Rapid detection method for lacidipine and fluvoxamine in accident site serum

Abstract

The invention discloses a rapid detection method for lacidipine and fluvoxamine in accident site serum, and relates to the technical field of trace drug detection in biological samples. The method comprises the following steps: preparing a surface enhanced Raman spectrum substrate with gold nanoparticles loaded on amino modified MIL-101 (Cr), and combining sample extraction, oscillation adsorption, centrifugal separation and Raman spectrum detection: mixing a sample solution and the substrate according to a volume ratio of 1: 3, carrying out oscillation adsorption at 35 DEG C for 30-40 minutes, then carrying out centrifugal collection, collecting a spectrum signal by adopting a 785nm laser, and calculating the Raman spectrum based on the spectrum signal. And the qualitative analysis of the target drug is realized by identifying the characteristic peaks of 1632 cm <-1 > + / -2 cm <-1 > and 1060 cm <-1 > + / -2 cm <-1 >. The method has the characteristics of rapidness, high efficiency, no need of complex pretreatment and high sensitivity, is suitable for rapid detection of lacidipine and fluvoxamine in a serum sample on an accident site, and provides technical support for clinical first aid and judicial expertise.

Description

Technical Field

[0001] The invention relates to the technical field of trace drug detection in biological samples, in particular to a rapid detection method for lacidipine and fluvoxamine in serum at an accident site. Background Art

[0002] In the first aid and forensic identification of accidents, the rapid and accurate detection of specific drug components in serum is of great significance for analyzing the cause of the accident and guiding the treatment plan. Lacidipine and fluvoxamine are commonly used clinical calcium channel blockers and selective serotonin reuptake inhibitors, respectively, and there is an urgent need for rapid analysis of their blood drug concentrations. However, the existing detection methods have the following technical difficulties: Although traditional chromatography-mass spectrometry technology (such as HPLC-MS / MS, GC-MS) can achieve accurate quantification, it requires complex sample pretreatment (such as liquid-liquid extraction, solid phase extraction), gradient elution and mass spectrometry ionization process. A single detection usually takes more than 2 hours, and the equipment is bulky and complicated to operate, which makes it difficult to meet the needs of rapid on-site detection. Although immunoassay has a certain degree of portability, its antibody specificity is easily interfered by the serum matrix, and the detection sensitivity (usually ng / mL level) is difficult to cope with the identification needs of low-concentration drug residues. Surface enhanced Raman spectroscopy (SERS) technology has shown application potential in the field of drug detection due to its advantages such as rapid response and no labeling. However, existing SERS substrate materials have problems such as low adsorption efficiency and poor stability. For example, conventional gold nanoparticle substrates are susceptible to nonspecific adsorption of biomacromolecules such as proteins in serum, resulting in the masking of target molecule signals; while MOFs materials have a high specific surface area, the unmodified pore structure has insufficient adsorption selectivity for drug molecules, and the metal sites are prone to coordination competition with the biological matrix, reducing the detection sensitivity. In addition, the traditional substrate materials have poor dispersibility in complex biological samples, making it difficult to achieve efficient enrichment of target molecules, resulting in a detection limit usually higher than 100 ng / mL, which cannot meet the requirements of trace analysis. To solve the above problems, researchers have tried to improve the performance of the substrate through material modification. For example, amino modification is introduced to enhance the affinity for polar drug molecules, but the amination process is prone to cause the collapse of the MOFs structure and affect the stability of the material; although the use of precious metal nanoparticle loading technology can enhance the Raman enhancement effect, the particle size distribution and loading amount of the nanoparticles are difficult to accurately control, resulting in poor detection repeatability of different batches of substrates. In addition, the existing methods often ignore the complexity of biological samples when optimizing adsorption conditions, and do not systematically examine the effects of parameters such as temperature and oscillation time on the interaction between drug molecules and substrates, resulting in insufficient reliability of actual detection results. These technical bottlenecks restrict the development of on-site rapid detection technology. There is an urgent need to develop a detection method that combines high sensitivity, anti-interference ability and ease of operation to meet the needs of instant analysis at the accident scene. Summary of the invention

[0003] One purpose of the present invention is to solve the problems of existing detection methods, such as complex operation, long detection cycle and insufficient sensitivity, which makes it difficult to meet the needs of rapid detection at the accident scene.

[0004] Another object of the present invention is to solve the problem that the amino modification degree and the precious metal loading amount of the base material are difficult to accurately control, which affects the material stability and detection repeatability.

[0005] Another purpose of the present invention is to solve the problem that the traditional amination process easily leads to the collapse of the MOFs structure and reduces the adsorption performance of the material.

[0006] Another object of the present invention is to solve the problem that the method for loading precious metal nanoparticles is difficult to achieve precise control of particle size and spacing, which affects the Raman enhancement effect.

[0007] Another object of the present invention is to solve the problem that the pre-treatment steps of serum samples are complicated and interfering substances are easily introduced.

[0008] Another object of the present invention is to solve the problems of low efficiency and uneven contact between target molecules and substrate materials in traditional adsorption methods.

[0009] Another object of the present invention is to solve the problem that the dispersion of the base material is poor during the vortex mixing process, which affects the adsorption effect.

[0010] Another object of the present invention is to solve the problem that the fluid mechanics conditions are not optimized, resulting in unstable adsorption process.

[0011] Another object of the present invention is to solve the problem that the dispersion of the substrate material on the glass slide is poor, which affects the collection of spectral signals.

[0012] Another object of the present invention is to solve the problem that the characteristic peak identification standard is unclear and prone to misjudgment.

[0013] In order to achieve the purpose and other advantages of the present invention, the present invention provides a rapid detection method of lacidipine and fluvoxamine in serum at an accident scene, comprising: Step 1: preparing a surface enhanced Raman spectroscopy substrate, wherein the substrate is composed of gold nanoparticles loaded on the surface of an amino-modified MIL-101 (Cr) metal organic framework material, wherein the particle size of the gold nanoparticles is 25-30 nm, and the pore size of the MIL-101 (Cr) is 3.4-3.6 nm; Step 2, extracting the components to be tested in the serum sample to obtain a sample solution; Step 3: Mix the sample solution and the substrate in a volume ratio of 1:3, and adsorb by shaking at a constant temperature of 35° C. for 30-40 minutes, and then centrifuge at 10,000 rpm for 5-8 minutes to collect the substrate material; Step 4: Evenly disperse the adsorbed substrate material on the surface of the quartz slide, use a 785 nm laser as an excitation light source, and collect Raman spectrum signals under the conditions of a laser power of 50 mW and an integration time of 10 s; Step 5: Identify whether there is a peak at 1632cm in the Raman spectrum -1 ±2 cm -1 and 1060 cm -1 ±2 cm -1 characteristic peaks to determine whether the serum sample contains lacidipine and / or fluvoxamine.

[0014] Preferably, in the method for rapid detection of lacidipine and fluvoxamine in serum at the scene of an accident of the present invention, in step 1, the amino modification degree of the metal organic framework material is controlled to contain 2-3 amino groups in each crystal unit.

[0015] Preferably, in the rapid detection method for lacidipine and fluvoxamine in serum at the accident scene of the present invention, in step one, the amino-modified MIL-101 (Cr) metal organic framework material is prepared by immersing MIL-101 (Cr) crystals in a mixed solution of 2-aminoterephthalic acid and N,N-dimethylformamide, and subjecting the mixture to a hydrothermal reaction at 120° C. for 12 hours, and the molar ratio of the 2-aminoterephthalic acid to MIL-101 (Cr) is 1:2.

[0016] Preferably, in the rapid detection method of lacidipine and fluvoxamine in serum at the accident scene of the present invention, in step 1, the gold nanoparticle loading adopts an in-situ reduction method, chloroauric acid and amino-modified MIL-101 (Cr) are dispersed in deionized water at a mass ratio of 1:20, and 0.1 mol / L sodium citrate solution is added as a reducing agent, the molar ratio of chloroauric acid solution to sodium citrate solution is 1:3, and the reaction is carried out at a constant temperature of 80°C for 30 minutes, and the distance between the gold nanoparticles is controlled to be 2-3 nm.

[0017] Preferably, in the rapid detection method for lacidipine and fluvoxamine in the serum at the accident scene of the present invention, step 2 specifically includes: adding ethyl acetate to the serum sample at a volume ratio of 1:2, vortexing for 30 seconds, centrifuging at 3000g for 10 minutes, carefully pipetting the upper ethyl acetate layer into a new centrifuge tube with a glass pipette, and then adding 1 mol / L sulfuric acid aqueous solution at 1 / 10 of the volume of the ethyl acetate layer, vortexing again for 30 seconds, and centrifuging at 3000g for 5 minutes. The resulting clear liquid is the sample solution.

[0018] Preferably, in the rapid detection method for lacidipine and fluvoxamine in serum at the accident scene of the present invention, in step three, the oscillation adsorption step adopts a two-stage mixing method, in the first stage, the substrate is added to the sample solution twice, 60% of the total volume of the substrate is added for the first time and vortexed at 2500 rpm for 30 seconds, and the remaining 40% of the substrate is added for the second time and vortexed at 1500 rpm for 60 seconds; in the second stage, the mixed solution is placed in a horizontal oscillator, the amplitude is set to 5 mm and the frequency is set to 200 times / min, and reciprocating oscillation is performed along the axial direction of the sample tube at a constant temperature of 35°C. The total duration of the two-stage mixing process is controlled to be 30 minutes, of which the first stage takes 2-3 minutes.

[0019] Preferably, in the rapid detection method for lacidipine and fluvoxamine in serum at the accident scene of the present invention, in step three, the inclination angle of the sample tube in the vortex mixing step is maintained at 45°, so that the base material spirally moves along the tube wall to form a liquid film with a thickness of 0.2-0.3 mm.

[0020] Preferably, in the rapid detection method for lacidipine and fluvoxamine in serum at the accident scene of the present invention, in step three, the Reynolds number of the mixed liquid during the reciprocating oscillation process is controlled within the range of 200-250, so that a stable velocity boundary layer is formed on the surface of the substrate material, and the boundary layer thickness is 50-80 μm.

[0021] Preferably, in the rapid detection method of lacidipine and fluvoxamine in serum at the scene of an accident of the present invention, in step 4, the substrate material is dispersed on the surface of a quartz glass slide by a microfluidic spotting method, including mixing the substrate material with a 0.05% polyoxyethylene lauryl ether surfactant solution with a pH value of 7.4 at a mass ratio of 1:50, and injecting into a microfluidic nozzle with an inner diameter of 100 μm; the surface of the quartz glass slide is provided with radial guide grooves with a depth of 0.2 μm and a width of 5 μm, and the guide groove spacing is the base. The sample was 2-3 times the average particle size of the base material. During spotting, the quartz slide rotated at a constant speed of 30rpm, and the nozzle movement speed was set to 2mm / s. The spotting process was carried out at a temperature of 25℃ and a humidity of 40%. The droplet volume was controlled to 0.5nL, and the spotting interval was set to 1.2 times the droplet diameter. The quartz slide after spotting was placed in a vacuum dryer and kept at a pressure of -10kPa for 3 minutes to remove residual solvents and increase the binding energy between the base material and the slide surface to 0.5-0.8J / m².

[0022] Preferably, in the rapid detection method of lacidipine and fluvoxamine in serum at the scene of an accident of the present invention, in step 5, when lacidipine is detected, the Raman spectrum at 1632 cm -1 Check whether there is a characteristic peak with a signal-to-noise ratio greater than 10 at the position. If so, it means that the serum sample contains lacidipine. When detecting fluvoxamine, observe the Raman spectrum at 1060cm-1 Whether there is a characteristic peak with a signal-to-noise ratio greater than 10 at the position, if so, it means that the serum sample contains fluvoxamine.

[0023] Beneficial effects: The present invention significantly improves the detection sensitivity and anti-interference ability by optimizing the particle size, pore size and adsorption conditions of the base material, realizes the rapid qualitative analysis of trace drugs in serum samples, and shortens the detection cycle to within 1 hour; accurately controls the amino modification degree and the gold nanoparticle loading ratio to ensure the adsorption selectivity and Raman enhancement effect of the base material, and improves the repeatability of detection of different batches; The amino-modified MOFs material is prepared by a hydrothermal reaction method, which enhances the affinity for polar drug molecules while maintaining the integrity of the crystal structure, and avoids the structural collapse problem caused by the traditional amination process; The in situ reduction method realizes the uniform loading of gold nanoparticles on the MOFs surface, and the nanoparticle spacing is precisely controlled by controlling the reaction conditions to maximize the Raman enhancement effect, and the detection limit can be as low as 10 ng / mL; The optimized liquid-liquid extraction process effectively removes interfering substances such as proteins in serum, simplifies the pretreatment steps, and improves the sample extraction efficiency and purity; The two-stage mixing method combines vortexing and oscillation to promote full contact between the base material and the target molecule, significantly improves the adsorption efficiency, and ensures the reliability of the detection results; The vortex mixing at an inclined angle forms a liquid film, which increases the contact area between the substrate material and the sample solution and further optimizes the uniformity of the adsorption process. By controlling the Reynolds number and the thickness of the boundary layer, the flow state of the mixed liquid is ensured to be stable, the agglomeration of the substrate material is avoided, and the controllability and repeatability of the adsorption process are improved. The microfluidic spotting method combined with the guide groove design achieves uniform dispersion of the substrate material on the glass slide, improves the consistency and intensity of the spectral signal, and reduces the background noise. The clear signal-to-noise ratio threshold standard provides a quantitative basis for the identification of characteristic peaks, reduces subjective misjudgment, and improves the accuracy and repeatability of the test results. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The Raman spectra of different concentrations of lacidipine in the present invention are shown in FIG. 1 , where, from bottom to top, the concentrations of lacidipine are 0 ng / mL, 10 ng / mL, 50 ng / mL, 100 ng / mL, and 500 ng / mL, respectively; Figure 2 1 is a Raman spectrum diagram corresponding to different concentrations of fluvoxamine of the present invention. In the diagram, from bottom to top, the concentrations of fluvoxamine are 0 ng / mL, 12 ng / mL, 24 ng / mL, 50 ng / mL, and 100 ng / mL, respectively. DETAILED DESCRIPTION

[0025] The present invention is described in further detail below so that those skilled in the art can implement it according to the description.

[0026] According to one embodiment of the present invention, during the preparation of the substrate, gold nanoparticles can be synthesized by chloroauric acid solution and sodium citrate reduction method, and the particle size can be controlled within the range of 25-30nm, which can be achieved by adjusting the amount of reducing agent and the reaction temperature (such as heating in a water bath at 80°C for 30 minutes). The amino-modified MIL-101 (Cr) metal organic framework material can be prepared by a hydrothermal method, and the MIL-101 (Cr) crystals and 2-aminoterephthalic acid are mixed in a 2:1 molar ratio, and reacted at 120°C for 12 hours to ensure that the amino group is evenly loaded on the surface of the material. During sample extraction, serum and ethyl acetate are mixed in a volume ratio of 1:2, vortexed for 30 seconds, and centrifuged at 3000g for 10 minutes. The upper organic phase is back-extracted with 1mol / L sulfuric acid, and the clear liquid is taken as the sample solution after centrifugation.

[0027] After the sample solution and the substrate are mixed in a volume ratio of 1:3, they are placed in a constant temperature oscillator at 35°C for 30-40 minutes, with the oscillation frequency set to 200 times / minute and an amplitude of 5mm. After adsorption is completed, the substrate material is collected by centrifugation at 10,000rpm for 5-8 minutes. The centrifuged substrate can be evenly dispersed on the surface of the quartz slide. A 785nm laser is used as the excitation light source, the laser power is set to 50mW, and the integration time is 10 seconds to ensure stable collection of spectral signals. Microfluidic spotting equipment can be used to assist in the dispersion process, and the uniform distribution of the substrate material can be achieved by controlling the nozzle movement speed (such as 2mm / s) and the slide rotation speed (30rpm).

[0028] By analyzing the Raman spectrum at 1632 cm -1 ±2cm -1 and 1060cm -1 ±2cm -1 The presence of characteristic peaks at the sample can be used to determine whether the sample contains the target drug. Experiments have shown that the detection limit of this method for lacidipine and fluvoxamine can reach 10 ng / mL, and a single detection takes less than 1 hour. The amino modification of the substrate material and the gold nanoparticle loading synergistically improve the adsorption selectivity and signal enhancement effect, and reduce serum matrix interference. The optimized oscillating adsorption conditions ensure that the drug molecules are in full contact with the substrate, and the centrifugation step effectively removes unbound substances, improving the detection sensitivity and accuracy. This method does not require complex pre-treatment equipment and is suitable for on-site rapid detection needs.

[0029] Specifically: When preparing the substrate, chloroauric acid and amino-modified MIL-101 (Cr) were dispersed in deionized water at a mass ratio of 1:20, so that the concentration of the chloroauric acid solution was 0.01 mol / L, and then 0.1 mol / L sodium citrate solution was added as a reducing agent, and the molar ratio of chloroauric acid solution to sodium citrate solution was 1:3, and the reaction was carried out at a constant temperature of 80°C for 30 minutes. When processing serum samples, ethyl acetate was added at a volume ratio of 1:2, vortexed for 30 seconds (such as using a VX-200 vortex mixer), centrifuged at 3000g for 10 minutes, and the upper organic phase was taken, and 1 / 10 volume of 1 mol / L sulfuric acid solution was added for back extraction, and the clear liquid was collected after centrifugation again for 5 minutes.

[0030] After the sample solution and substrate are mixed in a volume ratio of 1:3, they are placed in a 35°C constant temperature oscillator (such as MaxQ 4000) with an amplitude of 5 mm and a frequency of 200 times / min for 30 minutes. After adsorption is completed, the substrate material is collected by centrifugation at 10,000 rpm for 5 minutes (using an Eppendorf5424 centrifuge). When dispersing the substrate, a microfluidic spotter (such as Jetlab III) can be used to evenly coat the material on the surface of a quartz slide, and a 785 nm laser (such as Ocean Optics QE65000) is used for excitation. The laser power is set to 50 mW and the integration time is 10 seconds to collect Raman spectral signals. The spotting parameters are controlled as follows: the inner diameter of the nozzle is 100 μm, the rotation speed of the slide is 30 rpm, and the spotting spacing is 1.2 times the droplet diameter to ensure uniform distribution of the substrate.

[0031] Identify 1632cm using spectrum analysis software (such as WiRE 4.0) -1 ±2cm -1 (lacidipine) and 1060cm -1 ±2cm -1 The characteristic peak of fluvoxamine was detected, and the signal-to-noise ratio threshold was set to ≥10 as the positive judgment standard. Experimental data showed that the detection limit of this method could reach 10 ng / mL (such as Figure 1 and Figure 2 As shown). The amino modification of the substrate material and the gold nanoparticles work synergistically to reduce nonspecific adsorption in serum; the optimized oscillation conditions increase the contact area between the drug molecules and the substrate by 40%, and the centrifugal efficiency is increased to 95%. The entire detection process takes ≤1 hour, which is suitable for the needs of rapid screening at the accident site, and provides real-time data support for analyzing the cause of the accident and clinical emergency treatment.

[0032] According to another embodiment of the present invention, the degree of amino modification of the metal organic framework material is quantified by elemental analysis or infrared spectroscopy to ensure that each crystal unit contains 2-3 amino groups. During the preparation process, a hydrothermal reactor (such as Parr KH type) can be used for synthesis, and MIL-101 (Cr) crystals and 2-aminoterephthalic acid are mixed in a 2:1 molar ratio and reacted at 120°C for 12 hours. After the reaction is completed, the unreacted substances are removed by centrifugation (such as Eppendorf 5424 centrifuge, 3000g, 10 minutes) and deionized water washing, and the amino-modified MOFs material is obtained after drying.

[0033] X-ray photoelectron spectroscopy (XPS) was used to detect the degree of amino modification. Experiments showed that each crystal unit contained 2-3 amino groups. The precise degree of amino modification avoided the collapse of the MOFs structure.

[0034] According to another embodiment of the present invention, it is divided into the following aspects: raw material selection, mixing method, and reaction conditions. In terms of raw materials, MIL-101(Cr) crystals can be selected from metal organic framework materials commonly found on the market (such as Cr3O(BDC)3F·nH2O (BDC is terephthalate)), 2-aminoterephthalic acid can be purchased from a chemical reagent supplier, and N,N-dimethylformamide can also be selected as an analytical grade reagent. When mixing, a glass beaker can be used as a container and stirred with a glass rod. A magnetic stirrer (such as IKARCT basic) can be selected on the equipment to assist in mixing. MIL-101(Cr) crystals are added to a beaker containing a mixed solution of 2-aminoterephthalic acid and N,N-dimethylformamide. The molar ratio of 2-aminoterephthalic acid to MIL-101(Cr) is 1:2. The beaker is placed on a magnetic stirrer and stirred at a speed of 300-500 rpm for 10-15 minutes to fully mix the raw materials.

[0035] Transfer the mixed solution to the hydrothermal reactor, place the reactor in a high-temperature oven (such as DHG-9070A high-temperature oven produced by Shanghai Yiheng Scientific Instrument Co., Ltd.), set the reaction temperature to 120°C, and the reaction time to 12 hours. During the reaction, ensure that the temperature in the oven is stable to avoid temperature fluctuations that affect the reaction effect. After the reaction is completed, wait for the reactor to cool naturally to room temperature, then open the reactor and take out the product.

[0036] The crystal structure of the product was detected by X-ray diffraction (XRD) analysis (Bruker's D8 ADVANCE X-ray diffractometer can be used) to verify whether it is an amino-modified MIL-101(Cr) metal organic framework material. The specific surface area and pore size distribution of the material were measured by nitrogen adsorption-desorption experiment (such as ASAP2460 specific surface area and pore size analyzer of McMeretics) to confirm whether its pore size is within the appropriate range. The experimental object is the prepared amino-modified MIL-101(Cr) material. The experimental method is to test according to the operating procedures of the corresponding instrument. Statistical analysis can repeat the experiment many times to calculate the average value and standard deviation of the results. The amino-modified MIL-101(Cr) material prepared by this method has a good crystal structure and a suitable pore size, and can achieve uniform loading of amino groups without destroying the original pore structure, providing a stable basic material for subsequent loading of gold nanoparticles and detection of drugs, and improving the adsorption selectivity and affinity of the material for the target drug.

[0037] According to another embodiment of the present invention, it can be divided into the following aspects: raw material ratio, loading method, and reaction conditions. In terms of raw materials, chloroauric acid uses analytically pure chloroauric acid (HAuCl4); amino-modified MIL-101(Cr) can be prepared according to the previous embodiment. Sodium citrate solution can be prepared with analytically pure sodium citrate, with a concentration of 0.1 mol / L. When mixing, a glass beaker can be used as a container, and chloroauric acid and amino-modified MIL-101(Cr) are added to the beaker at a mass ratio of 1:20, and then deionized water is added to make the concentration of the chloroauric acid solution 0.01 mol / L. A magnetic stirrer (such as IKA RCT basic) can be used to assist in mixing, and the mixture can be stirred at a speed of 200-400 rpm for 5-10 minutes to fully disperse the two in water.

[0038] Place the mixed solution in a constant temperature water bath (such as HH-S2 constant temperature water bath produced by Shanghai Yiheng Scientific Instrument Co., Ltd.) and set the temperature to 80°C. Use a dropper to slowly add 0.1mol / L sodium citrate solution as a reducing agent, stirring while adding. The molar ratio of chloroauric acid solution to sodium citrate solution is 1:3, and the reaction time is controlled to be 30 minutes. During the reaction, ensure that the temperature of the water bath is stable and the stirring is uniform. After the reaction is completed, let the solution cool naturally to room temperature. Then you can use a centrifuge (such as an Eppendorf 5424 centrifuge) at 5000-8000 rpm for 10-15 minutes, collect the precipitate, and wash the precipitate with deionized water 2-3 times to remove excess reagents.

[0039] The particle size and spacing of gold nanoparticles were observed by transmission electron microscopy (TEM, such as JEOL JEM-2100). The experimental object was the prepared amino-modified MIL-101(Cr) material loaded with gold nanoparticles. The experimental method was to disperse a small amount of sample on a copper grid and put it into TEM for observation. Statistical analysis can measure gold nanoparticles in different areas multiple times, calculate the average and standard deviation of the particle size, and the results show that the particle size of gold nanoparticles is between 25-30nm and the spacing is 2-3nm. The Raman enhancement effect of the material was tested by Raman spectrometer (such as Renishaw inVia Raman spectrometer). The substrate material prepared by this method can produce uniform and efficient Raman enhancement hotspots due to the precise control of the particle size and spacing of gold nanoparticles, improve the sensitivity and stability of Raman spectroscopy detection, and provide a good basis for the subsequent rapid detection of lacidipine and fluvoxamine in serum.

[0040] According to another embodiment of the present invention, serum sample processing can be carried out by liquid-liquid extraction, and ethyl acetate is selected as the extraction solvent (analytical grade), and is added to the serum at a volume ratio of 2:1. A 15mL centrifuge tube (such as Corning brand) is used for mixing, and a vortex mixer (such as VX-200 model) can be used to oscillate at 2500rpm for 30 seconds to allow the two phases to fully contact. The centrifugation step can use a desktop centrifuge (such as Eppendorf 5424), set the centrifugal force to 3000g, and the separation time is 10 minutes to ensure that the organic phase and the aqueous phase are completely separated.

[0041] After the upper ethyl acetate phase was transferred to a new centrifuge tube, 1 mol / L sulfuric acid aqueous solution (prepared by diluting concentrated sulfuric acid) was added at a volume ratio of 1 / 10. Vortex again for 30 seconds at a speed of 1500 rpm to transfer the drug molecules from the organic phase to the aqueous phase. The centrifugation conditions were the same as before (3000 g, 5 minutes), and the lower clear liquid was carefully aspirated using a glass pipette to avoid inhaling impurities on the interface.

[0042] The extraction efficiency was verified by high performance liquid chromatography-mass spectrometry (HPLC-MS / MS) with serum samples spiked with known concentrations of lacidipine and fluvoxamine. The extraction recovery test showed that the method achieved a recovery of 85% - 92% for the two drugs, with a relative standard deviation (RSD) of ≤6%. The ethyl acetate extraction effectively removed macromolecular interferences such as proteins in serum, and the sulfuric acid back extraction step enriched the drugs in the aqueous phase, improving the signal-to-noise ratio of subsequent detection. The optimized centrifugation parameters ensured thorough phase separation, reduced the risk of cross-contamination, and made the sample solution meet the SERS substrate adsorption requirements, providing high-quality analytes for rapid detection.

[0043] According to another embodiment of the present invention, it can be divided into two-stage mixing methods, substrate addition ratios, oscillation parameters, etc. In the two-stage mixing, the substrate is added to the sample solution twice in the first stage. The substrate can be a gold nanoparticle prepared according to the above embodiment loaded on an amino-modified MIL-101 (Cr) metal organic framework material. A 1.5mL centrifuge tube can be used as a mixing container. For the first time, 60% of the total volume of the substrate is added, and a pipette (such as an Eppendorf pipette) can be used for precise addition. Then use a vortex mixer (such as a VX-200 model) to vortex mix at 2500rpm for 30 seconds to preliminarily mix the substrate and the sample solution. The remaining 40% of the substrate is added for the second time, and then vortex mixed at 1500rpm for 60 seconds to further promote uniform mixing.

[0044] In the second stage, the mixed solution is placed in a horizontal oscillator (such as the Thermo Scientific MaxQ 4000 horizontal oscillator). Set the amplitude to 5 mm and the frequency to 200 times / minute, and perform reciprocating oscillation along the axis of the sample tube at a constant temperature of 35°C. Constant temperature conditions can be achieved by placing the oscillator in a constant temperature incubator (such as the Shanghai Yiheng DHG-9070A constant temperature incubator). The total duration of the two-stage mixing process is controlled to 30 minutes, of which the first stage takes 2-3 minutes. During the oscillation process, ensure that the sample tube is fixed on the sample rack of the oscillator to avoid shaking or falling.

[0045] The adsorption effect was detected by Raman spectroscopy, and the experimental object was the substrate material after two-stage mixed adsorption treatment. The experimental method is to use a Raman spectrometer (such as Renishaw inVia Raman spectrometer) to collect Raman spectral signals and analyze the intensity of the characteristic peaks of the target drug. Statistical analysis can repeat the experiment many times to calculate the average and standard deviation of the characteristic peak intensity. This two-stage mixing method can make the substrate material fully contact with the sample solution and improve the adsorption efficiency of drug molecules. The vortex mixing in the first stage quickly disperses the substrate, and the horizontal oscillation in the second stage provides a stable adsorption environment. Compared with the traditional single oscillation method, the adsorption efficiency is increased by about 20% - 30%, which provides a more reliable sample for the subsequent accurate detection of lacidipine and fluvoxamine in serum and reduces the detection error.

[0046] According to another embodiment of the present invention, during the vortex mixing step, the inclination angle of the sample tube is fixed to 45° by a custom bracket (eg, using an Eppendorf tube rack adapter) to ensure that the liquid moves in a spiral along the tube wall during mixing.

[0047] During vortex mixing, the base material and the sample solution form a liquid film with a thickness of 0.2-0.3mm on the inner wall of the centrifuge tube. The mixing process is recorded by a high-speed camera (such as the Phantom Miro series), the thickness of the liquid film is measured and the parameters are optimized. Experiments show that when the sample tube is tilted at 45° for mixing, the liquid film is evenly distributed on the tube wall, and the contact area increases by about 30% compared to vertical mixing. The thickness of the liquid film is precisely controlled by adjusting the solution volume (such as 500μL mixed solution) and the inner diameter of the centrifuge tube (10mm).

[0048] The effect of the tilt angle on the adsorption efficiency was verified by comparative experiments: under the same conditions, the Raman signal intensity of the 45° tilted mixing group was 40% higher than that of the vertical mixing group, and the detection limit dropped from 15ng / mL to 10ng / mL. The thinning design of the liquid film reduces substrate agglomeration and increases the contact opportunities between drug molecules and gold nanoparticles. This method improves the uniformity of adsorption, reducing the RSD of different batches of detection from 12% to 8%. The optimized liquid film formation process ensures the efficient dispersion of the substrate material in the serum sample, providing a stable signal basis for subsequent spectral detection.

[0049] According to another embodiment of the present invention, it can be divided into the parameter setting of horizontal oscillation, oscillation direction and adsorption time. During the horizontal oscillation process, a horizontal oscillator (such as Thermo Scientific MaxQ 4000) can be used, and the amplitude is set to 5mm, which can be accurately set by the adjustment knob of the oscillator. The oscillation frequency is set to 200 times / minute, and the frequency can be adjusted according to the control panel of the oscillator. The oscillation direction is reciprocated along the axis of the sample tube. The 1.5mL centrifuge tube containing the sample and the substrate mixture needs to be placed on the sample rack of the oscillator to ensure that the centrifuge tube is consistent with the oscillation direction. The adsorption time is controlled at 30 minutes and can be set by the timing function of the oscillator.

[0050] Place the oscillator in a constant temperature incubator (such as Shanghai Yiheng DHG-9070A) and set the temperature to 35°C to maintain a stable environment for the adsorption process. During the oscillating adsorption process, the substrate material in the sample tube is in full contact with the drug molecules. As the oscillation proceeds, the amino groups on the surface of the substrate interact with the drug molecules, causing the drug molecules to be gradually adsorbed onto the substrate. Due to the combined effects of horizontal oscillation and constant temperature, this adsorption process is more stable and efficient. The mixing situation in the sample tube can be observed every 5 minutes to ensure that the adsorption process is proceeding normally.

[0051] The adsorption effect was detected by Raman spectroscopy, and the experimental object was the substrate material after horizontal oscillation adsorption treatment. The experimental method is to use a Raman spectrometer (such as Renishaw inVia) to collect Raman spectral signals and analyze the intensity of the characteristic peaks of the target drug. Perform multiple experiments (such as 10 times), record the intensity value of the characteristic peak in each experiment, and then calculate the average and standard deviation. The results of statistical analysis showed that the horizontal oscillation adsorption method increased the adsorption amount of drug molecules by about 25% compared with the traditional oscillation method, enhanced the adsorption stability, and reduced the standard deviation of the characteristic peak intensity by 30% compared with the traditional method. This adsorption method can more effectively enrich drug molecules, improve the sensitivity and accuracy of subsequent Raman spectroscopy detection, and provide a reliable sample processing basis for the rapid detection of lacidipine and fluvoxamine in serum.

[0052] According to another embodiment of the present invention, it can be divided into the preparation of the substrate material dispersion solution, the setting of the microfluidic spotting parameters, the design of the guide groove structure and the fixation of the substrate. The substrate material is mixed with a 0.05% polyoxyethylene lauryl ether surfactant solution with a pH value of 7.4 at a mass ratio of 1:50. Among them, the pH value can be adjusted by a precision pH meter (such as Mettler ToledoSevenMulti), and the surfactant can be polyoxyethylene lauryl ether (Brij-35) produced by Sigma-Aldrich. The mixing process uses a magnetic stirrer (such as IKARCT basic) to stir at 200rpm for 5 minutes to ensure uniform dispersion. The mixed solution is injected into a microfluidic nozzle with an inner diameter of 100μm (such as the MFCS series of MicroFab Technologies). The quartz slide (such as Marienfeld SuperFrost Plus) is fixed on a rotating platform (such as KDScientific's 100 series) and rotated at a constant speed of 30rpm. The nozzle movement speed is set to 2mm / s, which can be precisely adjusted by a motion control module (such as Newport ESP301). The spotting process is carried out in a constant temperature and humidity chamber, with the temperature controlled at 25℃±1℃ and the humidity at 40%±5%. The surface of the quartz slide is pre-etched with radial flow grooves with a depth of 0.2μm and a width of 5μm, and the groove spacing is 2-3 times the average particle size of the substrate material. The etching process can be electron beam lithography (such as JEOL JBX-9300FS) combined with plasma etching (such as Oxford Instruments Plasmalab80Plus). After spotting, the slide is placed in a vacuum dryer (such as Buchi V-700) and dried at -10kPa pressure for 3 minutes, and the pressure change is monitored by a vacuum pressure gauge (such as Keller PAA23X).

[0053] The mixed solution is evenly sprayed on a rotating quartz glass slide through a microfluidic system. The flow channel guides the droplets to flow along a radial path, and a uniformly distributed liquid film is formed under the action of centrifugal force. During the drying process, the vacuum environment accelerates the evaporation of the solvent and enhances the van der Waals force between the substrate material and the glass slide. The binding energy is confirmed to be within the range of 0.5-0.8J / m² by testing with a surface tension meter (such as Dataphysics OCA20).

[0054] By combining the microfluidic spotting method with a specially designed quartz slide flow channel, the substrate material is evenly dispersed on the slide, effectively improving the consistency and intensity of the spectral signal and reducing the background noise. The parameters of the spotting process and the drying conditions are precisely controlled to further enhance the binding energy between the substrate material and the slide surface, ensuring the stability and reliability of the detection process. Experimental verification shows that compared with the traditional drop coating method, this spotting method can increase the uniformity of the substrate material distribution to more than 95%, reduce the coefficient of variation (CV) of the characteristic peak intensity to 8%, and increase the sensitivity by 3 times. The flow channel structure design effectively reduces droplet splashing, and the vacuum drying process enhances the substrate stability, ensuring that the difference in spectral signals in different regions is less than 10%.

[0055] According to another embodiment of the present invention, Raman spectroscopy detection uses a 785 nm laser (such as Ocean Optics QE65000), the excitation power is set to 50 mW, and the integration time is 10 seconds. The spectral data is collected and analyzed by WiRE 4.0 software, and the 1632 cm -1 ±2 cm -1 (lacidipine) and 1060 cm -1 ±2 cm -1 The signal-to-noise ratio (S / N) was calculated based on the baseline noise standard deviation, with a threshold of ≥10, which can be automatically calculated by the software.

[0056] The adsorbed substrate material was evenly coated on a quartz glass slide and the spectral data was collected point by point. The spectra of at least 5 different regions were collected for each sample and the average value was taken for analysis. -1 S / N≥10 and peak position deviation≤2cm -1 , it is judged as positive for lacidipine; similarly, 1060 cm -1 If the conditions are met, it is judged as fluvoxamine positive. The experimental data are stored in a database (such as MySQL) to facilitate subsequent tracing and statistical analysis.

[0057] Through the standard solution test, the detection limit of this method for lacidipine is 10ng / mL (S / N=10), and that for fluvoxamine is 12ng / mL. Repeatability experiments show that the RSD of the characteristic peak intensity of repeated detection of the same substrate is ≤7%, and the RSD between different batches of substrates is ≤10%. The quantitative standard of the signal-to-noise ratio threshold effectively reduces subjective misjudgment, making the test results consistent with HPLC-MS / MS at a rate of 98%. This method provides a clear basis for positive judgment for rapid on-site screening and improves the credibility and consistency of the test results.

[0058] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to specific details.

Claims

1. A rapid detection method for lacidipine and fluvoxamine in serum at the scene of an accident, characterized in that: include: Step 1: preparing a surface enhanced Raman spectroscopy substrate, wherein the substrate is composed of gold nanoparticles loaded on the surface of an amino-modified MIL-101 (Cr) metal organic framework material, wherein the particle size of the gold nanoparticles is 25-30 nm, and the pore size of the MIL-101 (Cr) is 3.4-3.6 nm; Step 2, extracting the components to be tested in the serum sample to obtain a sample solution; Step 3: Mix the sample solution and the substrate in a volume ratio of 1:3, and adsorb by shaking at a constant temperature of 35° C. for 30-40 minutes, and then centrifuge at 10,000 rpm for 5-8 minutes to collect the substrate material; Step 4: Evenly disperse the adsorbed substrate material on the surface of the quartz slide, use a 785 nm laser as an excitation light source, and collect Raman spectrum signals under the conditions of a laser power of 50 mW and an integration time of 10 s; Step 5: Identify whether there is a peak at 1632cm in the Raman spectrum -1 ±2 cm -1 and 1060cm -1 ±2 cm -1 characteristic peaks to determine whether the serum sample contains lacidipine and / or fluvoxamine.

2. The rapid detection method for lacidipine and fluvoxamine in serum at the accident scene as claimed in claim 1, characterized in that: In step 1, the amino modification degree of the metal organic framework material is controlled to contain 2-3 amino groups in each crystal unit.

3. The rapid detection method for lacidipine and fluvoxamine in serum at the accident scene as claimed in claim 1, characterized in that: In step 1, the amino-modified MIL-101 (Cr) metal organic framework material is prepared by immersing MIL-101 (Cr) crystals in a mixed solution of 2-aminoterephthalic acid and N,N-dimethylformamide, and subjecting the mixture to a hydrothermal reaction at 120° C. for 12 hours, wherein the molar ratio of 2-aminoterephthalic acid to MIL-101 (Cr) is 1:

2.

4. The rapid detection method for lacidipine and fluvoxamine in serum at the accident scene as claimed in claim 1, characterized in that: In step 1, the gold nanoparticle loading adopts an in-situ reduction method, chloroauric acid and amino-modified MIL-101 (Cr) are dispersed in deionized water at a mass ratio of 1:20, and 0.1 mol / L sodium citrate solution is added as a reducing agent, the molar ratio of chloroauric acid solution to sodium citrate solution is 1:3, and the reaction is carried out at a constant temperature of 80°C for 30 minutes to control the distance between the gold nanoparticles to be 2-3 nm.

5. The rapid detection method for lacidipine and fluvoxamine in serum at the accident scene as claimed in claim 1, characterized in that: Step 2 specifically includes: adding ethyl acetate to the serum sample at a volume ratio of 1:2, vortexing for 30 seconds, centrifuging at 3000g for 10 minutes, carefully pipetting the upper ethyl acetate layer into a new centrifuge tube with a glass pipette, and then adding 1 mol / L sulfuric acid aqueous solution at 1 / 10 of the volume of the ethyl acetate layer, vortexing again for 30 seconds, and centrifuging at 3000g for 5 minutes. The resulting clear solution is the sample solution.

6. The rapid detection method for lacidipine and fluvoxamine in serum at the accident scene as claimed in claim 1, characterized in that: In step three, the oscillation adsorption step adopts a two-stage mixing method. In the first stage, the substrate is added to the sample solution twice. The first time, 60% of the total volume of the substrate is added and vortexed at 2500rpm for 30 seconds, and the second time, the remaining 40% of the substrate is added and vortexed at 1500rpm for 60 seconds. In the second stage, the mixed solution is placed in a horizontal oscillator, and the amplitude is set to 5mm and the frequency is set to 200 times / min. Reciprocating oscillation is performed along the axis of the sample tube at a constant temperature of 35°C. The total duration of the two-stage mixing process is controlled to be 30 minutes, of which the first stage takes 2-3 minutes.

7. The rapid detection method for lacidipine and fluvoxamine in serum at the accident scene as claimed in claim 6, characterized in that: In step three, the sample tube is tilted at a 45° angle during the vortex mixing step, so that the base material moves in a spiral along the tube wall to form a liquid film with a thickness of 0.2-0.3 mm.

8. The rapid detection method for lacidipine and fluvoxamine in serum at the accident scene as claimed in claim 6, characterized in that: In step three, the Reynolds number of the mixed liquid is controlled within the range of 200-250 during the reciprocating oscillation process, so that a stable velocity boundary layer is formed on the surface of the substrate material, and the thickness of the boundary layer is 50-80 μm.

9. The rapid detection method for lacidipine and fluvoxamine in serum at the accident scene as claimed in claim 1, characterized in that: In step 4, the substrate material is dispersed on the surface of a quartz slide by a microfluidic spotting method, including mixing the substrate material with a 0.05% polyoxyethylene lauryl ether surfactant solution with a pH value of 7.4 at a mass ratio of 1:50, and injecting it into a microfluidic nozzle with an inner diameter of 100 μm; a radial guide groove with a depth of 0.2 μm and a width of 5 μm is set on the surface of the quartz slide, and the guide groove spacing is 2-3 times the average particle size of the substrate material. During spotting, the quartz slide rotates at a constant speed of 30 rpm, and the nozzle movement speed is set to 2 mm / s; the spotting process is carried out at a temperature of 25°C and a humidity of 40%, the droplet volume is controlled to 0.5 nL, and the spotting spacing is set to 1.2 times the droplet diameter; the quartz slide after spotting is placed in a vacuum dryer and maintained at a pressure of -10 kPa for 3 minutes to remove residual solvents, so that the binding energy between the substrate material and the slide surface is increased to 0.5-0.8 J / m².

10. The rapid detection method for lacidipine and fluvoxamine in serum at the accident scene according to claim 1, characterized in that: In step 5, when detecting lacidipine, observe the Raman spectrum at 1632 cm -1 Check whether there is a characteristic peak with a signal-to-noise ratio greater than 10 at the position. If so, it means that the serum sample contains lacidipine. When detecting fluvoxamine, observe the Raman spectrum at 1060 cm -1 Whether there is a characteristic peak with a signal-to-noise ratio greater than 10 at the position, if so, it means that the serum sample contains fluvoxamine.

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