Rapid Detection Method for Lacidipine and Fluvoxamine in Serum at the Scene of Accident

Abstract

The present invention discloses a rapid detection method for lacidipine and fluvoxamine in serum at the accident scene, which relates to the technical field of trace drug detection in biological samples. The present invention prepares a surface-enhanced Raman spectroscopy substrate with gold nanoparticles loaded on amino-modified MIL-101(Cr), and combines the steps of sample extraction, oscillating adsorption, centrifugal separation and Raman spectroscopy detection: mixing the sample solution and the substrate at a volume ratio of 1:3, collecting by centrifugation after oscillating adsorption at 35 °C for 30-40 minutes, collecting by centrifugation, and collecting spectral signals with a 785 nm laser. Qualitative analysis of the target drug is achieved by identifying the characteristic peaks at 1632 cm<supgt;‑1< / supgt;±2 cm<supgt;‑1< / supgt> and 1060 cm<supgt;‑1< / supgt>±2 cm<supgt;‑1< / supgt>. This method has the characteristics of rapidity, high efficiency, no need for complex pretreatment and high sensitivity, and is suitable for the rapid detection of lacidipine and fluvoxamine in serum samples at the accident scene, providing technical support for clinical first aid and forensic identification.

Description

Technical Field

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

[0002] In first aid at the accident scene and forensic identification, 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, as commonly used calcium channel blockers and selective serotonin reuptake inhibitors in clinical practice respectively, have an urgent need for rapid analysis of their blood drug concentrations. However, the existing detection methods have the following technical problems: Although traditional chromatography-mass spectrometry techniques (such as HPLC-MS / MS, GC-MS) can achieve accurate quantification, they need to undergo complex sample pretreatment (such as liquid-liquid extraction, solid-phase extraction), gradient elution, and mass spectrometry ionization processes. The single detection usually takes more than 2 hours, and the equipment is large in volume and complex in operation, making it difficult to meet the requirements of on-site rapid detection. Although immunoassay has a certain portability, the specificity of its antibody is easily interfered by the serum matrix, and its detection sensitivity (usually at the ng / mL level) is difficult to meet the identification requirements for low-concentration drug residues. Surface-enhanced Raman spectroscopy (SERS) technology shows application potential in the field of drug detection due to its advantages such as rapid response and label-free. However, the existing SERS substrate materials have problems such as low adsorption efficiency and poor stability. For example, conventional gold nanoparticle substrates are easily affected by non-specific adsorption of biological macromolecules such as proteins in serum, resulting in the masking of the target molecule signal; while MOF materials have a high specific surface area, but 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 dispersion of traditional substrate materials in complex biological samples is poor, 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 substrate performance through material modification. For example, introducing amino modification to enhance the affinity for polar drug molecules, but the amination process is prone to cause the collapse of the MOF structure, affecting the material stability; using noble metal nanoparticle loading technology can improve the Raman enhancement effect, but 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 the adsorption conditions, and do not systematically investigate the influence of parameters such as temperature and oscillation time on the interaction between drug molecules and the substrate, resulting in insufficient reliability of the actual detection results. These technical bottlenecks restrict the development of on-site rapid detection technology, and there is an urgent need to develop a detection method with high sensitivity, anti-interference ability, and simplicity of operation to meet the needs of on-site instant analysis at the accident scene. Summary of the Invention

[0003] One object of the present invention is to solve the problems existing in the existing detection methods, such as complex operation, long detection period, and insufficient sensitivity, which are difficult to meet the rapid detection requirements at the accident scene.

[0004] Another object of the present invention is to solve the problem that it is difficult to precisely control the amino modification degree of the substrate material and the noble metal loading amount, which affects the material stability and detection repeatability.

[0005] Another object of the present invention is to solve the problem that the traditional amination process is prone to cause the collapse of the MOFs structure and reduce the material adsorption performance.

[0006] Another object of the present invention is to solve the problem that it is difficult to precisely control the particle size and spacing in the method for loading noble metal nanoparticles, 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 cumbersome and prone to introduce interfering substances.

[0008] Another object of the present invention is to solve the problem that the traditional adsorption method has low efficiency and the contact between the target molecule and the substrate material is uneven.

[0009] Another object of the present invention is to solve the problem that the dispersion of the substrate 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 hydrodynamic conditions are not optimized, resulting in an 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 spectral signal acquisition.

[0012] Another object of the present invention is to solve the problem that the characteristic peak recognition standard is not clear, which is prone to misjudgment.

[0013] To achieve the objects and other advantages of the present invention, the present invention provides a rapid detection method for lacidipine and fluvoxamine in serum at the accident scene, including:

[0014] Step 1: Prepare a surface-enhanced Raman spectroscopy substrate, which 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 MIL-101(Cr) is 3.4-3.6 nm;

[0015] Step 2: Extract the components to be detected in the serum sample to obtain a sample solution;

[0016] Step 3: Mix the sample solution and the substrate at a volume ratio of 1:3, oscillate and adsorb at a constant temperature of 35 °C for 30-40 minutes, and then centrifuge at 10000 rpm for 5-8 minutes to collect the substrate material;

[0017] Step 4: Uniformly disperse the adsorbed substrate material on the surface of a quartz slide, use a 785 nm laser as the excitation light source, and collect Raman spectral signals under the conditions of a laser power of 50 mW and an integration time of 10 s;

[0018] Step 5: Identify whether there are characteristic peaks at 1632 cm -1 ±2 cm -1 and 1060 cm -1 ±2 cm -1 to determine whether the serum sample contains lacidipine and / or fluvoxamine.

[0019] Preferably, in the rapid detection method for lacidipine and fluvoxamine in serum at the accident scene 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 per crystal unit.

[0020] Preferably, in the rapid detection method for lacidipine and fluvoxamine in serum at the accident scene of the present invention, 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 performing a hydrothermal reaction at 120°C for 12 hours. The molar ratio of 2-aminoterephthalic acid to MIL-101(Cr) is 1:2.

[0021] Preferably, in the rapid detection method for lacidipine and fluvoxamine in serum at the accident scene of the present invention, in Step 1, the gold nanoparticle loading is carried out by 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, 0.1 mol / L sodium citrate solution is added as a reducing agent, the molar ratio of the chloroauric acid solution to the 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 gold nanoparticles to be 2-3 nm.

[0022] Preferably, in the rapid detection method for lacidipine and fluvoxamine in serum at the accident scene of the present invention, in Step 2, it specifically includes: adding ethyl acetate to the serum sample at a volume ratio of 1:2, vortexing for 30 seconds, centrifuging at 3000 g for 10 minutes, carefully sucking the upper ethyl acetate layer to a new centrifuge tube with a glass pipette, 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 3000 g for 5 minutes. The obtained clear liquid is the sample solution.

[0023] Preferably, in the rapid detection method of lacidipine and fluvoxamine in accident scene serum of the present invention, in step three, the oscillating adsorption step adopts a two-stage mixing method. In the first stage, the substrate is added to the sample solution in two portions. For the first addition, 60% of the total volume of the substrate is added and vortex-mixed at 2500 rpm for 30 seconds. For the second addition, the remaining 40% of the substrate is added and vortex-mixed at 1500 rpm for 60 seconds. In the second stage, the mixture is placed on a horizontal shaker, with the amplitude set at 5 mm and the frequency at 200 times per minute, and reciprocally oscillated along the axial direction of the sample tube under the constant temperature condition of 35°C. The total duration of the two-stage mixing process is controlled to be 30 minutes, and the first stage takes 2 - 3 minutes.

[0024] Preferably, in the rapid detection method of lacidipine and fluvoxamine in accident scene serum of the present invention, in step three, in the vortex mixing step, the inclination angle of the sample tube is kept at 45°, so that the substrate material moves spirally along the tube wall to form a liquid film with a thickness of 0.2 - 0.3 mm.

[0025] Preferably, in the rapid detection method of lacidipine and fluvoxamine in accident scene serum of the present invention, in step three, during the reciprocating oscillation process, the Reynolds number of the mixture is controlled within the range of 200 - 250, so as to form a stable velocity boundary layer on the surface of the substrate material, and the boundary layer thickness is 50 - 80 μm.

[0026] Preferably, in the rapid detection method of lacidipine and fluvoxamine in accident scene serum of the present invention, in step four, the substrate material is dispersed on the surface of a quartz slide by microfluidic spotting, 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. The surface of the quartz slide is provided with radial diversion grooves with a depth of 0.2 μm and a width of 5 μm, and the distance between the diversion grooves is 2 - 3 times the average particle diameter of the substrate material. During spotting, the quartz slide rotates at a constant speed of 30 rpm, and the moving speed of the nozzle is set at 2 mm / s. The spotting process is carried out under the conditions of a temperature of 25°C and a humidity of 40%, the volume of the liquid droplet is controlled at 0.5 nL, and the spotting distance is set at 1.2 times the diameter of the liquid droplet. After spotting, the quartz slide is placed in a vacuum dryer and kept under a pressure of -10 kPa for 3 minutes to remove the residual solvent, so that the binding energy between the substrate material and the slide surface is increased to 0.5 - 0.8 J / m².

[0027] Preferably, in the rapid detection method of lacidipine and fluvoxamine in accident scene serum of the present invention, in step five, when detecting lacidipine, observe whether a characteristic peak with a signal-to-noise ratio greater than 10 appears at the position of 1632 cm -1 in the Raman spectrum. If it appears, it indicates 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 it appears, it indicates that the serum sample contains fluvoxamine.

[0028] Beneficial effects:

[0029] By optimizing the particle size, pore size, and adsorption conditions of the substrate material, the present invention significantly improves the detection sensitivity and anti-interference ability, realizes the rapid qualitative analysis of trace drugs in serum samples, and shortens the detection period to within 1 hour; precisely controls the amino modification degree and the loading ratio of gold nanoparticles to ensure the adsorption selectivity and Raman enhancement effect of the substrate material, and improves the repeatability of different batch detections; uses the hydrothermal reaction method to prepare amino-modified MOF materials, enhances the affinity for polar drug molecules while maintaining the integrity of the crystal structure, and avoids the problem of structural collapse caused by the traditional amination process; the in-situ reduction method realizes the uniform loading of gold nanoparticles on the surface of MOFs, precisely regulates the nanoparticle spacing by controlling the reaction conditions, maximizes 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 vortex and oscillation to promote the full contact between the substrate 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, 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 boundary layer thickness, the flow state of the mixed solution is ensured to be stable, the aggregation of the substrate material is avoided, and the controllability and repeatability of the adsorption process are improved; the microfluidic spotting method combined with the diversion groove design realizes the 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 characteristic peak identification, reduces subjective misjudgment, and improves the accuracy and repeatability of the detection results. Description of the drawings

[0030] Figure 1 It is the Raman spectrum diagram corresponding to different concentrations of lacidipine in the present invention. In the figure, 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;

[0031] Figure 2 It is the Raman spectrum diagram corresponding to different concentrations of fluvoxamine in the present invention. In the figure, 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 implementation manners

[0032] The following provides a further detailed description of the present invention so that those skilled in the art can implement it with reference to the text of the specification.

[0033] According to an embodiment of the present invention, during the preparation of the substrate, gold nanoparticles can be synthesized by the chloroauric acid solution and sodium citrate reduction method, with the particle size controlled within the range of 25 - 30 nm, which can be achieved by adjusting the dosage of the reducing agent and the reaction temperature (such as heating in a water bath at 80 °C for 30 minutes). The amino-functionalized MIL-101(Cr) metal-organic framework material can be prepared by the hydrothermal method. Mix MIL-101(Cr) crystals and 2-aminoterephthalic acid in a molar ratio of 2:1 and react at 120 °C for 12 hours to ensure that amino groups are uniformly loaded on the material surface. When extracting the sample, serum and ethyl acetate are mixed at a volume ratio of 1:2, vortexed for 30 seconds, and then centrifuged at 3000 g for 10 minutes. The upper organic phase is back-extracted with 1 mol / L sulfuric acid, and the supernatant is taken as the sample solution after centrifugation.

[0034] After mixing the sample solution and the substrate at a volume ratio of 1:3, place it in a constant temperature oscillator at 35 °C and oscillate for 30 - 40 minutes. The oscillation frequency is set at 200 times / minute, and the amplitude is 5 mm. After adsorption, centrifuge at 10000 rpm for 5 - 8 minutes to collect the substrate material. The centrifuged substrate can be evenly dispersed on the surface of a quartz slide. Use a 785 nm laser as the excitation light source, set the laser power to 50 mW, and the integration time to 10 seconds to ensure stable acquisition of spectral signals. During the dispersion process, a microfluidic spotting device can be used for assistance. By controlling the nozzle moving speed (such as 2 mm / s) and the slide rotation speed (30 rpm), uniform distribution of the substrate material can be achieved.

[0035] By analyzing the presence of characteristic peaks at 1632 cm -1 ±2 cm -1 and 1060 cm -1 ±2 cm -1 in the Raman spectrum, it is judged whether the target drug is contained in the sample. Experiments show that the detection limits of this method for lacidipine and fluvoxamine can reach 10 ng / mL, and the single detection time is less than 1 hour. The amino modification of the substrate material and the loading of gold nanoparticles synergistically improve the adsorption selectivity and signal enhancement effect, reducing serum matrix interference. The optimized oscillation adsorption conditions ensure sufficient contact between drug molecules and the substrate, and the centrifugation separation step effectively removes unbound substances, improving the detection sensitivity and accuracy. This method does not require complex pretreatment equipment and is suitable for on-site rapid detection requirements.

[0036] Specifically: When preparing the substrate, chloroauric acid and amino-modified MIL-101(Cr) are dispersed in deionized water at a mass ratio of 1:20 to make the concentration of the chloroauric acid solution 0.01 mol / L. Then, 0.1 mol / L sodium citrate solution is added as a reducing agent, and the molar ratio of the chloroauric acid solution to the sodium citrate solution is 1:3. The reaction is carried out at a constant temperature of 80 °C for 30 minutes. When processing the serum sample, ethyl acetate is added at a volume ratio of 1:2, vortexed for 30 seconds (such as using a VX-200 vortex mixer), and after centrifuging at 3000 g for 10 minutes, the upper organic phase is taken, and 1 / 10 volume of 1 mol / L sulfuric acid solution is added for back extraction. After centrifuging again for 5 minutes, the supernatant is collected.

[0037] After mixing the sample solution and the substrate at a volume ratio of 1:3, it is placed in a constant temperature oscillator at 35 °C (such as MaxQ 4000), and oscillated for 30 minutes with an amplitude of 5 mm and a frequency of 200 times per minute. After the adsorption is completed, the substrate material is collected by centrifuging at 10000 rpm for 5 minutes (using an Eppendorf 5424 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, excited by a 785 nm laser (such as Ocean Optics QE65000), with the laser power set at 50 mW and the integration time at 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.

[0038] Through a spectral analysis software (such as WiRE 4.0), identify the characteristic peaks at 1632 cm -1 ±2 cm -1 (lacidipine) and 1060 cm -1 ±2 cm -1 (fluvoxamine), and set the signal-to-noise ratio threshold ≥10 as the positive judgment criterion. Experimental data show that the detection limit of this method can reach 10 ng / mL (as shown in Figure 1 and Figure 2 ). The amino modification of the substrate material and the synergistic effect of gold nanoparticles reduce non-specific adsorption in the serum; the optimized oscillation conditions increase the contact area between drug molecules and the substrate by 40%, and the centrifugation efficiency is increased to 95%. The entire detection process takes ≤1 hour, which meets the rapid screening requirements at the accident scene and provides real-time data support for analyzing the accident cause and clinical first aid.

[0039] According to another embodiment of the present invention, the amino modification degree 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 reaction kettle (such as Parr KH type) can be used for synthesis. Mix MIL-101(Cr) crystals with 2-aminoterephthalic acid in a molar ratio of 2:1 and react at 120 °C for 12 hours. After the reaction, unreacted substances are removed by centrifugation (such as using an Eppendorf 5424 centrifuge at 3000g for 10 minutes) and washing with deionized water, and the amino-modified MOFs material is obtained after drying.

[0040] The amino modification degree is detected by X-ray photoelectron spectroscopy (XPS). Experiments show that each crystal unit contains 2-3 amino groups. The precise amino modification degree avoids the collapse of the MOFs structure.

[0041] According to yet another embodiment of the present invention, it is divided into aspects such as raw material selection, mixing method, and reaction conditions. In terms of raw materials, MIL-101(Cr) crystals can be selected from common metal-organic framework materials on the market (such as Cr 3 O(BDC) 3 F·nH 2 O (BDC is terephthalate)), 2-aminoterephthalic acid can be purchased from chemical reagent suppliers, 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. Add MIL-101(Cr) crystals to the beaker containing the mixed solution of 2-aminoterephthalic acid and N,N-dimethylformamide. The molar ratio of 2-aminoterephthalic acid to MIL-101(Cr) is 1:2. Place the beaker on the magnetic stirrer and stir at a speed of 300-500 revolutions per minute for 10-15 minutes to make the raw materials fully mixed and uniform.

[0042] Transfer the mixed solution to a hydrothermal reaction kettle, put the reaction kettle into a high-temperature oven (such as the DHG-9070A high-temperature oven produced by Shanghai Yiheng Scientific Instruments 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 the influence of temperature fluctuations on the reaction effect. After the reaction, wait for the reaction kettle to cool naturally to room temperature, then open the reaction kettle and take out the product.

[0043] The crystal structure of the product was detected by X-ray diffraction (XRD) analysis (using a D8 ADVANCE X-ray diffractometer from Bruker) to verify whether it was an amino-functionalized MIL-101(Cr) metal-organic framework material. The specific surface area and pore size distribution of the material were measured by nitrogen adsorption-desorption experiments (such as an ASAP2460 specific surface area and pore size analyzer from Micromeritics) to confirm whether its pore size was within the appropriate range. The experimental object was the prepared amino-functionalized MIL-101(Cr) material. The experimental method was to conduct tests according to the operating procedures of the corresponding instruments. Statistical analysis could repeat the experiment multiple times and calculate the average value and standard deviation of the results. The amino-functionalized MIL-101(Cr) material prepared by this method had a good crystal structure and appropriate pore size, and could achieve uniform loading of amino groups without damaging the original pore structure, providing a stable base material for subsequent loading of gold nanoparticles and drug detection, and improving the adsorption selectivity and affinity of the material for the target drug.

[0044] According to another embodiment of the present invention, it can be divided into aspects such as raw material ratio, loading method, and reaction conditions. In terms of raw materials, analytical grade chloroauric acid (HAuCl 4 ) was selected; the amino-functionalized MIL-101(Cr) could be prepared according to the previous embodiment. The sodium citrate solution could be prepared with analytical grade sodium citrate, and the concentration was 0.1 mol / L. When mixing, a glass beaker could be used as the container, and chloroauric acid and amino-functionalized MIL-101(Cr) were added to the beaker at a mass ratio of 1:20, and then deionized water was added to make the concentration of the chloroauric acid solution 0.01 mol / L. A magnetic stirrer (such as an IKA RCT basic) could be used to assist in mixing, and it was stirred at a speed of 200 - 400 revolutions per minute for 5 - 10 minutes to fully disperse the two in water.

[0045] The above mixed solution was placed in a constant temperature water bath (such as an HH-S2 constant temperature water bath produced by Shanghai Yiheng Scientific Instruments Co., Ltd.), and the temperature was set to 80 °C. A 0.1 mol / L sodium citrate solution was slowly added dropwise as a reducing agent with stirring. The molar ratio of the chloroauric acid solution to the sodium citrate solution was 1:3, and the reaction time was controlled for 30 minutes. During the reaction, it was necessary to ensure that the temperature of the water bath was stable and the stirring was uniform. After the reaction, the solution was allowed to cool naturally to room temperature. Then a centrifuge (such as an Eppendorf 5424 centrifuge) could be used to centrifuge at a speed of 5000 - 8000 revolutions per minute for 10 - 15 minutes to collect the precipitate, and the precipitate was washed with deionized water 2 - 3 times to remove the excess reagents.

[0046] The particle size and spacing of gold nanoparticles were observed by transmission electron microscopy (TEM, such as JEOL JEM - 2100), and 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 the sample on a copper grid and place it in the TEM for observation. Statistical analysis could measure the gold nanoparticles in different regions multiple times and calculate the average value and standard deviation of the particle size. The results showed that the particle size of the gold nanoparticles was between 25 - 30 nm and the spacing was between 2 - 3 nm. The Raman enhancement effect of the material was tested by a Raman spectrometer (such as Renishaw inVia Raman spectrometer). The substrate material prepared by this method, due to the precise control of the particle size and spacing of the gold nanoparticles, could generate uniform and efficient Raman enhancement hotspots, improving the sensitivity and stability of Raman spectroscopy detection, providing a good basis for the subsequent rapid detection of lacidipine and fluvoxamine in serum.

[0047] According to another embodiment of the present invention, liquid - liquid extraction can be used for serum sample treatment. Ethyl acetate was selected as the extraction solvent (analytical - pure grade) and added to the serum at a volume ratio of 2:1. A 15 mL centrifuge tube (such as Corning brand) was used for mixing, and it could be oscillated at 2500 rpm for 30 seconds by a vortex mixer (such as VX - 200 type) to make the two phases contact fully. For the centrifugation step, a tabletop centrifuge (such as Eppendorf 5424) could be used, setting a centrifugal force of 3000 g and a separation time of 10 minutes to ensure complete stratification of the organic phase and the aqueous phase.

[0048] After the upper ethyl acetate phase was transferred to a new centrifuge tube, a 1 mol / L sulfuric acid aqueous solution (prepared by diluting concentrated sulfuric acid) was added at a volume ratio of 1 / 10. It was vortex - oscillated again for 30 seconds with the rotation speed controlled at 1500 rpm to transfer the drug molecules from the organic phase to the aqueous phase. The centrifugation conditions were the same as the previous time (3000 g, 5 minutes), and a glass pipette was used to carefully aspirate the lower clear liquid, avoiding inhaling impurities at the interface.

[0049] The extraction efficiency was verified by high - performance liquid chromatography - tandem mass spectrometry (HPLC - MS / MS), and the experimental object was the serum samples added with known concentrations of lacidipine and fluvoxamine. The extraction recovery test showed that the recovery rates of the two drugs by this method reached 85% - 92%, and the relative standard deviation (RSD) ≤6%. Ethyl acetate extraction effectively removed macromolecular interferents such as proteins in the 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 complete phase separation, reduced the risk of cross - contamination, and made the sample solution meet the adsorption requirements of the SERS substrate, providing high - quality analytes for rapid detection.

[0050] According to another embodiment of the present invention, it can be divided into aspects such as two-stage mixing method, substrate addition ratio, oscillation parameters, etc. In the two-stage mixing, the substrate is added to the sample solution in two times in the first stage. The substrate can be gold nanoparticles loaded on amino-modified MIL-101(Cr) metal-organic framework material prepared according to the foregoing embodiment. A 1.5 mL centrifuge tube can be used as the mixing container. 60% of the total volume of the substrate is added for the first time, and a pipette (such as an Eppendorf pipette) can be used for precise addition. Then, a vortex mixer (such as a VX-200 type) is used to vortex mix at 2500 rpm 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 1500 rpm for 60 seconds to further promote uniform mixing.

[0051] In the second stage, the mixed solution is placed in a horizontal shaker (such as a Thermo Scientific MaxQ 4000 horizontal shaker). The amplitude is set to 5 mm and the frequency is 200 times / minute, and reciprocating oscillation is carried out along the axial direction of the sample tube under the constant temperature condition of 35°C. The constant temperature condition can be achieved by placing the shaker in a constant temperature incubator (such as a Shanghai Yiheng DHG-9070A constant temperature incubator). The total duration of the two-stage mixing process is controlled to be 30 minutes, and the first stage takes 2-3 minutes. During the oscillation process, ensure that the sample tube is fixed on the sample rack of the shaker to avoid shaking or falling.

[0052] The adsorption effect is detected by Raman spectroscopy, and the experimental object is the substrate material after two-stage mixing adsorption treatment. The experimental method is to use a Raman spectrometer (such as a 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 multiple times and calculate the average value 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%, providing a more reliable sample for the subsequent accurate detection of lercanidipine and fluvoxamine in serum and reducing the detection error.

[0053] According to another embodiment of the present invention, in the vortex mixing step, the inclination angle of the sample tube is fixed at 45° through a customized bracket (such as using an Eppendorf tube rack adapter) to ensure that the liquid moves spirally along the tube wall during mixing.

[0054] During vortex mixing, a liquid film with a thickness of 0.2 - 0.3 mm is formed between the substrate material and the sample solution 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 liquid film thickness 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 liquid film thickness is precisely controlled by adjusting the solution volume (such as 500 μL of the mixed solution) and the inner diameter of the centrifuge tube (10 mm).

[0055] The influence of the tilt angle on the adsorption efficiency is verified through comparative experiments: under the same conditions, the Raman signal intensity of the 45° tilt mixing group is 40% higher than that of the vertical mixing group, and the detection limit is reduced from 15 ng / mL to 10 ng / mL. The thinning design of the liquid film reduces the substrate aggregation and increases the contact opportunities between drug molecules and gold nanoparticles. This method improves the adsorption uniformity, reducing the RSD of different batch detections 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.

[0056] According to another embodiment of the present invention, it can be divided into parameter settings of horizontal oscillation, oscillation direction, and adsorption time. During horizontal oscillation, a horizontal oscillator (such as Thermo Scientific MaxQ 4000) can be used, and the amplitude is set to 5 mm, which can be precisely set through the adjustment knob of the oscillator. The oscillation frequency is set to 200 times per minute, and the frequency can be adjusted according to the control panel of the oscillator. The oscillation direction is reciprocating oscillation along the axial direction of the sample tube. The 1.5 mL centrifuge tube containing the sample and substrate mixed solution 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 through the timing function of the oscillator.

[0057] The oscillator is placed in a constant temperature incubator (such as Shanghai Yiheng DHG - 9070A), and the temperature is set to 35°C to maintain a stable environment for the adsorption process. During the oscillatory adsorption process, the substrate material in the sample tube is in full contact with the drug molecules. As the oscillation progresses, the amino groups on the substrate surface interact with the drug molecules, causing the drug molecules to be gradually adsorbed onto the substrate. Due to the combined action 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 the normal progress of the adsorption process.

[0058] The adsorption effect was detected by Raman spectroscopy. The experimental object was the substrate material after horizontal oscillation adsorption treatment. The experimental method was 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. Multiple experiments (such as 10 times) were carried out, and the intensity values of the characteristic peaks in each experiment were recorded, and then the average value and standard deviation were calculated. 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 the standard deviation of the characteristic peak intensity decreased by 30% compared with the traditional method. This adsorption method can enrich drug molecules more effectively, 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.

[0059] According to another embodiment of the present invention, it can be divided into the preparation of the substrate material dispersion solution, the setting of microfluidic spotting parameters, and the design of the diversion groove structure and the fixation of the substrate. The substrate material was 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 Toledo SevenMulti), and the surfactant can be polyoxyethylene lauryl ether (Brij-35) produced by Sigma-Aldrich. The mixing process was stirred with a magnetic stirrer (such as IKARCT basic) at 200 rpm for 5 minutes to ensure uniform dispersion. The mixed solution was injected into a microfluidic nozzle with an inner diameter of 100 μm (such as the MFCS series of MicroFab Technologies). A quartz slide (such as Marienfeld SuperFrost Plus) was fixed on a rotating platform (such as the 100 series of KDScientific) and rotated at a constant speed of 30 rpm. The nozzle moving speed was set at 2 mm / s, and precise adjustment can be achieved through a motion control module (such as Newport ESP301). The spotting process was carried out in a constant temperature and humidity chamber, with the temperature controlled at 25°C ± 1°C and the humidity at 40% ± 5%. Radial diversion grooves with a depth of 0.2 μm and a width of 5 μm were pre-etched on the surface of the quartz slide, and the groove spacing was 2-3 times the average particle diameter of the substrate material. The etching process can adopt electron beam lithography (such as JEOL JBX-9300FS) combined with plasma etching (such as Oxford Instruments Plasmalab80Plus). After spotting, the slide was placed in a vacuum dryer (such as Buchi V-700) and dried at a pressure of -10 kPa for 3 minutes, and the pressure change was monitored by a vacuum pressure gauge (such as Keller PAA23X).

[0060] The mixed solution is evenly sprayed onto a rotating quartz slide through a microfluidic system. The flow guiding groove 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 volatilization of the solvent, enhancing the van der Waals force between the substrate material and the slide. The binding energy is tested and confirmed to be in the range of 0.5 - 0.8 J / m² by a surface tensiometer (such as Dataphysics OCA20).

[0061] Through the microfluidic spotting method combined with a specifically designed flow guiding groove on the quartz slide, the uniform dispersion of the substrate material on the slide is achieved, effectively improving the consistency and intensity of the spectral signals and reducing the background noise. By precisely controlling the parameters of the spotting process and the drying treatment conditions, the binding energy between the substrate material and the slide surface is further enhanced, ensuring the stability and reliability of the detection process. Experimental verification shows that compared with the traditional drop coating method, this spotting method can improve the uniformity of the substrate material distribution to over 95%, reduce the coefficient of variation (CV) of the characteristic peak intensity to 8%, and increase the sensitivity by 3 times. The flow guiding groove structure design effectively reduces droplet splashing, and the vacuum drying process enhances the substrate stability, ensuring that the spectral signal differences in different regions are less than 10%.

[0062] According to another embodiment of the present invention, for Raman spectroscopy detection, a 785 nm laser (such as Ocean Optics QE65000) is used, 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 characteristic peaks at 1632 cm -1 ±2 cm -1 (lacidipine) and 1060 cm -1 ±2 cm -1 (fluvoxamine) are identified. The signal-to-noise ratio (S / N) is calculated based on the standard deviation of the baseline noise, and the threshold is set to ≥10, which can be automatically calculated by the software.

[0063] The adsorbed substrate material is evenly coated on the quartz slide, and the spectral data is collected point by point. At least 5 spectra from different regions of each sample are collected and averaged for analysis. If the S / N at 1632 cm -1 ≥10 and the peak position deviation ≤2 cm -1 , it is determined as positive for lacidipine; similarly, if the conditions are met at 1060 cm -1 , it is determined as positive for fluvoxamine. The experimental data is stored in a database (such as MySQL) for subsequent traceability and statistical analysis.

[0064] Through the standard solution test, the detection limit of this method for lacidipine is 10 ng / mL (S / N = 10), and for fluvoxamine is 12 ng / mL. The repeatability experiment shows that the RSD of the characteristic peak intensity for repeated detection on the same substrate is ≤7%, and the RSD between different batches of substrates is ≤10%. The quantization standard of the signal-to-noise ratio threshold effectively reduces the subjective misjudgment, and the coincidence rate of the detection results with HPLC - MS / MS reaches 98%. This method provides a clear positive judgment basis for on-site rapid screening, improving the credibility and consistency of the detection results.

[0065] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and embodiments. It can be fully applied to various fields suitable for the present invention. For those familiar with the field, additional modifications can be easily achieved. Therefore, without departing from the general concept defined by the claims and the equivalent scope, 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 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 of 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 of 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 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.

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