adding a precipitant, acetic acid (PinSHINs) means that the precipitant is acetic acid and PinSHINs prepared in the preparative example, and TFA (PinSHINs) means that the precipitant is trifluoroacetic acid and PinSHINs prepared in the preparative example; When the sample to be tested is a brain homogenate with a HI-6 concentration of 200 ppb, SERS detection diagram (a) of brain homogenate tissue treated with different protein precipitants on 1638 cm -1 The influence of the peak intensity at 1638cm (b); SERS detection spectra of HI-6 brain tissue samples with different concentrations when the precipitant is NaCl (c); and when the precipitant is NaCl, 1638cm -1 Working curve of SERS peak intensity-HI-6 concentration (10-500ng / ml) (d). DETAILED DESCRIPTION

[0085] Embodiments of the present invention are described in detail below. The embodiments described below are exemplary and intended to be used to explain the present invention, but should not be construed as limiting the present invention.

[0086] The present invention belongs to the field of analytical chemistry and relates to a rapid detection kit for oxime reactivators for organophosphorus nerve agent poisoning. Specifically, the present invention relates to the selection of SERS enhanced substrates; the optimization of the type and concentration of agglomerating agents; the optimization of protein precipitants for biological samples; the steps of applying detection reagents; the detection method in complex matrix samples and the preparation of related detection reagent packages.

[0087] The present invention belongs to the field of analytical chemistry, and relates to a rapid detection kit based on surface-enhanced Raman spectroscopy (SERS) technology and quaternary ammonium oximes (QAO) reactivators. Specifically, the present invention relates to the preparation of an enhanced substrate, the optimization of detection conditions and reagents, and a rapid detection method for a series of oxime reactivators in biological samples. The kit includes a detection reagent package such as a SERS substrate, an enhancement reagent, and simple sample pretreatment accessories. The present invention uses gold nanoparticles (AuNPs) as a substrate, utilizes the structural characteristics of the positively charged nitrogen atoms of quaternary ammonium oxime compounds, and constructs an Au-I based on the layer-by-layer self-assembly principle. - -QAO specific sensing system, by introducing I - The Au-I bond is formed with the Au core, which not only effectively removes the matrix interference adsorbed on the Au surface, but also utilizes the positively charged quaternary ammonium salt reactivator to bind to the coated I - The strong electrostatic interaction between Au and QAO greatly improves the detection sensitivity of QAO in biological samples and effectively simplifies the sample pretreatment process. The detection kit has many advantages such as easy operation, fast detection speed and high sensitivity. It can be used for rapid detection of quaternary ammonium oxime rescue drugs for nerve agent poisoning in biological samples.

[0088] The purpose of the present invention is to provide a rapid, simple and sensitive detection method for quaternary ammonium oxime reactivators in biological matrices, using Au-I - -QAO specific sensing system, using SERS detection means, to quickly detect QAO in biological matrices. Specifically, the present invention includes the following aspects:

[0089] A first aspect of the present invention relates to the preparation and optimization of an enhanced substrate, the method comprising the following steps:

[0090] Preparation of AuNPs: Au nanoparticles with a particle size of about 55 nm were prepared by Frens' sodium citrate reduction tetrachloroauric acid method. Accurately weigh 100 mg of tetrachloroauric acid trihydrate and dilute it to a 100 ml volumetric flask to obtain a 1 mg / ml mother solution (the solvent is water). Take 10 ml of the mother solution and dilute it to 100 ml. Pour it into a 250 ml three-necked flask and heat it in an oil bath. Heat it to boiling under rapid stirring. After condensation and reflux, quickly add 0.85 ml of 1% trisodium citrate aqueous solution, continue heating and stirring for 40 minutes, and prepare AuNPs with a particle size of about 55 nm.

[0091] Preparation of PinSHINs: Add 3-aminopropyltrimethoxysilane (APTMS) aqueous solution (1mM) to the prepared 55nm AuNPs, stir for 15min, add Na2SiO3 aqueous solution (0.54%, w / w) and stir for another 3min to coat the AuNPs surface with SiO2 shell. When the pH of Na2SiO3 aqueous solution is greater than 11, a porous SiO2 shell with a thickness of 1-2nm can be generated. After completion, store in a 4℃ refrigerator for use.

[0092] A second aspect of the present invention relates to the optimization of the type of enhancing agent, the method comprising the following steps:

[0093] KF, KI, NaCl, MgSO4, Na2CO3, Na2SO4, NaBr, NaHCO3, and Na2SO3 were selected as enhancing reagents and added to the enhanced substrate prepared above after centrifugation. 200 μL of 100 ppb QAO sample was added to the substrate to make the final concentration of inorganic salt 100 mmol / L. SERS detection was carried out, and the final results showed that KI had the best agglomeration effect.

[0094] A third aspect of the present invention relates to the optimization of the concentration of the agglomerating agent, the method comprising the following steps:

[0095] KI solution (solvent: water) was added to the enhanced substrate prepared above after centrifugation, and 200 μL of 100 ppb QAO sample was added to make the final KI concentration of 0, 20, 30, 50, 100, 200, 300, 400, and 500 mmol / L. Testing was carried out, and the final results showed that when the final KI concentration was 100 mmol / L, the agglomeration effect was optimal.

[0096] A fourth aspect of the present invention relates to the detection of QAO in a blood sample, the method comprising the following steps:

[0097] Take 1 ml of the enhanced substrate prepared above and put it in an EP tube. After centrifugation, discard the supernatant. Take another 10 μL of 2 mol / L potassium iodide solution and slowly add it to the enhanced substrate after centrifugation. After diluting the plasma sample with red blood cells discarded by centrifugation by multiples, take 200 μL of the sample solution and quickly add it to the enhanced substrate. After fully mixing, detect it with a handheld Raman spectrometer.

[0098] A fifth aspect of the present invention relates to the detection of QAO in a brain tissue sample, the method comprising the following steps:

[0099] Take 800μl of brain homogenate and add inorganic salt precipitant (NaCl). After protein is fully precipitated, centrifuge and take 200μL of supernatant. Take another 1ml of enhanced substrate, centrifuge and discard the supernatant, add 10μL of 2mol / L potassium iodide solution, mix well, then add 200μL of the supernatant sample after the previous centrifugation, mix well, and directly detect with a handheld Raman spectrometer.

[0100] Specifically, the present invention provides a SERS-based quaternary ammonium salt oxime anti-toxic agent rapid detection kit, characterized in that the kit includes a SERS enhancement substrate and an enhancement reagent. The operation steps include the following:

[0101] a) Preparation of nano-enhanced substrates;

[0102] b) optimization of enhancing agents;

[0103] c) Detection of quaternary ammonium salt oxime reactivators in solution samples: Take an appropriate amount of nano-enhanced substrate, centrifuge it in a centrifuge, suck out the supernatant, mix the enhancement reagent with the nanosol, add the sample, mix well, and detect it with a portable or handheld Raman spectrometer;

[0104] d) preparing quaternary ammonium salt oxime reactivator solutions of different concentrations, collecting characteristic Raman signal intensities of the quaternary ammonium salt oxime reactivator solutions of different concentrations as described in step c), and drawing a working curve according to the signal intensity and concentration;

[0105] e) Qualitative and quantitative detection of quaternary ammonium salt oxime reactivators in samples based on sample fingerprints and characteristic Raman peak intensities.

[0106] f) For the detection of various types of nerve agent poisoning reactivators in biological samples, in order to improve the detection sensitivity, the operation steps must be implemented in a predetermined order.

[0107] In some embodiments, the SERS nano-enhanced substrate is a gold nanosol, a silver nanosol, a gold magnetic nanosol or other forms of nano-detection substrates, preferably the substrate is pinhole shell-isolated nanoparticles (pinSHINs).

[0108] In some embodiments, the preferred part of the enhancing agent mainly involves the screening of various inorganic salt agglomerating agents and the optimization of the best application concentration.

[0109] In some embodiments, the inorganic salt includes KF, KI, NaCl, MgSO4, Na2CO3, Na2SO4, NaBr, NaHCO3, Na2SO3, and preferably the inorganic salt is KI.

[0110] In some embodiments, the optimal concentration optimization part of the inorganic salt agglomerating agent mainly refers to the final concentration after mixing with the nanosol and the sample, and the preferred concentration range is 50 to 200 mmol / L.

[0111] In some embodiments, the quaternary ammonium oxime reactivator detection part, the detection steps in the biological sample are: take an appropriate amount of enhanced substrate and centrifuge it, discard the supernatant, slowly add the enhancement reagent to the bottom of the enhanced substrate, dilute the biological sample by multiples, quickly add it to the enhanced substrate, mix it thoroughly, and then detect it with a portable or handheld Raman spectrometer.

[0112] In some embodiments, in order to present a good linear relationship in the working curve drawing part of the quaternary ammonium oxime reactivator of different concentrations, 1 ml of the enhanced substrate is taken in an EP tube and centrifuged to discard the supernatant, and the bottom nanogold sol is mixed evenly to maintain the uniformity of the morphology of the nanogold sol, and 10 μL of the mixed nanogold sol is taken and dispensed in an EP tube for use.

[0113] In some embodiments, the detection process is characterized in that the Raman spectrometer used is a portable Raman spectrometer, the laser power of which is 0-300 mW, preferably 60-120 mW, and the best detection substrate is pinSHINs nanosol.

[0114] In some embodiments, the optimal sequence of the steps for detecting the reactivating agent in the biological sample is: ① centrifuging the nanosol and aspirating the supernatant; ② adding the enhancing agent to the nanosol; ③ adding the sample to be tested; ④ collecting the SERS spectrum. This sequence should not be reversed or arbitrarily changed.

[0115] In some embodiments, for tissue samples, proteins and fat tissues therein need to be effectively precipitated first to avoid strong inhibition of the target detection signal. Commonly used protein precipitants include trifluoroacetic acid, formic acid, acetonitrile, sodium chloride, zinc sulfate, etc., preferably sodium chloride, with a concentration of 1 mol / L.

[0116] The present invention is further explained below with reference to specific embodiments.

[0117] Preliminary Examples

[0118] 1. Preparation of AuNPs:

[0119] Gold nanoparticles with a particle size of about 55 nm were prepared using the sodium citrate reduction tetrachloroauric acid method of Frens. 100 mg of tetrachloroauric acid trihydrate was accurately weighed and diluted to 100 ml in a 100 ml volumetric flask to obtain a 1 mg / ml mother solution (the solvent was water). 10 ml of the mother solution was diluted to 100 ml and poured into a 250 ml three-necked flask and heated in an oil bath. The mixture was heated to boiling under rapid stirring. After condensation and reflux, 0.85 ml of 1% trisodium citrate aqueous solution was quickly added. The mixture was heated and stirred for 40 min to prepare AuNPs with a particle size of about 55 nm.

[0120] 2. Preparation of PinSHINs:

[0121] Add 3-aminopropyltrimethoxysilane (APTMS) aqueous solution (1mM) to the prepared 55nm AuNPs, stir for 15min, then add Na2SiO3 aqueous solution (0.54%, w / w) and stir for another 3min to coat the surface of AuNPs to form a SiO2 shell. When the pH of the Na2SiO3 aqueous solution is greater than 11, a porous SiO2 shell with a thickness of 1-2nm can be generated, and then stored in a 4℃ refrigerator for use.

[0122] The prepared AuNPs and PinSHINs were Figure 1 As shown in a and 1b.

[0123] Example 1

[0124] KF, KI, NaCl, MgSO4, Na2CO3, Na2SO4, NaBr, NaHCO3, and Na2SO3 were taken as enhancement reagents and added to the nanosol (i.e., the AuNPs prepared in the preliminary example) after the supernatant was removed after centrifugation, and mixed. 200 μL of 100 ng / ml TMB-4 aqueous solution was added to make the final concentration of the aforementioned inorganic salts 100 mmol / L. After sufficient mixing, SERS detection was performed with a laser power of 60 mW and a detection time of 10 s. The results showed that KI had the best enhancement effect (e.g. Figure 2 a.2b.).

[0125] Example 2

[0126] KI was used as an enhancement reagent and added to the nanosol (i.e., the AuNPs prepared in the preliminary example) after the supernatant was removed after centrifugation, and then 200 μL of 100 ng / ml TMB-4 aqueous solution was added to make the final KI concentrations of 0, 20, 30, 50, 100, 200, 300, 400, and 500 mmol / L, respectively. SERS detection was performed with a laser power of 60 mW and a detection time of 10 s. The results showed that when the final KI concentration was 100 mmol / L, the enhancement effect was optimal (e.g., Figure 3 a.3b.).

[0127] Example 3

[0128] Ultrapure water was used to prepare 1 μg / ml TMB-4, HI-6, Obi, MMB-4 and 10 μg / ml PAM solutions.

[0129] KI was used as an enhancement reagent and added to the nanosol (i.e., the AuNPs prepared in the preliminary example) after the supernatant was removed after centrifugation, and then 200 μL of the aforementioned TMB-4, HI-6, Obi, MMB-4, and PAM aqueous solutions were added respectively to make the final concentration of KI 100 mmol / L. SERS detection was performed, and the detection laser power of TMB-4, HI-6, Obi, and MMB-4 was 60 mW, and the detection laser power of PAM was 180 mW, and the detection time was 10 s. The detection results are shown in Figure 2. Figure 4 As shown. Different reactivators have their own characteristic fingerprints, which can be clearly distinguished. This embodiment shows that the method of the present invention can perform rapid and accurate qualitative analysis on different quaternary ammonium salt oxime reactivators.

[0130] Example 4

[0131] Use ultrapure water to prepare gradient concentrations of TMB-4, HI-6, Obi, and MMB-4 solutions.

[0132] KI was used as an enhancing reagent and added to the nanosol (i.e., AuNPs prepared in the preliminary example) after the supernatant was removed after centrifugation, and then 200 μL of the aforementioned gradient concentrations of TMB-4, HI-6, Obi, and MMB-4 aqueous solutions were added respectively to make the final concentration of KI 100 mmol / L.

[0133] SERS detection was performed on QAO reactivators of different concentrations with a laser power of 120 mW and a detection time of 10 s to establish a standard curve at 1638 cm -1 The peak intensity is proportional to the concentration of QAO reactivator within a certain range. Figure 5a-5hTMB-4 (y = 1210x + 4567, R 2 =0.987), MMB-4(y=1023x+1363, R 2 =0.990), HI-6 (y=1673x+2092, R 2 =0.962), OBi(y=1180x+3110, R 2 =0.996) in the linear range of 0.5-20 ng / ml, and the detection limit was 0.5 ng / ml. This indicates that the method of the present invention can be used for highly sensitive quantitative detection of quaternary ammonium salt oxime reactivators, and does not require sample pretreatment. Its SERS detection only takes 10 s, which is very suitable for rapid detection of large quantities of samples.

[0134] Example 5

[0135] The plasma sample of 50 ppb HI-6 was measured using the self-made AuNPs and pinSHINs enhanced substrates (i.e., the AuNPs and pinSHINs prepared in the preliminary example). 10 μL of 2 mol / L enhancement reagent KI was added to the two enhanced substrates after centrifugation and the supernatant was removed, and 200 μL of the plasma sample was mixed with the enhanced substrates for SERS detection. The laser power was 120 mW and the detection time was 10 s. Figure 6 As shown in the figure, bare gold nanoparticles AuNPs have no signal response, while pinSHINs have obvious signal enhancement. This shows that the ultra-thin SiO2 shell of pinSHINs can effectively protect the Au core, making it more resistant to interference from complex biological matrices.

[0136] Example 6

[0137] PinSHINs (i.e., pinSHINs prepared in the preliminary example) were used as the enhancement substrate to detect HI-6 plasma samples. 10 μL of 2 mol / L enhancement reagent KI was added to the enhancement substrate after centrifugation and the supernatant was removed, and 200 μL of plasma sample was mixed with the enhancement substrate, and placed at room temperature for 0 min, 5 min, 10 min, 20 min, 30 min, 40 min, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, and 24 h. The SERS spectrum of the solution was collected at each time point for SERS detection. The laser power was 120 mW, and the detection time was 10 s. Figure 7 As shown, the plasma contains macromolecular substances and the core-shell structure of pinSHINs, which can stabilize the sample, and the SERS signal can remain unchanged for at least 12 hours.

[0138] Example 7

[0139] Using pinSHINs (ie, pinSHINs prepared in the preliminary example) as the enhanced substrate, the actual plasma sample was measured.

[0140] After injecting a loading dose of HI-6 reactivator solution into mice, blood was collected at 0, 5 min, 15 min, 30 min, and 1 h, and the supernatant was collected after centrifugation.

[0141] 10 μL of 2 mol / L enhancement reagent KI was added to the enhancement substrate after centrifugation and the supernatant was removed, and then 200 μL of the supernatant of the mouse plasma samples taken at the above time points after centrifugation was mixed with the enhancement substrate for SERS detection. The laser power was 120 mW and the detection time was 10 s. Figure 8 As shown, the plasma concentration of the drug is highest at 5 minutes. This method can achieve accurate qualitative and quantitative analysis in actual sample testing.

[0142] Example 8

[0143] Since the brain contains a large amount of complex matrix, which severely inhibits Raman signals, brain homogenate needs to be tested after protein precipitation. Prepare brain homogenate with HI-6 concentrations of 500ppb, 200ppb, 100ppb, 50ppb, 20ppb, and 10ppb, take 800μl of the above brain homogenate and add 200μl of different precipitants (the final concentration of each precipitant is 1M, and the precipitant is preferably NaCl with a final concentration of 1M), vortex for 3min, 12000rpm / min, and centrifuge for 5min to take the supernatant.

[0144] 10 μL of 2 mol / L enhancement reagent KI was added to the enhancement substrate (i.e., pinSHINs prepared in the preliminary example) after the supernatant was removed after centrifugation and mixed evenly. Then, 200 μL of the brain slurry homogenate after the centrifugation was taken and mixed evenly with the enhancement substrate, and SERS detection was performed. The laser power was 120 mW and the detection time was 10 s. The results are as follows: Fig. 9 ad. Considering the signal intensity and economic benefits, it is recommended to use NaCl as the precipitant. 2 =0.965) with a linear range of 10 to 500 ng / ml.

[0145] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for detecting quaternary ammonium oxime antitoxic agents, comprising: 1) Using surface enhanced Raman spectroscopy, detect the signal of quaternary ammonium salt oxime antitoxic agent in the sample to be tested; 2) determining the type and / or content of the quaternary ammonium oxime antitoxic agent in the sample to be tested based on the signal obtained by the detection; The enhanced substrate of the surface enhanced Raman spectroscopy is a porous shell-isolated gold nanoparticle, the porous shell-isolated gold nanoparticle comprises a core and a shell, the shell covers the core, the core is AuNPs, and the shell is a SiO2 shell; The surface enhanced Raman spectroscopy enhancement agent is KI; The sample to be tested is a brain tissue sample; The brain tissue sample is pre-precipitated to remove protein and / or fat tissue; The precipitant used in the precipitation treatment is sodium chloride.

2. The method of claim 1, wherein The concentration of the enhancing reagent is 50-200 mmol / L, wherein the concentration of the enhancing reagent refers to the final concentration of the enhancing reagent after the enhancing reagent is mixed with the enhancing substrate and the sample to be tested.

3. The method of claim 2, wherein: The concentration of the enhancing agent is 50-150 mmol / L.

4. The method of claim 2, wherein: The concentration of the enhancing agent is 100 mmol / L.

5. The method of claim 1, wherein: Step 1) includes: 1-1) Centrifuge the enhanced substrate and discard the supernatant; 1-2) mixing the enhancing agent with the product obtained in step 1-1); 1-3) mixing the sample to be tested with the product obtained in step 1-2); 1-4) Using a surface enhanced Raman spectrometer, the product obtained in step 1-3) is detected to obtain the signal of the quaternary ammonium salt oxime antitoxic agent in the sample to be tested.

6. The method of claim 5, wherein: Step 1-2) is to slowly add the enhancing reagent to the bottom of the product obtained in step 1-1).

7. The method of claim 1, wherein: The method has at least one of the following characteristics: (1) The laser power of the surface enhanced Raman spectroscopy is 0-300 mW; (2) The detection time of the detection is 8-12 seconds; (3) The detection wavelength of the detection is 785nm; (4) The surface enhanced Raman spectrometer is a portable or handheld Raman spectrometer.

8. The method of claim 7, wherein: The laser power of the surface enhanced Raman spectroscopy is 60-180 mW.

9. The method of claim 7, wherein: The laser power of the surface enhanced Raman spectroscopy is 60-120 mW.

10. The method of claim 7, wherein: The detection time of the detection is 10 s.

11. The method of claim 1, wherein: The concentration of the precipitant is 0.5-1.5 mol / L, wherein the concentration of the precipitant refers to the final concentration of the precipitant after the precipitant is mixed with the tissue sample.

12. The method of claim 11, wherein: The concentration of the precipitant is 1 mol / L.

13. The method of claim 1, wherein: The quaternary ammonium salt oxime antitoxic agent is selected from TMB-4, HI-6, Obi, MMB-4 and PAM.

14. The method of claim 1, wherein: The particle size of the AuNPs is 50-60 nm.

15. The method of claim 14, wherein: The particle size of the AuNPs is 55 nm.

16. The method of claim 1, wherein: The thickness of the SiO2 shell layer is 1-2 nm.

17. The method of claim 1, wherein: The porous shell-isolated gold nanoparticles are prepared by the following method: 1) first mixing AuNPs with 3-aminopropyltrimethoxysilane; 2) performing a second mixing of the first mixed product and Na2SiO3 to obtain the porous shell-isolated gold nanoparticles.

18. The method of claim 17, wherein: The 3-aminopropyltrimethoxysilane is provided in a form dissolved in water.

19. The method of claim 18, wherein: In the aqueous solution of 3-aminopropyltrimethoxysilane, the concentration of 3-aminopropyltrimethoxysilane is 0.5-1.5 mM.

20. The method of claim 19, wherein: The concentration of the 3-aminopropyltrimethoxysilane was 1 mM.

21. The method of claim 17, wherein: The Na2SiO3 is provided in a form dissolved in water.

22. The method of claim 21, wherein: In the Na2SiO3 aqueous solution, the concentration (w / w) of Na2SiO3 is 0.5%-0.6%.

23. The method of claim 22, wherein: In the Na2SiO3 aqueous solution, the concentration (w / w) of Na2SiO3 is 0.54%.

24. The method of claim 21, wherein: The pH of the Na2SiO3 aqueous solution is greater than 11.

25. The method of claim 17, wherein: The first mixing time is 10-20 minutes.

26. The method of claim 25, wherein: The first mixing time is 15 minutes.

27. The method of claim 17, wherein: The second mixing time is 2-5 minutes.

28. The method of claim 27, wherein: The second mixing time is 3 minutes.

29. The method of claim 1, wherein: The AuNPs are prepared by Frens' sodium citrate reduction of tetrachloroauric acid method.

30. The method of claim 1, wherein: The AuNPs are prepared by the following method: 1) mixing tetrachloroauric acid trihydrate with water to obtain a mother solution; 2) Take part of the mother liquor and dilute it with water; 3) The diluted mother liquor is heated to boiling, and after condensation and reflux, a trisodium citrate aqueous solution is added thereto, and heating is continued for 30-50 minutes to obtain the AuNPs.

31. The method of claim 30, wherein: Step 3) is to heat the diluted mother liquor to boiling, add trisodium citrate aqueous solution thereto after condensation and reflux, and continue heating for 40 minutes to obtain the AuNPs.

32. The method of claim 30, wherein: In the mother liquor, the concentration of tetrachloroauric acid trihydrate is 0.5-1.5 mg / mL.

33. The method of claim 32, wherein: In the mother solution, the concentration of tetrachloroauric acid trihydrate is 1 mg / mL.

34. The method of claim 30, wherein: When a portion of the mother liquor is diluted with water, the dilution multiple is 8-12 times.

35. The method of claim 34, wherein: When a portion of the mother liquor is diluted with water, the dilution multiple is 10 times.

36. The method of claim 30, wherein: In the trisodium citrate aqueous solution, the concentration (w / w) of the trisodium citrate is 0.5-1.5%.

37. The method of claim 36, wherein: In the trisodium citrate aqueous solution, the concentration (w / w) of the trisodium citrate is 1%.

38. The method of claim 30, wherein: The volume ratio of the diluted mother liquor to the trisodium citrate aqueous solution is 100:0.8-0.

9.

39. The method of claim 38, wherein: The volume ratio of the diluted mother liquor to the trisodium citrate aqueous solution is 100:0.

85.

40. The method of claim 1, wherein: The determination of the type of quaternary ammonium oxime antitoxic agent in the sample to be tested is carried out by the following steps: 1) Using surface enhanced Raman spectroscopy, detect the signal of quaternary ammonium salt oxime antitoxic agent in the sample to be tested; 2-1-A) comparing the detected signal with the characteristic fingerprint spectrum of each quaternary ammonium salt oxime antitoxic agent; 2-2-A) Based on the comparison results, determine the type of quaternary ammonium oxime antitoxic agent in the sample to be tested.

41. The method of claim 1, wherein: The determination of the content of quaternary ammonium oxime antitoxic agents in the sample to be tested is carried out by the following steps: 1) Using surface enhanced Raman spectroscopy, detect the signal of quaternary ammonium salt oxime antitoxic agent in the sample to be tested; 2-1-B) Using a surface enhanced Raman spectrometer, the signals of the quaternary ammonium salt oxime antitoxic agents of different preset concentrations are detected; 2-2-B) based on different preset concentrations of the quaternary ammonium salt oxime anti-toxic agent and the signal intensities of the different preset concentrations of the quaternary ammonium salt oxime anti-toxic agent obtained by detection, drawing a signal intensity-concentration working curve; 2-3-B) Based on the signal of the quaternary ammonium salt oxime anti-toxic agent in the sample to be tested obtained by detection and the signal intensity-concentration working curve drawn, the content of the quaternary ammonium salt oxime anti-toxic agent in the sample to be tested is determined.

42. The method of claim 41, wherein In step 2-1-B), when detecting the signals of quaternary ammonium oxime antitoxic agents of different preset concentrations, the enhanced substrate of each concentration is prepared by the following method: a first preset volume of the enhanced substrate is placed in an EP tube for centrifugation and the supernatant is discarded, and the enhanced substrate remaining at the bottom is mixed evenly, and then a second preset volume of the enhanced substrate is taken from the enhanced substrate remaining at the bottom and dispensed into an EP tube for standby use.

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