A method for detecting narcotic drugs based on SERS and magnetic solid-phase extraction, a kit and application thereof

Abstract

The present application relates to a kind of narcotic drug detection method based on SERS and magnetic solid phase extraction, kit and its application, belong to the technical field of perioperative narcotic drug monitoring.To solve the problem that existing plasma narcotic drug detection method is difficult to meet clinical needs, the present application provides a kind of narcotic drug detection method based on SERS and magnetic solid phase extraction, including the preparation of TiO2@Ag composite substrate, after weak cation exchange magnetic microbeads adsorb narcotic drugs, washing, elution, redissolution;The obtained sample to be tested and TiO2@Ag composite substrate are mixed and subjected to SERS detection, qualitative according to characteristic Raman shift, quantitative in combination with the linear relationship between peak intensity and concentration.The present application improves sensitivity through the double mechanism of electromagnetic enhancement of Ag and TiO2 interface charge transfer, combined with magnetic solid phase extraction to remove matrix interference efficiently, realizes the trace detection of multiple narcotic drugs, and is suitable for blood drug concentration monitoring of perioperative narcotic drugs.

Description

Technical Field

[0001] This invention belongs to the field of perioperative anesthetic drug monitoring technology, and particularly relates to a method, kit and application of anesthetic drug detection based on SERS and magnetic solid phase extraction. Background Technology

[0002] Precise control of perioperative anesthesia depth is crucial for ensuring patient safety and postoperative recovery quality. Currently, widely used clinical methods for monitoring anesthesia depth mainly rely on indirect physiological indicators such as heart rate, blood pressure, bispectral index (BPI) on electroencephalography (EEG), and four-wave stimulation. While these indicators can reflect sedation and muscle relaxation to some extent, they cannot directly quantify the true free concentration of anesthetic drugs in plasma, making precise closed-loop feedback control difficult.

[0003] Liquid chromatography-tandem mass spectrometry (LC-MS / MS) is currently the gold standard for detecting plasma anesthetic drug concentrations in laboratories. While this method boasts high sensitivity and specificity, its sample pretreatment steps are cumbersome and time-consuming, and it is highly dependent on large, sophisticated instruments and skilled technicians, resulting in high testing costs and failing to meet the clinical needs for rapid intraoperative bedside detection. Immunoassays, although relatively simple to operate, rely on the development of specific antibodies, leading to long development cycles, high costs, and a tendency to cross-react with drug metabolites or structural analogs, limiting their versatility in perioperative environments involving multiple drug administration.

[0004] Surface-enhanced Raman spectroscopy (SERS) has shown promise in the field of rapid biological sample detection due to its advantages such as fingerprint recognition capabilities, fast detection speed, low sample consumption, and resistance to aqueous phase interference. However, directly applying this technology to the detection of anesthetic drugs in plasma still faces two major bottlenecks: First, plasma is extremely complex, containing high concentrations of proteins, lipids, salts, and various endogenous small molecules. These matrix substances can non-specifically adsorb onto the surface of the SERS substrate, generating strong background signals that not only mask the characteristic Raman peaks of the target anesthetic drug but also contaminate the substrate, leading to a significant decrease in detection sensitivity and stability. Second, existing SERS substrates, such as single silver or gold nanoparticles, have limited enhancement mechanisms and insufficient sensitivity for detecting trace amounts of anesthetic drugs. Furthermore, the batch-to-batch reproducibility of these substrates is poor, making it difficult to support the stability requirements of clinical quantitative detection.

[0005] To address the aforementioned issues, recent studies have attempted to combine magnetic solid-phase extraction (SERS) with SERS to simplify pretreatment and reduce matrix interference through magnetic separation. However, existing combined methods still have the following shortcomings: (1) The magnetic materials used are mostly simple Fe3O4@SiO2 or unmodified nanoparticles, which have limited selective enrichment capabilities for weakly basic anesthetic drugs in plasma and have low recovery rates; (2) The SERS substrates used are still mainly single metal nanoparticles, lacking synergistic design of substrate enhancement mechanisms, making it difficult to achieve the sensitivity and resolution required for simultaneous detection of multiple anesthetic drugs in complex matrices; (3) Existing studies mostly focus on the detection of model drugs in environmental samples or buffer systems, lacking systematic validation for real intraoperative plasma samples, especially for the combined detection of multiple anesthetic drugs.

[0006] Therefore, developing a plasma anesthetic drug detection method that is rapid, highly sensitive, resistant to matrix interference, and adaptable to the needs of intraoperative bedside testing is of great clinical significance and application value for achieving precise perioperative medication and promoting the development of anesthesiology towards individualization and intelligence. Summary of the Invention

[0007] To address the challenge that existing plasma anesthetic drug detection methods cannot simultaneously meet the clinical requirements of "rapid, highly sensitive, resistant to matrix interference, and suitable for intraoperative bedside detection," this invention provides a method, kit, and application of anesthetic drug detection based on SERS and magnetic solid-phase extraction.

[0008] The technical solution of this invention:

[0009] A method for detecting anesthetic drugs based on SERS and magnetic solid-phase extraction includes the following steps:

[0010] Step 1: Using TiO2 nanoparticles as a carrier, silver nanoparticles are loaded by electrostatic self-assembly to prepare TiO2@Ag composite substrate;

[0011] Step 2: After protein precipitation, the plasma to be tested is centrifuged. The supernatant is added to weakly cation-exchange magnetic microbeads with carboxyl or sulfonic acid groups on the surface for incubation to complete the adsorption of anesthetic drugs. The magnetic microbeads are collected by magnetic separation, and the non-specifically adsorbed impurities on the magnetic microbeads are washed away. Then, the target anesthetic drug adsorbed on the magnetic microbeads is eluted, the eluent is collected, the solvent in the eluent is removed, and the residue is reconstituted to obtain the sample to be tested.

[0012] Step 3: Mix the sample to be tested with the TiO2@Ag composite substrate suspension, perform SERS detection, collect the SERS spectrum of the sample to be tested, qualitatively identify the anesthetic drug based on the characteristic Raman shift, and quantitatively detect the drug by combining the characteristic peak intensity with the linear relationship between the drug concentration.

[0013] Furthermore, the TiO2 nanoparticles mentioned in step one are rutile phase with a particle size of 260~280nm; the electrostatic self-assembly involves dispersing silver nanoparticles in a TiO2 nanoparticle suspension, such that the mass ratio of silver nanoparticles to TiO2 nanoparticles is 0.3~0.8:1, and immersing them at 40~60℃ and 150~300rpm for 120~240min, so that the silver nanoparticles are loaded onto the surface of the TiO2 nanoparticles through electrostatic self-assembly.

[0014] Furthermore, the silver nanoparticles in step one are prepared by chemical reduction: sodium citrate solution is added dropwise to silver nitrate aqueous solution under stirring, refluxed at 90~100℃ for 10~15min, refluxed at 80~90℃ for 30~40min, cooled to room temperature to obtain silver sol, the silver sol is centrifuged, and the precipitate is collected as silver nanoparticles.

[0015] Furthermore, the protein precipitation treatment in step two involves mixing plasma and acetonitrile at a volume ratio of 1:3~5 and vortexing for 1~2 minutes, followed by centrifugation at 2500~3500 rpm for 1~3 minutes.

[0016] Further, in step two, 2-3 mg of the weak cation exchange magnetic beads are added to every 1 mL of the supernatant, and the incubation time is 1-5 min; the washing involves sequentially washing the weak cation exchange magnetic beads with a 5% (v / v) methanol aqueous solution, a methanol-ammonia mixed aqueous solution, and deionized water, wherein the volume ratio of methanol, water, and ammonia in the methanol-ammonia mixed aqueous solution is 40:55:5; the elution reagent is a methanol-formic acid mixed aqueous solution, wherein the volume ratio of methanol, water, and formic acid is 90:8:2; and the resolution reagent is a 1 mmol / L ammonium bicarbonate aqueous solution.

[0017] Furthermore, in step three, the sample to be tested is mixed with the TiO2@Ag composite substrate suspension in equal volume, and the concentration of TiO2@Ag composite substrate in the TiO2@Ag composite substrate suspension is 1.5 mg / mL.

[0018] Furthermore, in step three, the pH value of the test system during SERS detection is 6.0~7.0; the SERS detection conditions are: excitation wavelength 638nm, laser power 53mW, integration time 10s, and spectral scanning range 300~1800cm². -1 .

[0019] Furthermore, the characteristic Raman shift of atracurium besylate was found to be 760 cm⁻¹. -1 The linear equation for the characteristic peak intensity versus concentration is y = 382.60x + 1375.40, with a linear detection range of 1 × 10⁻⁶. -7~1×10 -6 mol / L;

[0020] The characteristic Raman shift for quantitative detection of dexmedetomidine hydrochloride is 1240 cm⁻¹. -1 The linear equation for the characteristic peak intensity versus concentration is y = 313.81x + 256.70, with a linear detection range of 1 × 10⁻⁶. -6 ~1×10 -5 mol / L;

[0021] The characteristic Raman shift for quantitative detection of tramadol hydrochloride was 996 cm⁻¹. -1 The linear equation for the characteristic peak intensity versus concentration is y = 927.07x - 201.10, with a linear detection range of 1 × 10⁻⁶. -6 ~1×10 -5 mol / L;

[0022] The characteristic Raman shift for quantitative detection of fentanyl citrate was 1003 cm⁻¹. -1 The linear equation for the characteristic peak intensity versus concentration is y = 652.06x + 1149.73, with a linear detection range of 1 × 10⁻⁶. -7 ~1×10 -6 mol / L.

[0023] A kit for detecting anesthetic drugs to implement the detection method of the present invention comprises a TiO2@Ag composite substrate suspension and weakly cation exchange magnetic microbeads with carboxyl or sulfonic acid groups functionalized on their surface.

[0024] The application of an anesthetic drug detection kit provided by the present invention in the detection of anesthetic drugs for non-diagnostic purposes.

[0025] The beneficial effects of this invention are:

[0026] The TiO2@Ag composite substrate constructed in this invention utilizes localized surface plasmon resonance of silver nanoparticles to generate electromagnetic field enhancement, while simultaneously employing the interfacial charge transfer effect between TiO2 and Ag to generate chemical enhancement. This dual enhancement mechanism works synergistically to significantly improve the Raman signal intensity. Experimental results show that the detection limit for atracurium besylate in buffer solution is as low as 4.89 × 10⁻⁶. -9 mol / L, enabling trace detection of low concentrations of anesthetic drugs.

[0027] This invention innovatively applies magnetic solid-phase extraction (SPE) technology to the pretreatment of plasma for the detection of anesthetic drugs. Compared with traditional SPE columns, magnetic SPE is simpler to operate, requiring no centrifugation or column chromatography; phase separation can be rapidly achieved using an external magnetic field. Combined with a weak cation exchange mechanism, it can specifically enrich the target drug while efficiently removing strong background interference substances such as proteins and lipids from the plasma, obtaining a "clean" SERS spectrum and solving the problem of background interference in the direct detection of biological samples.

[0028] This invention successfully detects and differentiates four commonly used clinical anesthetic drugs: atracurium besylate, dexmedetomidine hydrochloride, tramadol hydrochloride, and fentanyl citrate. In the detection of mixed drugs, it exhibits characteristic Raman peaks that do not interfere with each other, demonstrating excellent chemical selectivity and making it suitable for perioperative scenarios involving multiple drugs.

[0029] The present invention achieves a spiked recovery rate of 84.2%–99.3% in complex fetal bovine serum matrices, demonstrating excellent accuracy and stability, and meeting the requirements of clinical quantitative analysis. Furthermore, the method is simple to operate and fast, making it suitable for the detection of trace amounts of anesthetic drugs in real plasma samples and for perioperative bedside blood drug concentration monitoring. Attached Figure Description

[0030] Figure 1 The images show transmission electron microscopy (TEM) images and field-of-view energy dispersive X-ray elemental distribution maps of the TiO2@Ag composite substrate prepared in Example 1. a is the TEM morphology image, b is the Ag elemental distribution map, c is the Ti elemental distribution map, and d is the O elemental distribution map.

[0031] Figure 2 The images show scanning electron microscope (SEM) images and field-of-view energy dispersive X-ray elemental distribution maps of the TiO2@Ag composite substrate prepared in Example 1. a is the SEM morphology image, b is the Ag elemental distribution map, c is the Ti elemental distribution map, and d is the O elemental distribution map.

[0032] Figure 3 The image shows the X-ray diffraction (XRD) pattern of the TiO2@Ag composite substrate prepared in Example 1.

[0033] Figure 4 Fourier transform infrared (FTIR) spectrum of the TiO2@Ag composite substrate material prepared in Example 1;

[0034] Figure 5 The above are surface-enhanced Raman spectra of four anesthetic drugs at different concentrations on the TiO2@Ag composite substrate in Example 2. a is atracurium besylate, b is dexmedetomidine hydrochloride, c is tramadol hydrochloride, and d is fentanyl citrate.

[0035] Figure 6The image shows a comparison of the intra-batch stability of the TiO2@Ag composite substrate to SERS detection of four anesthetic drugs in Example 3. a is atracurium besylate, b is dexmedetomidine hydrochloride, c is tramadol hydrochloride, and d is fentanyl citrate.

[0036] Figure 7 The following are the SERS quantitative detection results of four anesthetic drugs detected by the TiO2@Ag composite substrate in Example 4: a) SERS characteristic peak intensity-concentration relationship and linear fitting curve of atracurium besylate; b) SERS characteristic peak intensity-concentration relationship and linear fitting curve of dexmedetomidine hydrochloride; c) SERS characteristic peak intensity-concentration relationship and linear fitting curve of tramadol hydrochloride; d) SERS characteristic peak intensity-concentration relationship and linear fitting curve of fentanyl citrate.

[0037] Figure 8 The image shows the surface-enhanced Raman spectrum of atracurium benzenesulfonate and dexmedetomidine hydrochloride mixed solution detected on the TiO2@Ag composite substrate in Example 5.

[0038] Figure 9 This is a SERS detection result of atracurium besylate in fetal bovine serum matrix using a TiO2@Ag composite substrate combined with magnetic solid-phase extraction in Example 6. 'a' is the surface-enhanced Raman spectrum, and the curves from bottom to top represent 1×10⁻⁶ spectral density. -7 mol / L, 2×10 -7 mol / L, 3×10 -7 mol / L, 4×10 -7 mol / L, 5×10 -7 The spiking concentration is mol / L, and b is 760 cm⁻¹. -1 Linear fitting curve of characteristic peak intensity versus spiked concentration;

[0039] Figure 10 The above are surface-enhanced Raman spectra of atracurium besylate in patient plasma samples detected by TiO2@Ag composite substrate combined with magnetic solid-phase extraction in Example 7. Curve A is the atracurium besylate standard solution sample, curve B is the plasma sample after patient administration, curve C is the blank plasma matrix spiked sample, curve D is the blank plasma sample after magnetic solid-phase extraction, and curve E is the original untreated blank plasma sample.

[0040] Figure 11 The graphs show a comparison of the SERS detection performance of the TiO2@Ag composite substrate against atracurium besylate under different pH conditions in Example 8. a is the surface-enhanced Raman spectrum, and b is the 760 cm⁻¹ spectrum. -1 Characteristic peak intensity as a function of pH value;

[0041] Figure 12This is a comparison of the SERS detection performance of TiO2@Ag composite substrates with different TiO2 particle sizes against atracurium cissulfonate in Example 9;

[0042] Figure 13 This is a comparison chart of the spiked recoveries of atracurium besylate by magnetic solid-phase extraction and conventional solid-phase extraction in Example 10. Detailed Implementation

[0043] The technical solution of the present invention will be further described below with reference to embodiments, but it is not limited thereto. Any modifications or equivalent substitutions to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention should be covered within the protection scope of the present invention. In the following embodiments, the process equipment or apparatus not specifically specified are all conventional equipment or apparatus in the art. Unless otherwise specified, the raw materials used in the embodiments of the present invention are all commercially available; unless otherwise specified, the technical means used in the embodiments of the present invention are all conventional means well known to those skilled in the art.

[0044] Example 1

[0045] This embodiment provides a method for preparing and characterizing a titanium dioxide-supported silver nanoparticle (TiO2@Ag) composite substrate.

[0046] The preparation method of the TiO2@Ag composite substrate in this embodiment is as follows:

[0047] Prepare 200 mL of 0.20 g / L silver nitrate aqueous solution, add 4 mL of 10 g / L sodium citrate solution dropwise while stirring at 2000 rpm, reflux at 95 °C for 12 min, then reflux at 85 °C for 35 min, and cool to room temperature to obtain silver sol; centrifuge the obtained silver sol at 25 °C and 5000 rpm for 20 min, and collect the precipitate, which is silver nanoparticles with a mass of about 25 mg.

[0048] A TiO2 nanoparticle suspension with a concentration of 1 g / L was prepared using rutile TiO2 nanoparticles with a particle size of 260~280 nm. The resulting silver nanoparticles were dispersed in the TiO2 nanoparticle suspension at a mass ratio of 0.5:1 to silver nanoparticles. The suspension was then immersed at 50 °C and 200 rpm for 180 min. After the reaction was completed, the precipitate was collected by centrifugation at 8000 rpm for 10 min, washed twice with ultrapure water, and redispersed in 50 mL of ultrapure water to prepare a TiO2@Ag composite substrate suspension with a concentration of 1.5 mg / mL. The suspension was stored at 4 °C for later use.

[0049] In this embodiment, transmission electron microscopy, scanning electron microscopy, X-ray diffraction, and Fourier transform infrared spectroscopy were used to characterize the microstructure, elemental distribution, crystal phase structure, and surface functional groups of the prepared TiO2@Ag composite substrate material.

[0050] (a) Transmission electron microscopy

[0051] The TiO2@Ag composite substrate was ultrasonically dispersed in ethanol, dropped onto a carbon-supported copper mesh, dried, and then observed under a transmission electron microscope with an accelerating voltage of 200 kV.

[0052] The results are as follows Figure 1 As shown, silver nanoparticles are uniformly loaded on the surface and between the TiO2 nanoparticles, and the silver nanoparticles have a spherical or near-spherical morphology. The same field energy dispersive X-ray elemental distribution map shows that Ag, Ti and O elements are uniformly distributed on the substrate, confirming that the silver nanoparticles are successfully loaded on the TiO2 surface.

[0053] (ii) Scanning electron microscope

[0054] The overall morphology and elemental composition of the TiO2@Ag composite substrate were analyzed using scanning electron microscopy and energy-dispersive X-ray elemental mapping.

[0055] The results are as follows Figure 2 As shown, the TiO2@Ag composite substrate has a uniform morphology, and the Ag element signal is uniformly distributed on the surface of the TiO2 substrate. Furthermore, the Ti and O element signals are highly consistent with the morphology of the substrate, proving that the elements in the composite substrate are uniformly distributed and the silver nanoparticle loading effect is stable.

[0056] (iii) X-ray diffraction

[0057] The crystal structure of the TiO2@Ag composite substrate was analyzed by X-ray diffraction under the following conditions: Cu Kα radiation (λ=1.5406 Å), scanning range 10°~80°, step size 0.02°.

[0058] The results are as follows Figure 3 The diffraction peaks show the characteristic rutile phase of TiO2 and the typical characteristic peaks of metallic silver. The peak positions are free of obvious impurities, indicating that the composite substrate has good crystallinity.

[0059] (iv) Fourier transform infrared spectroscopy

[0060] Fourier transform infrared spectroscopy was used to analyze the surface functional groups of the TiO2@Ag composite substrate. After drying, the samples were tested using the KBr pellet method (sample and KBr were mixed at a mass ratio of 1:100 and pelleted), with wavenumbers ranging from 400 to 4000 cm⁻¹. -1 4cm resolution -1 .

[0061] The results are as follows Figure 4 As shown, 3321cm -1 and 1614cm -1 The absorption peak at 1383 cm⁻¹ is attributed to the abundance of hydroxyl groups and adsorbed water on the TiO₂ surface, which facilitates the anchoring of silver nanoparticles; -1 The absorption peak at that point originates from the carboxylate group, proving that Ag and TiO2 are linked by coordinate bonds.

[0062] Example 2

[0063] This embodiment verifies the SERS detection performance of the TiO2@Ag composite substrate prepared in Example 1 for four common anesthetic drugs: atracurium besylate, dexmedetomidine hydrochloride, tramadol hydrochloride, and fentanyl citrate, achieving qualitative identification and quantitative analysis at different concentrations.

[0064] The detection method is as follows:

[0065] Prepare anesthetic drug solutions of different concentrations:

[0066] The concentration of atracurium besylate solution is 1×10⁻⁶. -7 mol / L, 5×10 -7 mol / L, 1×10 -6 mol / L, 5×10 -6 mol / L, 1×10 -5 mol / L, 5×10 -5 mol / L, 1×10 -4 mol / L;

[0067] The concentration of dexmedetomidine hydrochloride solution is 5 × 10⁻⁶. -7 mol / L, 1×10 -6 mol / L, 5×10 -6 mol / L, 1×10 -5 mol / L, 5×10 -5 mol / L, 1×10 -4 mol / L;

[0068] The concentration of tramadol hydrochloride solution is 5 × 10⁻⁶. -7 mol / L, 1×10 -6 mol / L, 5×10 -6 mol / L, 1×10 -5 mol / L, 5×10 -5 mol / L, 1×10 -4 mol / L;

[0069] The concentration of fentanyl citrate solution is 1×10⁻⁶. -7 mol / L, 5×10-7 mol / L, 1×10 -6 mol / L, 5×10 -6 mol / L, 1×10 -5 mol / L, 5×10 -5 mol / L.

[0070] The TiO2@Ag composite substrate suspension prepared in Example 1 was mixed with the anesthetic drug solution to be tested at a volume ratio of 1:1. The mixture was incubated at 25°C with shaking at 1000 rpm for 5 min, followed by surface-enhanced Raman spectroscopy. The detection parameters were set as follows: excitation wavelength 638 nm, laser power 53 mW, integration time 10 s, and spectral scanning range 300–1800 cm⁻¹. -1 For each sample, the spectrum was collected three times in parallel and the average value was taken.

[0071] Monitoring results as follows Figure 5 As shown, SERS spectra of the four anesthetic drugs at different concentration gradients were obtained with high signal-to-noise ratios and clear characteristic peaks. The intensity of the characteristic peaks showed a significant positive correlation with increasing drug concentration, demonstrating good concentration response. Among them, atracurium besylate showed a high signal-to-noise ratio at 760 cm⁻¹. -1 A characteristic Raman shift peak appeared at 1240 cm⁻¹, with dexmedetomidine hydrochloride showing a peak at 1240 cm⁻¹. -1 A characteristic Raman shift peak appears at 996 cm⁻¹, with tramadol hydrochloride showing a peak at 996 cm⁻¹. -1 A characteristic Raman shift peak appears at 1003 cm⁻¹, with fentanyl citrate showing a peak at 1003 cm⁻¹. -1 Characteristic Raman shift peaks appear at [specific locations]; these characteristic peaks are observed in the concentration range of 1×10 [units]. -7 mol / L ~ 1×10 -4 The intensity within mol / L increases in an orderly manner with increasing drug concentration, without significant interference from other peaks, and can be used as a specific marker peak for qualitative identification and quantitative detection of four anesthetic drugs.

[0072] Example 3

[0073] This embodiment evaluates the intra-batch stability and reproducibility of the TiO2@Ag composite substrate prepared in the same batch of Example 1 for SERS detection of four anesthetic drugs by repeated testing, verifying the consistency of substrate performance and providing a reliable basis for subsequent quantitative analysis.

[0074] Detection method:

[0075] Take the TiO2@Ag composite substrate prepared in Example 1 from the same batch, and mix it with a concentration of 1×10 -6 Atracurium besylate and fentanyl citrate at concentrations of mol / L, and 1×10 mol / L. -5Dexmedetomidine hydrochloride and tramadol hydrochloride standard solutions of mol / L were mixed and incubated according to the method described in Example 2. Under the same experimental conditions, the characteristic Raman peak intensities of each drug were collected in parallel eight times, and the relative standard deviation (RSD) of the characteristic peak intensities of each drug was calculated.

[0076] Test results as follows Figure 6 As shown, the peak intensities of the four anesthetic drugs at their respective characteristic Raman shifts all exhibit good consistency: atracurium besylate at 760 cm⁻¹ -1 The characteristic peak RSD was 8.07%, and that of dexmedetomidine hydrochloride was at 1240 cm⁻¹. -1 The characteristic peak RSD was 4.02%, and tramadol hydrochloride peaked at 996 cm⁻¹. -1 The characteristic peak RSD was 7.16%, and fentanyl citrate had an RSD of 1003 cm⁻¹. -1 The RSD of the characteristic peaks was 6.92%. The relative standard deviation of the characteristic peak intensity for all drugs was less than 10%, indicating that the prepared TiO2@Ag composite substrate has excellent intra-batch detection reproducibility and signal stability, and can meet the stability requirements for quantitative analysis of anesthetic drugs.

[0077] Example 4

[0078] This embodiment establishes a linear relationship between the intensity of characteristic peaks and drug concentration by performing SERS detection on a series of anesthetic drug standards with varying concentration gradients. It verifies the quantitative analytical capability of the TiO2@Ag composite substrate prepared in Example 1 for four anesthetic drugs and calculates the limit of detection of the method.

[0079] Standard solutions of atracurium besylate, dexmedetomidine hydrochloride, tramadol hydrochloride, and fentanyl citrate with different concentration gradients set in Example 2 were mixed and incubated with the TiO2@Ag composite substrate prepared in Example 1 according to the method described in Example 2 and SERS detection was performed. Peak intensity data at the characteristic Raman shifts of each drug were collected, and the drug concentration was linearly fitted with the characteristic peak intensity to establish a quantitative standard curve.

[0080] The results are as follows Figure 7 As shown:

[0081] Atracurium besylate at 1×10 -7 ~1×10 -6 Within the concentration range of mol / L, 760cm -1 The characteristic peak intensity showed a good linear relationship with the concentration, with the linear equation being y = 382.60x + 1375.40 and a correlation coefficient R. 2 =0.973, and based on a signal-to-noise ratio (S / N) of 3, its detection limit in the buffer solution is 4.89 × 10⁻⁶. -9 mol / L;

[0082] Dexmedetomidine hydrochloride at 1×10 -6 ~1×10 -5 Within the concentration range of mol / L, 1240cm -1 The characteristic peak intensity showed a significant linear relationship with the concentration, with the linear equation being y = 313.81x + 256.70 and a correlation coefficient R0. 2 =0.994, detection limit is 1.11×10 -7 mol / L;

[0083] Tramadol hydrochloride at 1×10 -6 ~1×10 -5 Within the concentration range of mol / L, 996cm -1 The characteristic peak intensity showed a good linear relationship with the concentration, with the linear equation being y = 927.07x - 201.10 and a correlation coefficient R0. 2 =0.987;

[0084] Fentanyl citrate at 1×10 -7 ~1×10 -6 Within the concentration range of mol / L, 1003 cm -1 The characteristic peak intensity shows a linear relationship with concentration, with the linear equation being y = 652.06x + 1149.73 and a correlation coefficient R0. 2 =0.916.

[0085] The above results show that the method of the present invention has a wide linear detection range, high correlation coefficient and low detection limit for all four anesthetic drugs, and has good quantitative analysis capability, which can meet the quantitative detection requirements of anesthetic drugs in plasma samples.

[0086] Example 5

[0087] This embodiment verifies the ability of the TiO2@Ag composite substrate prepared in Example 1 to simultaneously detect multiple anesthetic drug coexisting systems by SERS detection of atracurium besylate and dexmedetomidine hydrochloride mixed solution, and examines the selectivity and anti-interference performance of the method.

[0088] Prepare a series of mixed standard solutions, wherein the concentration of dexmedetomidine hydrochloride is fixed at 1×10⁻⁶. -6 The concentration of atracurium besylate was set to 1 × 10 mol / L. -7 mol / L, 2×10 -7 mol / L, 3×10 -7 mol / L, 4×10 -7 mol / L, 5×10 -7By using a gradient of mol / L, mixed samples with different concentration ratios were obtained. Following the method described in Example 2, the mixed solutions were incubated with the TiO2@Ag composite substrate prepared in Example 1, followed by SERS detection.

[0089] The results are as follows Figure 8 As shown, in the mixed system, atracurium cissulfonate at 760 cm⁻¹ -1 The characteristic peak at 1240 cm⁻¹ is similar to that of dexmedetomidine hydrochloride. -1 The characteristic peaks at each location are independent and distinguishable, with no obvious peak overlap; simultaneously, as the concentration of atracurium besylate increases from 1×10⁻⁶, the characteristic peaks at each location do not interfere with each other and are independently distinguishable. -7 Increased to 5 × 10 mol / L -7 mol / L, at 760cm -1 The characteristic peak intensity at 1240 cm⁻¹ increases systematically with increasing concentration, while that of dexmedetomidine hydrochloride increases systematically with increasing concentration. -1 The characteristic peak intensity remained stable. These results demonstrate that the method of this invention can achieve simultaneous qualitative and quantitative identification of mixed anesthetic drugs, exhibiting good selectivity and anti-interference capabilities, and is suitable for analyzing samples containing multiple coexisting anesthetic drugs.

[0090] Example 6

[0091] This embodiment uses fetal bovine serum as a complex biological matrix model to evaluate the application performance of the detection method prepared in Example 1, which combines TiO2@Ag composite substrate with magnetic solid phase extraction, in actual biological samples, including matrix interference elimination ability, quantitative accuracy, and spiked recovery rate.

[0092] Both fetal bovine serum and plasma contain high concentrations of proteins, lipids, and endogenous small molecules, exhibit similar matrix complexity, and neither contains blood cells. For the detection of small-molecule anesthetic drugs, both show high consistency in drug presentation and matrix interference characteristics. Therefore, this embodiment uses fetal bovine serum as a complex biological matrix model to verify the applicability of the method of this invention in complex biological samples.

[0093] Different concentration gradients of atracurium besylate standards were added to fetal bovine serum to prepare spiked concentrations of 1×10⁻⁶. -7 mol / L, 2×10 -7 mol / L, 3×10 -7 mol / L, 4×10 -7 mol / L, 5×10 -7 A series of mol / L samples were pretreated by magnetic solid-phase extraction and then subjected to spectral acquisition under the SERS detection conditions described in Example 2. At the same time, spiked recovery experiments were carried out.

[0094] The adsorbent used in the magnetic solid-phase extraction pretreatment in this embodiment is BeaverBeads® WCX, a weak cation exchange magnetic microbead with surface phenyl carboxyl functionalization, with a particle size range of 15~50μm, purchased from Suzhou Beaver Biotechnology Co., Ltd., product number 71111.

[0095] The specific pretreatment method for magnetic solid-phase extraction is as follows:

[0096] Take the prepared spiked fetal bovine serum sample stock solution, add acetonitrile at a volume ratio of 1:4 for protein precipitation, vortex for 1 min, centrifuge at 3000 rpm for 2 min to completely remove protein impurities from the serum, and collect the supernatant for later use; take 1 mL of supernatant, add 2 mg of surface phenyl carboxyl functionalized weak cation exchange magnetic beads, and incubate for 2 min to adsorb the target drug; achieve magnetic separation with an external magnetic field, and discard the supernatant; wash the magnetic beads sequentially with 5% methanol aqueous solution, methanol-ammonia mixed aqueous solution (methanol, water and ammonia in a volume ratio of 40:55:5), and deionized water, and perform magnetic separation after each 30 s wash; after washing, add methanol-formic acid mixed aqueous solution with a volume ratio of 90:8:2 (methanol, water and formic acid) to elute the target drug for 1 min; collect the eluent, dry it at low temperature, and redissolve the residue with 10 μL of 1 mmol / L ammonium bicarbonate aqueous solution to obtain the sample to be tested. Take 10 μL of the TiO2@Ag composite substrate suspension prepared in Example 1 and mix it with 10 μL of the sample to be tested and vortex it. Collect Raman spectra according to the SERS detection conditions described in Example 2.

[0097] SERS test results are as follows Figure 9 As shown, after pretreatment with magnetic solid-phase extraction, the background signal of the serum matrix was effectively removed, and atracurium besylate was extracted at 760 cm⁻¹. -1 The characteristic Raman peaks at the spiking point were clearly distinguishable, with no obvious matrix interference; the intensity of the characteristic peaks showed a good linear relationship with the spiked concentration, with the linear equation being y = 185.72x + 122.79 and a correlation coefficient R. 2 =0.987; the spiked recovery rate was in the range of 84.2% to 99.3%, indicating that the detection method of TiO2@Ag composite substrate combined with magnetic solid phase extraction of the present invention has good accuracy and stability in complex biological matrices, and can be used for the quantitative detection of anesthetic drugs in actual plasma / serum samples.

[0098] Example 7

[0099] This embodiment verifies the feasibility of the detection method of TiO2@Ag composite substrate combined with magnetic solid phase extraction in clinical complex matrices by detecting real patient plasma samples during surgery, and evaluates its ability to detect anesthetic drugs in real biological samples and its matrix tolerance.

[0100] In this embodiment, the following test samples and control samples are set simultaneously:

[0101] (1) Standard control sample: concentration 1×10 -6 A pure aqueous solution of atracurium besylate (mol / L) standard was used to determine the position of the characteristic Raman peak of the drug.

[0102] (2) Clinical real samples (magnetic solid phase extraction): fresh plasma samples from patients who received intravenous infusion of atracurium besylate during surgery;

[0103] (3) Matrix-spiked control samples (magnetic solid-phase extraction): Atracurium besylate was added to blank plasma at a concentration of 1×10⁻⁶. -6 mol / L was used to verify the effectiveness of the plasma matrix pretreatment and detection methods;

[0104] (4) Blank plasma control samples (magnetically solid-phase extraction): Blank plasma from patients who have not used atracurium besylate anesthetic drugs and has been magnetically solid-phase extracted is used to exclude false positive signals generated by magnetic solid-phase processing;

[0105] (5) Unprocessed blank plasma samples (without magnetic solid phase extraction): blank plasma from patients who have not used atracurium besylate anesthetic drugs without magnetic solid phase extraction is used to exclude interference from the plasma substrate’s own spectral signal.

[0106] The preprocessing methods for real clinical samples are as follows:

[0107] Whole blood samples were collected from patients who received atracurium besylate during surgery. Plasma was obtained by centrifugation at 3000 rpm for 10 min. Acetonitrile was added to the plasma at a volume ratio of 1:4 to precipitate proteins. After vortexing for 1 min, plasma proteins were removed by centrifugation at 3000 rpm for 2 min. 1 mL of the supernatant was collected, and 2 mg of weakly cation-exchange magnetic beads with phenyl carboxyl groups on their surface were added and incubated for 2 min to adsorb the target drug. Magnetic separation was achieved using an external magnetic field, and the supernatant was discarded. The magnetic beads were washed sequentially with 5% methanol aqueous solution, a methanol-ammonia mixed aqueous solution (methanol, water, and ammonia volume ratio of 40:55:5), and deionized water, with magnetic separation performed after each 30 s wash. After washing, the target drug was eluted with a methanol-formic acid mixed aqueous solution (methanol, water, and formic acid volume ratio of 90:8:2) for 1 min. The eluent was collected, dried at low temperature, and the residue was reconstituted with 10 μL of 1 mmol / L ammonium bicarbonate aqueous solution to obtain the test sample.

[0108] The adsorbent used in the magnetic solid-phase extraction pretreatment in this embodiment is BeaverBeads® WCX, a weak cation exchange magnetic microbead with surface phenyl carboxyl functionalization, with a particle size range of 15~50μm, purchased from Suzhou Beaver Biotechnology Co., Ltd., product number 71111.

[0109] 10 μL of the TiO2@Ag composite substrate suspension prepared in Example 1 was mixed with 10 μL of the sample to be tested and vortexed. Raman spectra were acquired under the SERS detection conditions described in Example 2. Raman spectra of the remaining four groups of samples were acquired under the same SERS detection conditions.

[0110] Test results as follows Figure 10 As shown, the SERS spectrum (curve B) of a real patient plasma sample at 760 cm⁻¹ -1 The method produced a characteristic Raman peak at a position consistent with that of atracurium besylate standard (curve A) and matrix-spiked control sample (curve C). Compared to blank plasma control sample (curve D) and untreated blank plasma sample (curve E), there were no significant matrix interference peaks, and the target drug's characteristic peak was clearly distinguishable. This indicates that the method of the present invention can effectively process real clinical plasma samples, removing complex matrix background interference while stably detecting the target anesthetic drug. These results verify the applicability of the present invention in complex clinical biological samples and demonstrate its potential for real-time monitoring of intraoperative anesthetic drug concentrations.

[0111] Example 8

[0112] This embodiment systematically investigates the effect of pH on the detection of atracurium besylate on TiO2@Ag composite substrate, clarifies the optimal detection pH conditions, and provides experimental basis for the optimization of buffer system for subsequent actual sample detection.

[0113] The TiO2@Ag composite substrate suspension prepared in Example 1 was placed in a series of buffer solution systems with pH values ​​of 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0, respectively, and then mixed with a 1×10⁻⁶ solution. -5 A mol / L atracurium besylate standard solution was mixed at a 1:1 volume ratio, vortexed, and Raman spectra were acquired under the SERS detection conditions described in Example 2. Three parallel samples were set for each pH condition, and spectral data were acquired simultaneously, and the average value and standard deviation of the characteristic peak intensities were calculated.

[0114] Test results as follows Figure 11 As shown, atracurium cissulfonate at 760 cm⁻¹ under different pH conditions -1Significant differences were observed in the intensity of the characteristic Raman peak at different locations. Under acidic conditions (pH 3–5), the peak intensity decreased with increasing pH, reaching its lowest value at pH 5. As the pH continued to rise, the peak intensity increased rapidly, reaching its peak value at neutral conditions (pH 7.0), and then decreased under alkaline conditions (pH 8–9). This trend indicates that pH directly affects the adsorption efficiency of drug molecules on the substrate surface and the SERS enhancement effect by regulating the charge state of the TiO2@Ag composite substrate surface and the ionization degree of atracurium besylate molecules. Under neutral pH (pH 7.0) conditions, the interaction between drug molecules and the substrate was most complete, resulting in the optimal SERS enhancement effect. These results clarify the optimal detection pH conditions of this invention, providing crucial experimental basis for the selection and optimization of the buffer system in actual sample detection.

[0115] Example 9

[0116] This embodiment systematically investigates the effect of TiO2 nanoparticle size on the SERS enhancement performance of TiO2@Ag composite substrate.

[0117] TiO2 nanoparticles with particle sizes of 70-90 nm, 180-200 nm, and 260-280 nm were selected, respectively, to prepare TiO2@Ag composite substrates according to the method described in Example 1. Pure substrates containing 260-280 nm TiO2 nanoparticles and pure substrates containing silver nanoparticles (AgNPs) were used as controls. Each substrate was compared with a concentration of 1×10⁻⁶ g / L. -4 A mol / L atracurium besylate standard solution was mixed at a 1:1 volume ratio, vortexed, and then Raman spectra were collected under the SERS detection conditions described in Example 2.

[0118] Test results as follows Figure 12 As shown, the SERS signal intensities of TiO2@Ag composite substrates prepared with different TiO2 particle sizes differ significantly for atracurium besylate. Specifically, the composite substrates prepared with 260–280 nm TiO2 nanoparticles showed the highest SERS signal intensity at 760 cm⁻¹. -1 The characteristic Raman peak intensity at the chromatic spectroscopy point was significantly higher than that of TiO2@Ag substrates of other particle sizes, pure TiO2 nanoparticle substrates, and pure silver nanoparticle substrates, exhibiting the best SERS enhancement effect. This result indicates that the particle size of TiO2 nanoparticles is a key factor affecting the performance of the composite substrate: TiO2 nanoparticles in the 260–280 nm particle size range have a more suitable specific surface area and surface morphology, enabling high-density uniform loading of silver nanoparticles and forming more "hot spot" structures, thereby significantly improving the SERS enhancement efficiency of atracurium besylate. These results clarify the optimal preparation parameters for the TiO2@Ag composite substrate of this invention, providing crucial experimental basis for subsequent large-scale preparation and performance optimization of the substrate.

[0119] Example 10

[0120] This embodiment systematically compares the recovery rate and operational performance of the magnetic solid-phase extraction pretreatment method used in this invention with the conventional solid-phase extraction column method in extracting atracurium besylate from fetal bovine serum matrix, verifying the superiority of the pretreatment process of this invention.

[0121] Add 1×10 to fetal bovine serum. -7 mol / L, 5×10 -7 mol / L, 1×10 -6 A series of spiked serum samples were prepared using a mol / L atracurium besylate standard; two pretreatment methods were used for extraction and purification:

[0122] (1) Magnetic solid phase extraction method: The process described in Example 6 was carried out, and BeaverBeads®WCX weak cation exchange magnetic microbeads (Suzhou Beaver Biotechnology, product number 71111, particle size 15~50μm) were used as the solid phase extraction medium.

[0123] (2) Traditional solid phase extraction column method: In accordance with the conventional operating procedures in this field, the commercially available WCX mixed weak cation exchange solid phase extraction column (Waters Oasis WCX) was used for pretreatment, and the target drug was extracted by sequentially going through activation, loading, rinsing and elution steps.

[0124] Raman spectra of the eluents after treatment by both methods were collected under the SERS detection conditions described in Example 2. The spiked recoveries at each concentration were calculated based on the standard curve, and the operation procedures and reproducibility of the two methods were compared.

[0125] Test results as follows Figure 13 As shown, in 1×10 -7 mol / L, 5×10 -7 mol / L, 1×10 -6At three spiking concentrations (mol / L), the recoveries of magnetic solid-phase extraction (MSE) were 84.2%, 99.3%, and 94.5%, respectively, with an overall recovery rate ranging from 84.2% to 99.3%. In contrast, the recoveries of traditional solid-phase extraction (SPE) column methods were 103.6%, 89.6%, and 95.0%, respectively, showing significant fluctuations in overall recovery. The comparative results demonstrate that the magnetic SPE method employed in this invention can rapidly achieve separation using an external magnetic field, eliminating the need for complex steps such as column activation and equilibration. This method is more convenient, less time-consuming, and offers stable and reproducible recovery rates. It also avoids the problems of column clogging, high column pressure, and high organic solvent consumption associated with traditional SPE methods. These results validate the comprehensive advantages of the magnetic SPE pretreatment method of this invention in terms of extraction efficiency, stability, and ease of operation, making it more suitable for the rapid pretreatment of anesthetic drugs in complex biological samples.

Claims

1. A method for detecting anesthetic drugs based on SERS and magnetic solid-phase extraction, characterized in that, Includes the following steps: Step 1: Using TiO2 nanoparticles as a carrier, silver nanoparticles are loaded by electrostatic self-assembly to prepare TiO2@Ag composite substrate; Step 2: After protein precipitation, the plasma to be tested is centrifuged. The supernatant is added to weakly cation-exchange magnetic microbeads with carboxyl or sulfonic acid groups on the surface for incubation to complete the adsorption of anesthetic drugs. The magnetic microbeads are collected by magnetic separation, and the non-specifically adsorbed impurities on the magnetic microbeads are washed away. Then, the target anesthetic drug adsorbed on the magnetic microbeads is eluted, the eluent is collected, the solvent in the eluent is removed, and the residue is reconstituted to obtain the sample to be tested. Step 3: Mix the sample to be tested with the TiO2@Ag composite substrate suspension, perform SERS detection, collect the SERS spectrum of the sample to be tested, qualitatively identify the anesthetic drug based on the characteristic Raman shift, and quantitatively detect the drug by combining the characteristic peak intensity with the linear relationship between the drug concentration.

2. The detection method according to claim 1, characterized in that, The TiO2 nanoparticles mentioned in step one are rutile phase with a particle size of 260~280nm; the electrostatic self-assembly involves dispersing silver nanoparticles in a TiO2 nanoparticle suspension with a mass ratio of silver nanoparticles to TiO2 nanoparticles of 0.3~0.8:1, and immersing them at 40~60℃ and 150~300rpm for 120~240min, so that the silver nanoparticles are loaded onto the surface of the TiO2 nanoparticles through electrostatic self-assembly.

3. The detection method according to claim 1 or 2, characterized in that, The silver nanoparticles described in step one are prepared by chemical reduction: sodium citrate solution is added dropwise to silver nitrate aqueous solution under stirring, refluxed at 90~100℃ for 10~15 min, refluxed at 80~90℃ for 30~40 min, cooled to room temperature to obtain silver sol, the silver sol is centrifuged, and the precipitate is collected as silver nanoparticles.

4. The detection method according to claim 3, characterized in that, The protein precipitation process in step two involves mixing plasma and acetonitrile at a volume ratio of 1:3-5 and vortexing for 1-2 minutes, followed by centrifugation at 2500-3500 rpm for 1-3 minutes.

5. The detection method according to claim 4, characterized in that, In step two, 2-3 mg of the weak cation exchange magnetic beads are added to each 1 mL of the supernatant, and the incubation time is 1-5 min. The washing involves sequentially washing the weak cation exchange magnetic beads with a 5% (v / v) methanol aqueous solution, a methanol-ammonia mixed aqueous solution, and deionized water. The volume ratio of methanol, water, and ammonia in the methanol-ammonia mixed aqueous solution is 40:55:

5. The elution reagent is a methanol-formic acid mixed aqueous solution with a volume ratio of methanol, water, and formic acid of 90:8:

2. The resolvation reagent is a 1 mmol / L ammonium bicarbonate aqueous solution.

6. The detection method according to claim 5, characterized in that, In step three, the sample to be tested is mixed with an equal volume of the TiO2@Ag composite substrate suspension, wherein the concentration of the TiO2@Ag composite substrate in the TiO2@Ag composite substrate suspension is 1.5 mg / mL.

7. The detection method according to claim 6, characterized in that, In step three, the pH value of the test system during SERS detection is 6.0~7.0; the SERS detection conditions are: excitation wavelength 638nm, laser power 53mW, integration time 10s, and spectral scanning range 300~1800cm². -1 .

8. The detection method according to claim 7, characterized in that, The characteristic Raman shift for quantitative detection of atracurium besylate is 760 cm⁻¹. -1 The linear equation for the characteristic peak intensity versus concentration is y = 382.60x + 1375.40, with a linear detection range of 1 × 10⁻⁶. -7 ~1×10 -6 mol / L; The characteristic Raman shift for quantitative detection of dexmedetomidine hydrochloride is 1240 cm⁻¹. -1 The linear equation for the characteristic peak intensity versus concentration is y = 313.81x + 256.70, with a linear detection range of 1 × 10⁻⁶. -6 ~1×10 -5 mol / L; The characteristic Raman shift for quantitative detection of tramadol hydrochloride was 996 cm⁻¹. -1 The linear equation for the characteristic peak intensity versus concentration is y = 927.07x - 201.10, with a linear detection range of 1 × 10⁻⁶. -6 ~1×10 -5 mol / L; The characteristic Raman shift for quantitative detection of fentanyl citrate was 1003 cm⁻¹. -1 The linear equation for the characteristic peak intensity versus concentration is y = 652.06x + 1149.73, with a linear detection range of 1 × 10⁻⁶. -7 ~1×10 -6 mol / L.

9. A kit for detecting anesthetic drugs to implement the detection method according to any one of claims 1-8, characterized in that, It contains a TiO2@Ag composite substrate suspension and weakly cation-exchange magnetic microbeads with surface functionalized carboxyl or sulfonic acid groups.

10. The application of the anesthetic drug detection kit as described in claim 9 in the detection of anesthetic drugs for non-diagnostic purposes.

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