A levocarnitine-imprinted MOF-Au Raman substrate, its preparation method and application

Abstract

This invention belongs to the field of surface-enhanced Raman spectroscopy (SERS) detection, and provides a levocarnitine-imprinted MOF-Au Raman substrate, its preparation method, and its applications. This invention employs a composite film design of a dual-anchored MOF-loaded gold nanoislands. The gold nanoislands are stably held in place through a synergistic anchoring mechanism of Au–S and Au–O–P bonds. The imprinted channels provide selective recognition sites, constructing a high-density nano-interstitial hotspot enhancement system. This Raman substrate exhibits excellent selective enrichment ability, high-sensitivity SERS signal, good structural stability, and signal repeatability, enabling rapid and accurate detection of impurities in levocarnitine oral solution. This invention solves the problem of balancing film stability, anti-interference ability, and long-term signal consistency in existing substrates, and has broad application value in the field of rapid drug quality detection.

Description

Technical Field

[0001] This invention relates to the field of surface-enhanced Raman spectroscopy, specifically to a levocarnitine-imprinted MOF-Au Raman substrate, its preparation method, and its application. Background Technology

[0002] L-carnitine, a widely used clinical treatment for cardiovascular and metabolic diseases, is prone to developing impurity I (E / Z isomer of 4-trimethylammonium-2-butenoic acid inner salt) during the production and storage of its oral liquid formulation. The presence of this impurity not only affects the stability of the drug's quality but may also pose a risk to medication safety. Therefore, establishing a rapid, sensitive, and highly selective impurity detection method is crucial for quality control and batch release during the production process. Surface-enhanced Raman spectroscopy (SERS) has shown great potential in pharmaceutical analysis due to its strong fingerprint recognition capabilities, lack of complex pretreatment, and fast detection speed. However, achieving efficient application of this technology in the complex aqueous matrix of L-carnitine oral liquid places stringent requirements on the SERS substrate material: the substrate must simultaneously possess high selective enrichment capabilities for target molecules to reduce matrix interference, a stable nano-interstitial hotspot structure to ensure reproducible signal enhancement, and excellent film structure stability to adapt to the liquid phase detection environment. The synergistic optimization of these multi-dimensional performance requirements has become a key bottleneck in propelling SERS technology from the laboratory to practical applications.

[0003] Currently, research on SERS substrates mainly focuses on the construction and enhancement mechanisms of gold / silver nanostructures, but significant shortcomings remain when dealing with the detection of complex real-world samples. Chinese patent CN114414548B discloses an SERS substrate and its application, but it uses a simple physical adsorption method to immobilize gold nanoparticles, which is prone to nanoparticle aggregation and detachment in liquid-phase detection environments, leading to hotspot structural instability and signal attenuation. Chinese patent CN107913741A discloses a method for preparing MOF-199-loaded nanoparticle composite materials. While utilizing the high specific surface area of ​​MOFs to improve the dispersion of noble metals, it lacks a selective recognition mechanism for target molecules, resulting in severe non-specific adsorption in complex matrices such as oral liquids and high background interference signals. Furthermore, existing substrates often rely on a single chemical bonding method for metal-carrier interface anchoring, making it difficult to maintain the accessibility of plasmon activity on the metal surface while ensuring strong anchoring force, leading to poor signal repeatability after long-term use. The root cause of these technical bottlenecks lies in the failure to achieve systematic synergistic optimization among the structural stability, selective recognition capability, nano-gap hotspot density, and long-term signal consistency of composite films. Summary of the Invention

[0004] The purpose of this invention is to provide a levocarnitine-imprinted MOF-Au Raman substrate, its preparation method, and its application, thereby solving the technical problems of current SERS substrates in the process of composite membrane construction and use, such as the difficulty in balancing membrane stability and nano-interstitial hotspot density, the difficulty in balancing high enrichment capacity and resistance to matrix interference, and the difficulty in synergistically coordinating strong anchoring and long-term signal consistency.

[0005] This invention employs a design concept of dual-anchor imprinted MOF synergistic fixation of gold nanoislands. Through the synergistic anchoring effect of Au-S and Au-OP bonds, the gold nanoislands are firmly fixed and the nano-gap is precisely controlled. The selective recognition sites provided by the molecular imprinted channels enhance the enrichment of target molecules and suppress non-specific adsorption, thus constructing a multifunctional SERS substrate that integrates structural stability, selective recognition, high-density hotspots, and long-term signal reliability. This results in a significantly improved comprehensive performance compared to traditional single-anchored or non-imprinted substrates.

[0006] Technical solution

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] The term "imprinted MOF" as used in this article refers to a zirconium-based MOF containing imprinted channels, formed using a template molecule and then eluted to remove the template molecule. The term "dual-anchor imprinted MOF" as used in this article refers to an imprinted MOF in which sulfur and phosphorus sites are introduced by surface treatment with 3-mercaptopropionic acid and phosphoric acid on the basis of the aforementioned imprinted MOF. The term "imprinted MOF-Au composite intermediate" as used in this article refers to a composite obtained by loading gold nanoislands onto the aforementioned dual-anchor imprinted MOF.

[0009] A levocarnitine-imprinted MOF-Au Raman substrate includes a substrate and a composite film loaded on the surface of the substrate;

[0010] The substrate is selected from either a silicon dioxide substrate or a silicon substrate;

[0011] The composite film comprises an imprinted MOF and gold nano islands anchored to the imprinted MOF via both Au-S and Au-OP bonds.

[0012] The imprinted MOF contains zirconium and terephthalic acid ligands, and has imprinted channels formed using template molecules as templates and obtained by elution to remove the template molecules;

[0013] The specific surface area of ​​the imprinted MOF is 500-1500 m². 2 / g, with a pore size of 0.5-2.0 nm;

[0014] The gold nano islands have a particle size of 10-40 nm and the nano gaps between adjacent gold nano islands are less than 10 nm.

[0015] The mass fraction of gold in the composite film is 20-60 wt% based on the dry basis of the composite film, and the area coverage of the composite film on the substrate surface is 40-80%.

[0016] Furthermore, the imprinted MOF is prepared under the following conditions:

[0017] A1) Raw material preparation: Zirconium tetrachloride, terephthalic acid, template molecule, glacial acetic acid and N,N-dimethylformamide are provided, wherein the molar ratio of zirconium tetrachloride to terephthalic acid is 0.40–0.60:1, the molar ratio of template molecule to terephthalic acid is 0.05–0.60:1, the molar ratio of glacial acetic acid to zirconium tetrachloride is 10–60:1, and N,N-dimethylformamide is added to make the molar concentration of zirconium tetrachloride in the mixed system 0.02–0.20 mol / L;

[0018] A2) Solvent-thermal reaction: The mixture obtained in A1) is placed in a closed reaction vessel and reacted at 90-120 °C for 6-18 h;

[0019] A3) Post-treatment and template elution: The solid obtained in A2) was washed sequentially with N,N-dimethylformamide and ethanol, 2-6 times with N,N-dimethylformamide and 2-6 times with ethanol. The solid-liquid ratio for each wash was 1:5–1:50 g / mL and the washing time was 5-30 min. Then, the template molecules were removed by elution with a mixed solvent of ethanol and deionized water, with a volume ratio of ethanol to deionized water of 30:70–70:30. The elution was repeated 3-10 times, with a solid-liquid ratio of 1:5–1:50 g / mL and the elution time was 10-60 min. Finally, the solid was dried at 40-120 °C for 4-12 h.

[0020] A4) Endpoint Criteria and Quality Control: When the specific surface area of ​​the obtained MOF is 500-1500 m² 2 When the pore size is 0.5-2.0 nm, the pore size is calculated using the DFT method based on nitrogen adsorption-desorption data to obtain the imprinted MOF.

[0021] Furthermore, the imprinted MOF in the composite film is a dual-anchor positioning point imprinted MOF, which is prepared under the following conditions:

[0022] B1) Raw material supply: Providing imprinted MOF, 3-mercaptopropionic acid, phosphoric acid, ethanol, and deionized water;

[0023] B2) Introduction of sulfide sites: The imprinted MOF is dispersed in a mixed solvent of ethanol and deionized water, wherein the volume ratio of ethanol to deionized water is 10:90-90:10, and the mass concentration of the imprinted MOF in the mixed solvent is 1-50 mg / mL. The dispersion is achieved by ultrasonic treatment, wherein the ultrasonic power is 100-800 W and the ultrasonic time is 1-30 min. Then, 3-mercaptopropionic acid is added to a concentration of 0.01-0.20 mol / L, and the reaction is carried out at 25-70 ℃ for 1-8 h.

[0024] B3) Phosphorus site introduction: Phosphoric acid is added to the dispersion system obtained in B2) to make the concentration of phosphoric acid in the system 0.005-0.10 mol / L, and then the reaction is carried out at 25-70 °C for 1-8 h;

[0025] B4) Post-treatment: The obtained solid was washed with ethanol and deionized water 2-6 times and 2-6 times with deionized water. The solid-liquid ratio of each wash was 1:5-1:50 g / mL and the washing time was 5-30 min. Then it was dried at 40-120 ℃ for 4-12 h.

[0026] B5) Quality control: When the mass fraction of sulfur in the obtained solid is 0.10-2.00 wt% and the mass fraction of phosphorus is 0.05-1.50 wt%, the double anchor positioning point imprint MOF is obtained.

[0027] Furthermore, the imprinted MOF in the composite film is formed from an imprinted MOF-Au composite intermediate, which is prepared under the following conditions:

[0028] C1) Raw material supply: Provide dual-anchor positioning point imprint MOF, tetrachloroauric acid, L-ascorbic acid, ethanol and deionized water;

[0029] C2) Adsorption: The dual-anchor positioning point imprinted MOF is dispersed in a mixed solvent of ethanol and deionized water, wherein the volume ratio of ethanol to deionized water is 10:90-90:10, and the mass concentration of the dual-anchor positioning point imprinted MOF in the mixed solvent is 1-50 mg / mL. Tetrachloroauric acid is then added to adjust the gold ion concentration to Au. 3+ The concentration was calculated to be 0.2-5.0 mmol / L, and the addition method was dropwise addition at a rate of 0.05-5 mL / min, followed by adsorption at 5-40 °C for 5-60 min;

[0030] C3) Reduction and nucleation: L-ascorbic acid is added to the system obtained in C2) to make the concentration of L-ascorbic acid 0.5-20 mmol / L, and then the reaction is carried out at 5-40 ℃ for 5-60 min to obtain the imprinted MOF-Au complex intermediate;

[0031] C4) Endpoint Criteria and Quality Control: When the mass fraction of gold in the obtained composite intermediate is 20-60 wt%, and the particle size of the gold nano islands is 10-40 nm, and the nano-gap between adjacent gold nano islands is less than 10 nm, it is judged to be qualified.

[0032] Furthermore, the composite film is constructed on the surface of the substrate through the following steps:

[0033] D1) Substrate provision: providing the silicon dioxide substrate or the silicon substrate;

[0034] D2) Coupling treatment: The substrate is immersed in an ethanol solution of 3-aminopropyltriethoxysilane, wherein the volume fraction of 3-aminopropyltriethoxysilane is 0.5-5.0 vol%, and the solvent of the ethanol solution is ethanol with a volume fraction of 95.0-99.5 vol%. The reaction is then carried out for 5-60 min and rinsed with ethanol. Finally, the substrate is dried at 60-120 °C for 10-60 min.

[0035] D3) Film Formation: The imprinted MOF-Au composite intermediate is dispersed in a mixed solvent of ethanol and deionized water to form a dispersion, wherein the volume ratio of ethanol to deionized water is 10:90-90:10, and the mass concentration of the dispersion is 0.5-10 mg / mL. The dispersion is achieved by ultrasonic treatment with an ultrasonic power of 100-800 W and an ultrasonic time of 1-30 min. Subsequently, a composite film is formed on the substrate surface by dip coating or spin coating. The dip coating speed is 0.5-10 mm / s and then dried at 25-60 ℃ for 10-120 min, or the spin coating speed is 500-4000 rpm and the spin coating time is 10-60 s and then dried at 25-60 ℃ for 10-120 min.

[0036] D4) Endpoint criterion: When the area coverage of the composite film is 40-80%, the Raman substrate is obtained.

[0037] Furthermore, the thickness of the composite membrane is 50-500 nm, and the thickness is obtained by measuring the cross-sectional scanning electron microscope image of the composite membrane and taking the average value of at least 5 different positions.

[0038] As a concept of this invention, the design of using imprinted MOFs as a carrier and employing dual anchoring points to synergistically hold gold nanoislands is primarily intended to enhance the structural stability, selective recognition capability, and signal reliability of SERS substrates. The high chemical stability and open-pore structure of zirconium-based MOFs (UiO-66 type) provide a stable loading framework for the gold nanoislands, with a specific surface area of ​​500–1500 m². 2 The range of / g ensures sufficient dispersion sites for gold nanoislands and adsorption capacity for target molecules. By constructing imprinted channels using template molecules (levocarnitine or its impurity molecules) as templates, recognition sites complementary to the spatial configuration and chemical functional groups of the target molecules are formed in the MOF framework, achieving selective enrichment of levocarnitine and its impurities, effectively suppressing the non-specific adsorption of excipients and coexisting substances in the complex matrix of oral liquids. The design of dual anchoring sites, through surface treatment with 3-mercaptopropionic acid and phosphoric acid, simultaneously introduces thiol (–SH) and phosphate (–PO3H2) functional groups onto the MOF framework, enabling gold nanoislands to be anchored to the MOF surface through both Au–S and Au–O–P bonds. The synergistic effect significantly enhances the bonding strength at the gold-support interface, preventing the detachment and aggregation of gold nanoislands in the liquid phase detection environment. The precise design of gold nanoislands with a particle size of 10–40 nm and a gap of less than 10 nm ensures the formation of high-density electromagnetic field enhancement hotspots between adjacent gold nanoparticles. The gold mass fraction range of 20–60 wt% ensures the hotspot density while avoiding the disappearance of nano-gap and signal reduction caused by excessive gold loading.

[0039] This invention also discloses a method for preparing a levocarnitine-imprinted MOF-Au Raman substrate, comprising the following steps:

[0040] S1) Preparation of imprinted MOF: Using zirconium tetrachloride and terephthalic acid as raw materials, in the presence of template molecules, a solvothermal reaction is carried out in a mixed solvent system of N,N-dimethylformamide and glacial acetic acid to obtain imprinted MOF;

[0041] S2) Preparation of MOF with dual anchor points: The surface of the imprinted MOF was treated with 3-mercaptopropionic acid and phosphoric acid to obtain MOF with dual anchor points.

[0042] S3) Preparation of composite intermediate: Tetrachloroauric acid is adsorbed onto the surface of the double anchoring site imprinted MOF and reduced with L-ascorbic acid to form gold nano islands on the surface of the double anchoring site imprinted MOF, thus obtaining the imprinted MOF–Au composite intermediate.

[0043] S4) Film formation and substrate construction: The imprinted MOF-Au composite intermediate is coated on the surface of a silicon dioxide substrate or a silicon substrate, and then dried to obtain a Raman substrate.

[0044] Further, in step S1, the molar ratio of zirconium tetrachloride to terephthalic acid is 0.40–0.60:1, the molar ratio of template molecule to terephthalic acid is 0.05–0.60:1, the molar ratio of glacial acetic acid to zirconium tetrachloride is 10–60:1, and N,N-dimethylformamide is added to make the molar concentration of zirconium tetrachloride in the mixed system 0.02–0.20 mol / L.

[0045] Further, in step S2, the concentration of 3-mercaptopropionic acid is 0.01–0.20 mol / L, the concentration of phosphoric acid is 0.005–0.10 mol / L, and the mass concentration of the imprinted MOF in the mixed solvent of ethanol and deionized water is 1–50 mg / mL, and the volume ratio of ethanol to deionized water is 10:90–90:10.

[0046] In step S3, the gold ion concentration is expressed as Au. 3+ The concentration of L-ascorbic acid is 0.5–20 mmol / L, and the mass concentration of the dual-anchored positioning point imprinted MOF in a mixed solvent of ethanol and deionized water is 1–50 mg / mL, the volume ratio of ethanol to deionized water is 10:90–90:10, and tetrachloroauric acid is added dropwise at a rate of 0.05–5 mL / min.

[0047] As another aspect of this invention, a step-by-step controllable preparation method is employed to enhance the precise construction of imprinted channels, the uniform introduction of dual anchoring points, and the precise control of the particle size / gap of gold nanoislands. In the solvothermal preparation of imprinted MOFs, the preferred range of a template molecule to terephthalic acid molar ratio of 0.05–0.60:1 ensures a balance between imprinted channel density and MOF crystal integrity. Controlling the reaction temperature to 90–120℃ and the reaction time to 6–18 h achieves high crystallinity and good channel openness. Subsequent template elution utilizes an ethanol-deionized water mixed solvent system. Solvent polarity control improves template molecule removal efficiency and avoids interference from residual template on subsequent recognition performance. The introduction of dual anchoring points employed a stepwise treatment strategy, first sulfur-based and then phosphorus-based. A ratio of 0.01–0.20 mol / L 3-mercaptopropionic acid and 0.005–0.10 mol / L phosphate ensured the uniform distribution of both functional groups on the MOF surface. The endpoint criteria of 0.10–2.00 wt% sulfur and 0.05–1.50 wt% phosphorus guaranteed the sufficient introduction of dual anchoring points. Gold nanoislands were grown using an in-situ reduction nucleation strategy. A ratio of 0.2–5.0 mmol / L gold ions and 0.5–20 mmol / L L-ascorbic acid controlled the nucleation rate and particle size growth. The dropwise addition method and low-temperature reaction at 5–40 °C inhibited excessive growth and aggregation of gold nanoparticles, achieving precise control over the 10–40 nm particle size range and internanoscale gaps smaller than 10 nm.

[0048] This invention also discloses the application of a L-carnitine imprinted MOF-Au Raman substrate in the rapid detection of impurity I in L-carnitine oral solution, wherein impurity I is (E)-4-(trimethylammonium)-2-butenoic acid inner salt and / or its (Z)-isomer or a mixture thereof; by contacting the L-carnitine oral solution sample to be tested with the imprinted MOF-Au Raman substrate and acquiring the surface-enhanced Raman spectral signal, the qualitative identification or quantitative detection of impurity I can be achieved, which can be used for product pre-inspection in the production process, thereby reducing or replacing the complex sample pretreatment required by high-performance liquid chromatography and shortening the detection time.

[0049] As the third concept of this invention, the detection strategy combining imprinting recognition and surface-enhanced Raman spectroscopy is primarily used to improve the detection sensitivity and selectivity of impurity I in L-carnitine oral solution. The spatial matching and chemical complementarity of the imprinted channels to the target molecule preferentially enriches impurity I within the MOF channels, directly translating the increased enrichment factor into enhanced Raman signal, resulting in a significantly lower detection limit compared to non-imprinted substrates. Simultaneously, the molecular sieving effect of the imprinted channels effectively eliminates interference from excipient molecules such as methylparaben, propylparaben, and sodium saccharin in the oral solution, suppressing background signals caused by non-specific adsorption. The high-density nano-interstic hotspot structure of the gold nanoislands provides strong electromagnetic field enhancement, which, combined with the synergistic effect of imprinting enrichment, achieves highly sensitive detection of trace impurities. The structural stability of the substrate in the liquid-phase detection environment ensures batch-to-batch repeatability of the signal, meeting the requirements of production process quality control for rapid, accurate, and stable detection methods, and providing a new technical means for real-time monitoring of drug quality.

[0050] Furthermore, the template molecule is selected from at least one of levocarnitine, E-configuration 4-trimethylammonium-2-butenoic acid inner salt, and Z-configuration 4-trimethylammonium-2-butenoic acid inner salt.

[0051] Furthermore, the specific surface area of ​​the imprinted MOF was determined by vacuum degassing the sample at 120 °C for 12 h and measuring it using the BET method based on nitrogen adsorption-desorption data.

[0052] Furthermore, the pore size of the imprinted MOF was obtained by vacuum degassing the sample at 120 °C for 12 h and calculating it using the DFT method based on nitrogen adsorption-desorption data.

[0053] Furthermore, the mass fraction of gold in the composite film is based on a dry basis after the composite film has been vacuum dried to constant weight at 60 °C.

[0054] Furthermore, the mass fraction of gold in the composite intermediate is based on a dry basis after the obtained composite intermediate has been vacuum dried to constant weight at 60 °C.

[0055] Furthermore, the mass fractions of sulfur and phosphorus in the dual-anchor positioning point imprint MOF are based on the dry basis after the obtained solid is vacuum dried to constant weight at 60 °C.

[0056] Furthermore, the mass fraction of gold was determined by inductively coupled plasma atomic emission spectrometry.

[0057] Furthermore, the Au–S and Au–O–P bonds were characterized by X-ray photoelectron spectroscopy.

[0058] Furthermore, the particle size and nano-gap of the gold nanoislands were obtained by statistical analysis of electron microscope images, with a sample size of no less than 200 gold nanoislands.

[0059] Furthermore, the area coverage of the composite film on the substrate surface is obtained by threshold segmentation statistics of the scanning electron microscope image of the substrate surface.

[0060] Furthermore, in step A2, the sealed reaction vessel is a stainless steel autoclave with a polytetrafluoroethylene liner and a filling degree of 40–80%.

[0061] Furthermore, in step A3, the endpoint criterion for template elution is that the concentration of template molecules in the eluent is less than 0.5 μg / mL as detected by high performance liquid chromatography.

[0062] Furthermore, in step B2, the dispersion system is stirred after the addition of 3-mercaptopropionic acid, and the stirring method is magnetic stirring or mechanical stirring.

[0063] Furthermore, in step B3, the phosphoric acid is added dropwise or in batches.

[0064] Furthermore, in step C2, the adsorption process is carried out under light-protected conditions.

[0065] Furthermore, in step C3, the reduction reaction is carried out under light-protected conditions and is performed using magnetic stirring or mechanical stirring.

[0066] Furthermore, in step C3, the reduction reaction is considered complete when the system color turns purplish-red or wine-red.

[0067] Furthermore, the Raman substrate is used for quality control pre-inspection during the production process of L-carnitine oral solution, particularly for detecting impurity I in L-carnitine oral solution, wherein impurity I is E-configuration 4-trimethylammonium-2-butenoic acid inner salt or Z-configuration 4-trimethylammonium-2-butenoic acid inner salt.

[0068] Furthermore, in step D1, the substrate is cleaned before use. The cleaning process includes ultrasonic cleaning with acetone, ethanol and deionized water in sequence, with each ultrasonic cleaning lasting 5–30 min. The substrate is then dried under a nitrogen flow or at 60–120 °C.

[0069] Furthermore, in step S1, the reaction is a solvothermal reaction with a reaction temperature of 90–120 °C and a reaction time of 6–18 h.

[0070] Furthermore, in step S1, after the zirconium tetrachloride, terephthalic acid, and template molecule are mixed evenly in a mixed solvent of N,N-dimethylformamide and glacial acetic acid, they are transferred to a reaction vessel for a solvothermal reaction.

[0071] Furthermore, after step S1 is completed, the reaction product is washed sequentially with N,N-dimethylformamide and ethanol, and then eluted with a mixed solvent of ethanol and deionized water to remove template molecules, followed by drying to obtain the imprinted MOF.

[0072] Furthermore, in step S2, the surface treatment includes, in one embodiment, introducing sulfur-based sites first and then introducing phosphorus-based sites; in another embodiment, introducing phosphorus-based sites first and then introducing sulfur-based sites.

[0073] Furthermore, in step S4, the coating method is dip coating or spin coating, the drying temperature is 25–60 ℃, and the drying time is 10–120 min.

[0074] The preparation process of the levocarnitine oral solution of the present invention includes the following steps:

[0075] P1. Add 472 kg of purified water to the concentration tank, start stirring at a frequency of 40 Hz, and start heating to heat the purified water to 80°C for later use.

[0076] P2. Turn on the external circulation system. After the circulation reflux is stable, add 0.679 kg of methylparaben and 0.192 kg of propylparaben in sequence. Rinse the feeding container with purified water and spray the tank wall. Continue stirring for 30 min until the solution is clear. Turn off the heating and external circulation system, turn on the cooling device, and lower the solution temperature to 25°C.

[0077] P3. Add 1.888 kg of DL-malic acid, rinse the feeding container with purified water and spray the tank wall, and continue stirring for 10 minutes until the solution is clear;

[0078] P4. Add 59 kg of L-carnitine, rinse the feeding container with purified water and spray the tank wall, and continue stirring for 15 min until the solution is clear after the L-carnitine is completely dissolved;

[0079] P5. Add 2.832 kg of sodium saccharin, rinse the feeding container with purified water and spray the tank wall, and continue stirring for 10 min until the solution is clear;

[0080] P6. After stirring, take a sample of the concentrated solution and send it for testing;

[0081] P7. Add purified water to a total volume of 590 L, circulate and stir for 30 min, and take the intermediate product to test its properties, pH value, relative density, and content;

[0082] P8. The drug solution is circulated and filtered for 30 minutes through a 5 μm titanium rod filter, and the clarity of the solution is monitored and found to be within acceptable limits. After the intermediate product passes inspection, the drug solution is transferred to the filling and sealing production line, the filling volume is adjusted to 118 mL per bottle, and filling and sealing begins.

[0083] P9. Label and package the selected qualified products and put them into storage.

[0084] The synergistic mechanism of imprinted MOF and gold nanoislands in the composite system of this invention is manifested in multi-level coupling of structural support, selective recognition, and signal enhancement. As a structural framework, the imprinted MOF, with its high specific surface area and porous structure, provides a three-dimensional space for the dispersed loading of gold nanoislands, preventing the disordered aggregation of gold nanoparticles. Simultaneously, the selective recognition function of the imprinted channels precisely guides target molecules to the electromagnetic field hotspots on the surface of the gold nanoislands, achieving a synergistic effect of enrichment and enhancement. The gold nanoislands are firmly anchored to the MOF framework through dual anchoring points. The strong covalent interaction of Au–S bonds and the coordination / ionic bonds of Au–O–P bonds form a multi-anchoring network, significantly enhancing the bonding strength at the gold-support interface and preventing the detachment of gold nanoislands in the liquid phase. Furthermore, the localized surface plasmon resonance effect of the gold nanoislands generates a strong Raman signal enhancement for the target molecules enriched within the imprinted channels. This three-dimensional synergistic effect of structural stability, selective recognition, and electromagnetic field enhancement enables the substrate of this invention to exhibit significantly improved comprehensive performance compared to single-function materials when facing complex matrix samples, achieving a unity of high sensitivity, high selectivity, and long-term stability.

[0085] Beneficial technical effects

[0086] 1. Significantly improves structural stability and wash resistance: By introducing thiol and phosphate functional groups simultaneously on the surface of the imprinted MOF through a dual anchoring point design, gold nano islands are firmly held in place by the MOF framework through the synergistic anchoring mechanism of Au–S and Au–O–P bonds. The formation of multiple chemical bonding networks effectively inhibits the shedding, aggregation, and instability of the nano-interstitial structure of gold nanoparticles in the liquid phase detection environment, ensuring that the substrate maintains a stable hot spot structure and signal intensity even after long-term use and repeated washing, thus solving the problem of easy failure of traditional physical adsorption or single anchoring substrates.

[0087] 2. Achieving high selectivity and resistance to matrix interference: Molecular imprinting technology is used to construct imprinted channels in a zirconium-based MOF framework that are complementary to the spatial configuration and chemical functional groups of L-carnitine and its impurity molecules. The specific recognition function of the imprinted channels enables the preferential enrichment of target molecules. At the same time, the molecular sieving effect of the imprinted channels effectively eliminates the non-specific adsorption of excipients and coexisting substances such as methylparaben, propylparaben, and sodium saccharin in oral liquids, significantly reducing background interference from complex matrices and improving detection selectivity and signal-to-noise ratio.

[0088] 3. Construction of high-density nano-interstic hotspots and strong signal enhancement: By precisely controlling the particle size of gold nanoislands (10–40 nm) and the gap between adjacent nanoislands (less than 10 nm), a high-density electromagnetic field-enhanced hotspot array is formed on the surface of the MOF support. Combined with the enrichment effect of imprinted channels guiding target molecules to the hotspot region, a dual enhancement of the Raman signal is achieved. The detection sensitivity is significantly improved compared with non-imprinted or low hotspot density substrates, meeting the needs of trace impurity detection.

[0089] 4. Ensuring long-term signal reliability and batch consistency: The high chemical stability and structural robustness of zirconium-based MOFs, coupled with their dual anchoring mechanism, ensure the stable maintenance of the nano-interstitial hotspot structure during repeated use and storage. The long-term retention of plasmon activity on the surface of gold nanoislands guarantees the batch-to-batch repeatability of Raman signal intensity and characteristic peak positions. This provides a stable and reliable detection tool for quality control in the production process, reducing quality control costs and the risk of false negatives / false positives.

[0090] 5. Simplified detection process and shortened analysis time: The simple operation of Raman spectroscopy acquisition after direct immersion or drop addition of sample eliminates the need for complex sample pretreatment (deproteinization, extraction, derivatization, etc.) required by high performance liquid chromatography. The detection time is reduced from several hours in traditional methods to several minutes. It is suitable for rapid online / offline pre-inspection of production lines, realizing a technological leap from offline detection to real-time quality control, and improving production efficiency and product release speed. Attached Figure Description

[0091] Figure 1 XPS overlay of Au-S and Au-OP bonding states to provide in-depth analysis of the dual-anchoring synergistic enhancement of film stability and gold nanoisland retention capacity.

[0092] Figure 2 High-resolution peak fitting comparison of Au 4f spectra in depth analysis of Au-S and Au-OP bonding states to enhance film stability and gold nanoisland retention through dual-anchoring synergistic enhancement.

[0093] Figure 3 X-ray photoelectron spectroscopy (XPS) analysis of the S 2p high-resolution spectra of Au-S and Au-OP bonding states to enhance film stability and gold nanoisland retention through dual-anchoring synergistic enhancement.

[0094] Figure 4 X-ray photoelectron spectroscopy (XPS) analysis of the P 2p high-resolution peak fitting of Au-S and Au-OP bonding states to enhance film stability and gold nanoisland retention through dual-anchoring synergistic enhancement.

[0095] Figure 5The graph shows the relationship between the morphology and distribution stability of gold nanoislands before and after cyclic rinsing, and the number of rinsing cycles, in order to enhance the stability of the film layer and the retention capacity of gold nanoislands through dual anchoring.

[0096] Figure 6 The difference in particle size distribution of gold nanoislands before and after cyclic rinsing demonstrates the morphology and distribution stability of the gold nanoislands before and after the synergistic enhancement of film stability and gold nanoisland retention capacity through dual anchoring.

[0097] Figure 7 The nano-gap difference distribution curves show the morphology and distribution stability of gold nano-islands before and after cyclic rinsing to enhance the stability of the film and the retention capacity of gold nano-islands through dual anchoring.

[0098] Figure 8 The cumulative particle size distribution of gold nanoislands in Example 1, Comparative Example 8, and Comparative Example 7 is shown.

[0099] Figure 9 The cumulative distribution curves of nano-gap morphology and distribution stability of gold nano-islands before and after cyclic rinsing to enhance the stability of the film layer and the retention capacity of gold nano-islands through dual anchoring synergistic enhancement.

[0100] Figure 10 This is a superimposed diagram of nitrogen adsorption-desorption isotherms and pore size distribution isotherms to demonstrate the selective identification and high enrichment capacity of imprinted pores in complex matrices, exhibiting strong resistance to interference.

[0101] Figure 11 A comparison of nitrogen adsorption-desorption isotherms and DFT pore size distribution curves for selective identification and high enrichment of imprinted pores in complex matrices, demonstrating their anti-interference properties.

[0102] Figure 12 Raman spectral overlays are used to verify the selective adsorption of imprinted channels and their high enrichment capacity in complex matrices against interference. Detailed Implementation

[0103] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. The high-performance liquid chromatography method for detecting the concentration of impurity I is described in the Pharmacopoeia of the People's Republic of China.

[0104] Example 1

[0105] This embodiment provides a levocarnitine-imprinted MOF-Au Raman substrate, comprising a silica substrate and a composite film loaded on the substrate surface. The composite film contains an imprinted MOF and gold nanoislands doubly anchored to the imprinted MOF via Au-S and Au-OP bonds.

[0106] Preparation of S1 imprinted MOF

[0107] A1) Raw material preparation: Weigh 3.24 g of zirconium tetrachloride and 5.09 g of terephthalic acid, with a molar ratio of 0.45:1. Weigh 1.22 g of the template molecule L-carnitine, with a molar ratio of 0.25:1 to terephthalic acid. Weigh 18.01 g of glacial acetic acid, with a molar ratio of 22:1 to zirconium tetrachloride. Add N,N-dimethylformamide to a total volume of 139 mL, so that the molar concentration of zirconium tetrachloride in the mixed system is 0.10 mol / L. Stir and mix evenly at room temperature.

[0108] A2) Solvothermal reaction: The above mixture was transferred to a stainless steel autoclave lined with polytetrafluoroethylene, with a filling degree of 60%, and then sealed and placed in an oven at 105°C for 12 h.

[0109] A3) Post-treatment and template elution: After the reaction, the product was allowed to cool naturally to room temperature and collected by centrifugation. It was washed four times with N,N-dimethylformamide at a solid-liquid ratio of 1:20 g / mL for 15 min each time, followed by four washes with ethanol at the same solid-liquid ratio and time. Template elution was performed using a 50:50 mixture of ethanol and deionized water for six washes at a solid-liquid ratio of 1:20 g / mL for 30 min each time. The concentration of L-carnitine in the eluent was determined by high-performance liquid chromatography (HPLC), and elution was considered complete when the concentration was below 0.5 μg / mL. Finally, the solid was vacuum-dried at 80 °C for 8 h.

[0110] A4) Quality control: The obtained sample was degassed under vacuum at 120℃ for 12 h. Based on nitrogen adsorption-desorption data, the specific surface area was determined to be 1000 m² / g by BET method, and the pore size was calculated to be 1.2 nm by DFT method, thus obtaining the imprinted MOF.

[0111] Preparation of S2 dual-anchor positioning point imprinted MOF

[0112] B1) Raw material supply: Take 2.0 g of the above-mentioned imprinted MOF, 3-mercaptopropionic acid, phosphoric acid, ethanol and deionized water.

[0113] B2) Introduction of sulfide sites: The imprinted MOF was dispersed in a mixed solvent of ethanol and deionized water at a volume ratio of 50:50, with a MOF mass concentration of 20 mg / mL. The MOF was sonicated at a power of 400 W for 15 min to ensure thorough dispersion. Subsequently, 3-mercaptopropionic acid was added to a concentration of 0.10 mol / L, and the mixture was magnetically stirred at 45 °C for 4 h.

[0114] B3) Phosphorus site introduction: Phosphoric acid was added dropwise to the above dispersion system to make the concentration of phosphoric acid in the system 0.05 mol / L, and then the reaction was carried out at 45℃ with magnetic stirring for 4 h.

[0115] B4) Post-processing: The solid was collected by centrifugation, washed 4 times with ethanol and 4 times with deionized water, with a solid-liquid ratio of 1:20 g / mL each time and a washing time of 15 min each time. It was then vacuum dried at 80℃ for 8 h.

[0116] B5) Quality control: After the obtained solid was vacuum dried to constant weight at 60℃, the mass fraction of sulfur was determined by elemental analysis to be 1.0 wt% and the mass fraction of phosphorus was 0.7 wt%. X-ray photoelectron spectroscopy confirmed the successful introduction of sulfur and phosphate groups, and a double anchoring site imprint MOF was obtained.

[0117] Preparation of S3 Imprinted MOF-Au Complex Intermediate

[0118] C1) Raw material supply: Take 1.5 g of the above-mentioned double anchor positioning point imprint MOF, tetrachloroauric acid, L-ascorbic acid, ethanol and deionized water.

[0119] C2) Adsorption: The dual-anchored MOF was dispersed in a mixed solvent of ethanol and deionized water at a volume ratio of 50:50, with a mass concentration of 20 mg / mL. Tetrachloroauric acid solution was added under light-protected conditions at a dropping rate of 2 mL / min to increase the gold ion concentration by Au. 3+ The concentration was calculated to be 2.5 mmol / L, and then adsorption was carried out at 20℃ with magnetic stirring for 30 min.

[0120] C3) Reduction and nucleation: Add L-ascorbic acid to the above system to a concentration of 10 mmol / L, and stir magnetically at 20°C for 30 min under light-protected conditions. The reduction reaction is considered complete when the system turns wine-red.

[0121] C4) Quality Control: The solid was collected by centrifugation and washed alternately with ethanol and deionized water until the supernatant was colorless. It was then vacuum dried at 60℃ to constant weight. The mass fraction of gold in the composite intermediate was determined to be 40 wt% by inductively coupled plasma atomic emission spectrometry. More than 200 gold nanoislands were identified by transmission electron microscopy, and the particle size of the gold nanoislands was measured to be 25 nm, with a nanometer gap of 5 nm between adjacent gold nanoislands, thus obtaining the imprinted MOF-Au composite intermediate.

[0122] S4 film formation and substrate construction

[0123] D1) Substrate provision: Take a silicon dioxide substrate and ultrasonically clean it in sequence with acetone, ethanol and deionized water, each time for 15 minutes, and then blow it dry under nitrogen flow.

[0124] D2) Coupling treatment: The cleaned substrate was immersed in an ethanol solution of 3-aminopropyltriethoxysilane with a volume fraction of 2.5 vol% and a volume fraction of 97.5 vol% of ethanol. The reaction was carried out at room temperature for 30 min, followed by rinsing with ethanol and drying at 90 °C for 30 min.

[0125] D3) Film Formation: The imprinted MOF-Au composite intermediate was dispersed in a mixed solvent of ethanol and deionized water at a volume ratio of 50:50, with a dispersion concentration of 5 mg / mL. The ultrasonic power was 400 W, and the ultrasonic time was 15 min. The film was formed by spin coating at a speed of 2000 rpm for 30 s, followed by drying at 40℃ for 60 min.

[0126] D4) Quality Control: The substrate surface was observed using a scanning electron microscope (SEM), and threshold segmentation statistics were performed on the SEM images. The composite film area coverage was 60%. The cross-section of the composite film was observed using an SEM, and the film thickness was measured at at least 5 different locations and the average value was taken, resulting in a film thickness of 250 nm.

[0127] In this embodiment, the imprinted MOF specific surface area of ​​the Raman substrate composite film is 1000 m². 2 The composite membrane has a pore size of 1.2 nm, a gold nanoisland size of 25 nm, a spacing of 5 nm between adjacent gold nanoislands, a gold mass fraction of 40 wt%, a composite membrane coverage of 60%, and a membrane thickness of 250 nm. The solid component of the composite membrane is derived from an imprinted MOF–Au composite intermediate; therefore, the gold mass fraction in the composite membrane is expressed as the dry basis value determined after the composite intermediate has been vacuum dried to constant weight at 60 °C.

[0128] Features of this embodiment: This embodiment employs a moderate parameter design, with a zirconium tetrachloride to terephthalic acid molar ratio of 0.45:1, a template molecule molar ratio of 0.25:1, a glacial acetic acid molar ratio of 22:1, a solvothermal reaction temperature of 105℃, and a reaction time of 12 h, to prepare an imprinted MOF with a specific surface area of ​​1000 m² / g and a pore size of 1.2 nm. The composite membrane contains 40 wt% gold, gold nanoisland size of 25 nm, nano-intervals of 5 nm, a composite membrane coverage of 60%, and a membrane thickness of 250 nm. This parameter combination exhibits good process stability and reproducibility, making it suitable for large-scale production. This scheme is applicable to routine quality control pre-inspection in the production process of L-carnitine oral solution, demonstrating stable detection sensitivity for impurity I, and can meet the needs of rapid screening of large batches of samples.

[0129] Example 2

[0130] This embodiment provides a levocarnitine-imprinted MOF-Au Raman substrate, comprising a silicon substrate and a composite film loaded on the substrate surface. The composite film contains an imprinted MOF and gold nanoislands doubly anchored to the imprinted MOF via Au-S and Au-OP bonds.

[0131] Preparation of S1 imprinted MOF

[0132] A1) Raw material preparation: Weigh 6.49 g of zirconium tetrachloride and 10.72 g of terephthalic acid, with a molar ratio of 0.43:1. Weigh 1.12 g of the template molecule E-configuration 4-trimethylammonium-2-butenoic acid inner salt, with a molar ratio of 0.12:1 to terephthalic acid. Weigh 77.81 g of glacial acetic acid, with a molar ratio of 47:1 to zirconium tetrachloride. Add N,N-dimethylformamide to a total volume of 558 mL, so that the molar concentration of zirconium tetrachloride in the mixed system is 0.05 mol / L. Stir and mix evenly at room temperature.

[0133] A2) Solvothermal reaction: The above mixture was transferred to a stainless steel autoclave lined with polytetrafluoroethylene, with a filling degree of 65%, and then sealed and placed in an oven at 110°C for 15 h.

[0134] A3) Post-treatment and template elution: After the reaction, the product was allowed to cool naturally to room temperature and collected by centrifugation. It was washed five times with N,N-dimethylformamide at a solid-liquid ratio of 1:30 g / mL for 20 min each time, followed by five washes with ethanol at the same solid-liquid ratio and time. Template elution was performed eight times with a solid-liquid ratio of 1:30 g / mL for 45 min each time. The concentration of template molecules in the eluent was determined by high-performance liquid chromatography (HPLC), and elution was considered complete when the concentration was below 0.5 μg / mL. Finally, the solid was vacuum-dried at 100℃ for 10 h.

[0135] A4) Quality control: The obtained sample was degassed under vacuum at 120℃ for 12 h. Based on nitrogen adsorption-desorption data, the specific surface area was determined to be 1300 m² / g by BET method, and the pore size was calculated to be 1.7 nm by DFT method, thus obtaining the imprinted MOF.

[0136] Preparation of S2 dual-anchor positioning point imprinted MOF

[0137] B1) Raw material supply: Take 1.5 g of the above-mentioned imprinted MOF, 3-mercaptopropionic acid, phosphoric acid, ethanol and deionized water.

[0138] B2) Introduction of sulfide sites: The imprinted MOF was dispersed in a mixed solvent of ethanol and deionized water at a volume ratio of 70:30, with a MOF mass concentration of 10 mg / mL. The MOF was sonicated at 600 W for 20 min to ensure thorough dispersion. Subsequently, 3-mercaptopropionic acid was added to a concentration of 0.15 mol / L, and the reaction was carried out at 60 °C with magnetic stirring for 6 h.

[0139] B3) Phosphorus site introduction: Phosphoric acid was added dropwise to the above dispersion system to make the concentration of phosphoric acid in the system 0.08 mol / L, and then the reaction was carried out at 60℃ with magnetic stirring for 6 h.

[0140] B4) Post-processing: The solid was collected by centrifugation, washed 5 times with ethanol and 5 times with deionized water, with a solid-liquid ratio of 1:30 g / mL each time and a washing time of 20 min each time. It was then dried under vacuum at 100℃ for 10 h.

[0141] B5) Quality control: After the obtained solid was vacuum dried to constant weight at 60℃, the mass fraction of sulfur was determined by elemental analysis to be 1.5 wt% and the mass fraction of phosphorus was 1.1 wt%. X-ray photoelectron spectroscopy confirmed the successful introduction of sulfur and phosphate groups, and a double anchoring site imprinted MOF was obtained.

[0142] Preparation of S3 Imprinted MOF-Au Complex Intermediate

[0143] C1) Raw material supply: Take 1.2 g of the above-mentioned double anchor positioning point imprint MOF, tetrachloroauric acid, L-ascorbic acid, ethanol and deionized water.

[0144] C2) Adsorption: The dual-anchored MOF was dispersed in a mixed solvent of ethanol and deionized water at a volume ratio of 70:30, with a mass concentration of 10 mg / mL. Tetrachloroauric acid solution was added at a dropping rate of 1 mL / min under light-protected conditions to increase the gold ion concentration by Au. 3+ The concentration was calculated to be 1.5 mmol / L, and then adsorption was carried out at 15 °C with magnetic stirring for 45 min.

[0145] C3) Reduction and nucleation: L-ascorbic acid was added to the above system to a concentration of 6 mmol / L, and the mixture was magnetically stirred at 15°C for 45 min under light-protected conditions. The reduction reaction was considered complete when the system turned purple-red.

[0146] C4) Quality Control: The solid was collected by centrifugation and washed alternately with ethanol and deionized water until the supernatant was colorless. It was then dried under vacuum at 60°C to constant weight. The mass fraction of gold in the composite intermediate was determined to be 28 wt% by inductively coupled plasma atomic emission spectrometry. More than 200 gold nanoislands were identified by transmission electron microscopy, and the particle size of the gold nanoislands was measured to be 18 nm, with a nanometer gap of 7 nm between adjacent gold nanoislands, thus obtaining the imprinted MOF-Au composite intermediate.

[0147] S4 film formation and substrate construction

[0148] D1) Substrate provision: Take a silicon substrate and ultrasonically clean it in sequence with acetone, ethanol and deionized water for 15 minutes each time, and then dry it under nitrogen flow.

[0149] D2) Coupling treatment: The cleaned substrate was immersed in an ethanol solution of 3-aminopropyltriethoxysilane with a volume fraction of 3.5 vol% and a volume fraction of 96.5 vol% of ethanol. The reaction was carried out at room temperature for 45 min, followed by rinsing with ethanol and drying at 100 °C for 40 min.

[0150] D3) Film Formation: The imprinted MOF-Au composite intermediate was dispersed in a mixed solvent of ethanol and deionized water at a volume ratio of 70:30, with a dispersion concentration of 3 mg / mL. The ultrasonic power was 600 W, and the ultrasonic time was 20 min. The film was formed by dip coating at a pulling speed of 3 mm / s, and then dried at 45℃ for 90 min.

[0151] D4) Quality Control: The substrate surface was observed using a scanning electron microscope (SEM), and threshold segmentation statistics were performed on the SEM images. The composite film area coverage was 55%. The cross-section of the composite film was observed using an SEM, and the film thickness was measured at at least 5 different locations and the average value was taken, resulting in a film thickness of 150 nm.

[0152] In this embodiment, the imprinted MOF specific surface area of ​​the Raman substrate composite film is 1300 m². 2 / g, pore size of 1.7nm, gold nanoisland size of 18nm, nano-gap between adjacent gold nanoislands of 7nm, gold mass fraction of 28 wt%, composite film coverage of 55%, film thickness of 150 nm.

[0153] Features of this embodiment: This embodiment employs a low-gold-content, high-specific-surface-area imprinted MOF design, with a zirconium tetrachloride to terephthalic acid molar ratio of 0.43:1, a template molecule molar ratio of 0.12:1, a glacial acetic acid molar ratio of 47:1, a solvothermal reaction temperature of 110℃, and a reaction time of 15 h, resulting in a MOF with a specific surface area of ​​1300 m². 2A 1.7 nm pore size imprinted MOF was developed. The MOF with dual anchoring points contained 1.5 wt% sulfur and 1.1 wt% phosphorus. The composite membrane contained 28 wt% gold, with gold nanoislands of 18 nm in size and 7 nm inter-nanospaces. The composite membrane had 55% coverage and a thickness of 150 nm. This method enhances the recognition and adsorption capacity for target molecules through its high specific surface area and pore size. Simultaneously, the smaller gold nanoisland size and moderate inter-nanospaces facilitate the formation of more hotspot regions, making it suitable for high-sensitivity detection of low-concentration impurities (I). It is particularly suitable for incoming inspection of L-carnitine oral liquid raw materials and trace analysis of minor impurities.

[0154] Example 3

[0155] This embodiment provides a levocarnitine-imprinted MOF-Au Raman substrate, comprising a silica substrate and a composite film loaded on the substrate surface. The composite film contains an imprinted MOF and gold nanoislands doubly anchored to the imprinted MOF via Au-S and Au-OP bonds.

[0156] Preparation of S1 imprinted MOF

[0157] A1) Raw material preparation: Weigh 4.98 g of zirconium tetrachloride and 6.78 g of terephthalic acid, with a molar ratio of 0.52:1. Weigh 3.46 g of the template molecule Z-configuration 4-trimethylammonium-2-butenoic acid inner salt, with a molar ratio of 0.60:1 to terephthalic acid. Weigh 19.92 g of glacial acetic acid, with a molar ratio of 16:1 to zirconium tetrachloride. Add N,N-dimethylformamide to a total volume of 144 mL, so that the molar concentration of zirconium tetrachloride in the mixed system is 0.15 mol / L. Stir and mix evenly at room temperature.

[0158] A2) Solvothermal reaction: The above mixture was transferred to a stainless steel autoclave lined with polytetrafluoroethylene, with a filling degree of 55%, and then sealed and placed in an oven at 95°C for 9 h.

[0159] A3) Post-treatment and template elution: After the reaction, the product was allowed to cool naturally to room temperature and collected by centrifugation. It was washed three times with N,N-dimethylformamide at a solid-liquid ratio of 1:10 g / mL for 10 min each time, followed by three washes with ethanol at the same solid-liquid ratio and time. Template elution was performed five times with a solid-liquid ratio of 1:10 g / mL for 20 min each time, using a mixed solvent of ethanol and deionized water at a volume ratio of 40:60. The template molecule concentration in the eluent was determined by high-performance liquid chromatography (HPLC), and elution was considered complete when the concentration was below 0.5 μg / mL. Finally, the solid was vacuum-dried at 60℃ for 6 h.

[0160] A4) Quality control: The obtained sample was degassed under vacuum at 120℃ for 12 h. Based on nitrogen adsorption-desorption data, the specific surface area was determined to be 750 m² / g by BET method, and the pore size was calculated to be 0.8 nm by DFT method, thus obtaining the imprinted MOF.

[0161] Preparation of S2 dual-anchor positioning point imprinted MOF

[0162] B1) Raw material supply: Take 2.5 g of the above-mentioned imprinted MOF, 3-mercaptopropionic acid, phosphoric acid, ethanol and deionized water.

[0163] B2) Introduction of sulfide sites: The imprinted MOF was dispersed in a mixed solvent of ethanol and deionized water at a volume ratio of 30:70, with a MOF mass concentration of 35 mg / mL. The MOF was sonicated at 250 W for 10 min to ensure thorough dispersion. Subsequently, 3-mercaptopropionic acid was added to a concentration of 0.06 mol / L, and the reaction was carried out at 35 °C with magnetic stirring for 2 h.

[0164] B3) Phosphorus site introduction: Phosphoric acid was added dropwise to the above dispersion system to make the concentration of phosphoric acid in the system 0.03 mol / L, and then the reaction was carried out at 35℃ with magnetic stirring for 2 h.

[0165] B4) Post-processing: The solid was collected by centrifugation, washed three times with ethanol and three times with deionized water, with a solid-liquid ratio of 1:10 g / mL each time and a washing time of 10 min each time. It was then vacuum dried at 60℃ for 6 h.

[0166] B5) Quality control: After the obtained solid was vacuum dried to constant weight at 60℃, the mass fraction of sulfur was determined by elemental analysis to be 0.5 wt% and the mass fraction of phosphorus was 0.3 wt%. X-ray photoelectron spectroscopy confirmed the successful introduction of sulfur and phosphate groups, and a double anchoring site imprint MOF was obtained.

[0167] Preparation of S3 Imprinted MOF-Au Complex Intermediate

[0168] C1) Raw material supply: Take 2.0 g of the above-mentioned double anchor positioning point imprint MOF, tetrachloroauric acid, L-ascorbic acid, ethanol and deionized water.

[0169] C2) Adsorption: The dual-anchored MOF was dispersed in a mixed solvent of ethanol and deionized water at a volume ratio of 30:70, with a mass concentration of 35 mg / mL. Tetrachloroauric acid solution was added under light-protected conditions at a dropping rate of 4 mL / min to increase the gold ion concentration by Au. 3+ The concentration was calculated to be 4.0 mmol / L, and then adsorption was carried out at 35℃ with magnetic stirring for 15 min.

[0170] C3) Reduction and nucleation: Add L-ascorbic acid to the above system to a concentration of 16 mmol / L, and react with magnetic stirring at 35°C for 15 min under light-protected conditions. When the system turns wine-red, the reduction reaction is considered complete.

[0171] C4) Quality Control: The solid was collected by centrifugation and washed alternately with ethanol and deionized water until the supernatant was colorless. It was then vacuum dried at 60℃ to constant weight. The mass fraction of gold in the composite intermediate was determined to be 52 wt% by inductively coupled plasma atomic emission spectrometry. More than 200 gold nanoislands were identified by transmission electron microscopy, and the particle size of the gold nanoislands was measured to be 35 nm, with a nanometer gap of 4 nm between adjacent gold nanoislands, thus obtaining the imprinted MOF-Au composite intermediate.

[0172] S4 film formation and substrate construction

[0173] D1) Substrate provision: Take a silicon dioxide substrate and ultrasonically clean it in sequence with acetone, ethanol and deionized water, each time for 15 minutes, and then blow it dry under nitrogen flow.

[0174] D2) Coupling treatment: The cleaned substrate was immersed in an ethanol solution of 3-aminopropyltriethoxysilane with a volume fraction of 1.5 vol% and a volume fraction of 98.5 vol% of ethanol. The reaction was carried out at room temperature for 20 min, followed by rinsing with ethanol and drying at 75 °C for 20 min.

[0175] D3) Film Formation: The imprinted MOF-Au composite intermediate was dispersed in a mixed solvent of ethanol and deionized water at a volume ratio of 30:70, with a dispersion concentration of 7 mg / mL. The ultrasonic power was 250 W, and the ultrasonic time was 10 min. The film was formed by spin coating at a speed of 3000 rpm for 20 s, followed by drying at 50℃ for 45 min.

[0176] D4) Quality Control: The substrate surface was observed using a scanning electron microscope (SEM), and threshold segmentation statistics were performed on the SEM images. The composite film area coverage was 70%. The cross-section of the composite film was observed using an SEM, and the film thickness was measured at at least 5 different locations and the average value was taken, resulting in a film thickness of 350 nm.

[0177] In the Raman substrate composite film obtained in this embodiment, the imprinted MOF has a specific surface area of ​​750 m² / g, a pore size of 0.8 nm, a gold nanoisland size of 35 nm, a nano-gap between adjacent gold nanoislands of 4 nm, a gold mass fraction of 52 wt%, a composite film coverage of 70%, and a film thickness of 350 nm.

[0178] Features of this embodiment: This embodiment employs a high gold content, large gold nanoisland design, a zirconium tetrachloride to terephthalic acid molar ratio of 0.52:1, a template molecule molar ratio of 0.60:1, a glacial acetic acid molar ratio of 16:1, a solvothermal reaction temperature of 95℃, and a reaction time of 9 h, resulting in a preparation with a specific surface area of ​​750 m². 2 A 0.8 nm pore size imprinted MOF was constructed. The MOF with dual anchoring points contained 0.5 wt% sulfur and 0.3 wt% phosphorus. The composite membrane contained 52 wt% gold, with gold nanoislands of 35 nm in size and 4 nm in spacing. The composite membrane had 70% coverage and a thickness of 350 nm. This method significantly enhances Raman signal enhancement through high gold content and large gold nanoisland size. The smaller spacing and higher membrane coverage further strengthen the electromagnetic field enhancement effect. It is suitable for rapid qualitative identification and semi-quantitative analysis of medium-concentration impurity I, and is particularly suitable for online rapid monitoring and rapid screening before batch release in L-carnitine oral solution production lines.

[0179] Example 4

[0180] This embodiment provides a levocarnitine-imprinted MOF-Au Raman substrate, comprising a silicon substrate and a composite film loaded on the substrate surface. The composite film contains an imprinted MOF and gold nanoislands doubly anchored to the imprinted MOF via Au-S and Au-OP bonds.

[0181] Preparation of S1 imprinted MOF

[0182] A1) Raw material preparation: Weigh 5.87 g of zirconium tetrachloride and 8.35 g of terephthalic acid, with a molar ratio of 0.50:1. The template molecule is a mixture of 1.85 g of L-carnitine and 1.91 g of E-configuration 4-trimethylammonium-2-butenoic acid inner salt (mass ratio 1:1), with a molar ratio of the total amount of template molecules to terephthalic acid of 0.49:1. The molar ratio of 85.74 g of glacial acetic acid to zirconium tetrachloride is 57:1. Add N,N-dimethylformamide to a total volume of 180 mL, so that the molar concentration of zirconium tetrachloride in the mixed system is 0.14 mol / L. Stir and mix evenly at room temperature.

[0183] A2) Solvent-thermal reaction: The above mixture was transferred to a stainless steel autoclave lined with polytetrafluoroethylene, with a filling degree of 70%, and then sealed and placed in an oven at 117°C for 7 h.

[0184] A3) Post-treatment and template elution: After the reaction, the product was allowed to cool naturally to room temperature and collected by centrifugation. It was washed twice with N,N-dimethylformamide at a solid-liquid ratio of 1:45 g / mL for 28 min each time, followed by six washes with ethanol at the same solid-liquid ratio and time. Template elution was performed nine times using a mixed solvent of ethanol and deionized water at a volume ratio of 68:32, with a solid-liquid ratio of 1:45 g / mL and a elution time of 55 min each time. The concentration of template molecules in the eluent was determined by high-performance liquid chromatography (HPLC), and elution was considered complete when the concentration was below 0.5 μg / mL. Finally, the solid was vacuum-dried at 110℃ for 4.5 h.

[0185] A4) Quality control: The obtained sample was degassed under vacuum at 120℃ for 12 h. Based on nitrogen adsorption-desorption data, the specific surface area was determined to be 1380 m² / g by BET method, and the pore size was calculated to be 1.85 nm by DFT method, thus obtaining the imprinted MOF.

[0186] Preparation of S2 dual-anchor positioning point imprinted MOF

[0187] B1) Raw material supply: Take 3.5 g of the above-mentioned imprinted MOF, 3-mercaptopropionic acid, phosphoric acid, ethanol and deionized water.

[0188] B2) Introduction of sulfide sites: The imprinted MOF was dispersed in a mixed solvent of ethanol and deionized water at a volume ratio of 85:15, with a MOF mass concentration of 46 mg / mL. The MOF was sonicated at a power of 730 W for 27 min to ensure thorough dispersion. Subsequently, 3-mercaptopropionic acid was added to a concentration of 0.18 mol / L, and the reaction was carried out at 65 °C with magnetic stirring for 7.5 h.

[0189] B3) Phosphorus site introduction: Phosphoric acid was added dropwise to the above dispersion system to make the concentration of phosphoric acid in the system 0.092 mol / L, and then the reaction was carried out at 65℃ with magnetic stirring for 7.5 h.

[0190] B4) Post-processing: The solid was collected by centrifugation, washed 5 times with ethanol and 5 times with deionized water, with a solid-liquid ratio of 1:45 g / mL each time and a washing time of 28 min each time. It was then vacuum dried at 110℃ for 4.5 h.

[0191] B5) Quality control: After the obtained solid was vacuum dried to constant weight at 60℃, the mass fraction of sulfur was determined by elemental analysis to be 1.85 wt% and the mass fraction of phosphorus was 1.38 wt%. X-ray photoelectron spectroscopy confirmed the successful introduction of sulfur and phosphate groups, and a double anchoring site imprinted MOF was obtained.

[0192] Preparation of S3 Imprinted MOF-Au Complex Intermediate

[0193] C1) Raw material supply: Take 3.0 g of the above-mentioned double anchor positioning point imprint MOF, tetrachloroauric acid, L-ascorbic acid, ethanol and deionized water.

[0194] C2) Adsorption: The dual-anchored MOF was dispersed in a mixed solvent of ethanol and deionized water at a volume ratio of 85:15, with a mass concentration of 46 mg / mL. Tetrachloroauric acid solution was added under light-protected conditions at a dropping rate of 4.6 mL / min to increase the gold ion concentration by Au. 3+ The concentration was calculated to be 4.6 mmol / L, and then adsorption was carried out at 37 °C with magnetic stirring for 55 min.

[0195] C3) Reduction and nucleation: L-ascorbic acid was added to the above system to a concentration of 18.5 mmol / L, and the mixture was magnetically stirred at 37°C for 55 min under light-protected conditions. The reduction reaction was considered complete when the system turned wine-red.

[0196] C4) Quality Control: The solid was collected by centrifugation and washed alternately with ethanol and deionized water until the supernatant was colorless. It was then vacuum dried at 60℃ to constant weight. The mass fraction of gold in the composite intermediate was determined to be 55 wt% by inductively coupled plasma atomic emission spectrometry. More than 200 gold nanoislands were identified by transmission electron microscopy, and the particle size of the gold nanoislands was measured to be 37 nm, with a nanometer gap of 8.5 nm between adjacent gold nanoislands, thus obtaining the imprinted MOF-Au composite intermediate.

[0197] S4 film formation and substrate construction

[0198] D1) Substrate provision: Take a silicon substrate and ultrasonically clean it in sequence with acetone, ethanol and deionized water for 15 minutes each time, and then dry it under nitrogen flow.

[0199] D2) Coupling treatment: The cleaned substrate was immersed in an ethanol solution of 3-aminopropyltriethoxysilane with a volume fraction of 4.6 vol% and a volume fraction of 95.4 vol% of ethanol. The reaction was carried out at room temperature for 55 min, followed by rinsing with ethanol and drying at 110 °C for 55 min.

[0200] D3) Film Formation: The imprinted MOF-Au composite intermediate was dispersed in a mixed solvent of ethanol and deionized water at a volume ratio of 85:15, with a dispersion concentration of 9.2 mg / mL. The ultrasonic power was 730 W, and the ultrasonication time was 27 min. The film was formed by spin coating at a speed of 3700 rpm for 55 s, followed by drying at 55℃ for 110 min.

[0201] D4) Quality Control: The substrate surface was observed using a scanning electron microscope (SEM), and threshold segmentation statistics were performed on the SEM images. The composite film area coverage was 74%. The cross-section of the composite film was observed using an SEM, and the film thickness was measured at at least 5 different locations and the average value was taken, resulting in a film thickness of 460 nm.

[0202] In the composite film obtained from the Raman substrate in this embodiment, the imprinted MOF has a specific surface area of ​​1380 m². 2 / g, pore size of 1.85nm, gold nanoisland size of 37nm, nano-gap between adjacent gold nanoislands of 8.5nm, gold mass fraction of 55 wt%, composite film coverage of 74%, film thickness of 460 nm.

[0203] Features of this embodiment: This embodiment employs a multi-parameter design close to the technical solution boundary, with a zirconium tetrachloride to terephthalic acid molar ratio of 0.50:1, a template molecule molar ratio of 0.49:1, a glacial acetic acid molar ratio of 57:1, a zirconium tetrachloride molar concentration of 0.14 mol / L, and a solvothermal reaction temperature of 117℃, resulting in a prepared material with a specific surface area of ​​1380 m². 2 A 1.85 nm pore size imprinted MOF was prepared. The MOF with dual anchoring points had an ethanol-to-water volume ratio of 85:15, a MOF mass concentration of 46 mg / mL, an ultrasonic power of 730 W, a 3-mercaptopropionic acid concentration of 0.18 mol / L, a phosphoric acid concentration of 0.092 mol / L, a sulfur mass fraction of 1.85 wt%, and a phosphorus mass fraction of 1.38 wt%. During the preparation of the composite intermediate, the gold ion concentration was 4.6 mmol / L, the L-ascorbic acid concentration was 18.5 mmol / L, and the reaction temperature was 37℃. The composite membrane contained 55 wt% gold, gold nanoisland size of 37 nm, nano-intervals of 8.5 nm, a composite membrane coverage of 74%, and a membrane thickness of 460 nm. This scheme fully verifies the feasibility of the technical solution within a wide parameter range, while maintaining a reasonable process safety margin. Through the synergistic effect of high specific surface area, large pore size, high gold content and high film coverage, it achieves ultra-high sensitivity detection of impurity I, and is suitable for the limit detection of trace impurities in L-carnitine oral solution, methodological validation and precise control of key quality attributes.

[0204] Comparative Example 1: Basically the same as Example 1, except that the specific surface area of ​​the imprinted MOF is 420 m². 2 / g, achieved by adjusting the solvothermal reaction time to 4 h, while keeping the amounts of other components and preparation conditions unchanged.

[0205] Comparative Example 2: It is basically the same as Example 1, except that the pore size of the imprinted MOF is 2.4 nm, which is achieved by adjusting the molar ratio of zirconium tetrachloride to terephthalic acid to 0.85:1. The other steps and conditions remain unchanged, and except that the amount of zirconium tetrachloride and / or terephthalic acid is adjusted accordingly to meet the above molar ratio, the amount of other raw materials remains unchanged.

[0206] Comparative Example 3: It is basically the same as Example 1, except that the gold nanoisland particle size is 8 nm, which is achieved by reducing the gold ion concentration to 0.15 mmol / L and shortening the reduction time to 10 min. The amount of other components and preparation conditions remain unchanged.

[0207] Comparative Example 4: Basically the same as Example 1, except that the nano-gap between adjacent gold nanoislands is 12 nm, which is achieved by reducing the gold ion concentration to 1.0 mmol / L. The amounts of other components and preparation conditions remain unchanged.

[0208] Comparative Example 5: It is basically the same as Example 1, except that the mass fraction of gold in the composite film is 16 wt%, which is achieved by reducing the gold ion concentration to 0.8 mmol / L and shortening the reduction time to 15 min. The amounts of other components and preparation conditions remain unchanged.

[0209] Comparative Example 6: Basically the same as Example 1, except that the composite membrane area coverage is 35%, which is achieved by reducing the mass concentration of the dispersion to 0.3 mg / mL and adjusting the spin coating speed to 1000 rpm. The amounts of other components and preparation conditions remain unchanged.

[0210] Comparative Example 7: It is basically the same as Example 1, except that the dual anchoring point modification was not performed, that is, step S2 was skipped, and the imprinted MOF was directly reacted with tetrachloroauric acid to prepare a composite intermediate. The amount of other components and the preparation conditions remained unchanged.

[0211] Comparative Example 8: It is basically the same as Example 1, except that only sulfur sites are introduced and phosphorus sites are not introduced. That is, step C1 is carried out directly after step B2, skipping the phosphoric acid treatment in step B3. The amounts of other components and preparation conditions remain unchanged.

[0212] Performance testing:

[0213] Experiment 1: Composite film adhesion and wash resistance stability test

[0214] Test Subject: Raman-modified composite membrane. Test Objective: To evaluate the adhesion strength of the composite membrane to the substrate surface and its structural stability under simulated oral liquid rinsing conditions. Test Principle: The membrane adhesion was quantitatively assessed using the tape peeling method, combined with a cyclic rinsing experiment to examine the membrane mass loss rate and Raman signal retention rate. Experimental Method: 3M 600 tape was applied to the substrate surface at a 45° angle, 2 kg pressure was applied and held for 10 s, followed by rapid peeling. This was repeated 5 times. The peeling rate was observed using scanning electron microscopy, and the adhesion grade was calculated. The rinsing experiment used simulated L-carnitine oral solution (pH 3.5, 25℃) to circulate and rinse the substrate surface at a flow rate of 5 mL / min. Each rinse lasted 5 min, with a 30 min interval, for a total of 10 cycles. Key Parameters: Tape pressure 2 kg, peel angle 45°, rinsing pH 3.5, flow rate 5 mL / min. Data processing: Adhesion grade is rated from 0 to 5. Film mass loss rate = (initial mass - mass after rinsing) / initial mass × 100%. Raman signal retention rate = signal intensity after rinsing / initial signal intensity × 100%. n ≥ 3.

[0215] Experiment 2: Measurement of hotspot density and electromagnetic field enhancement factor in nano-gap

[0216] Test Subject: Raman-based gold nanoisland array. Test Objective: To quantitatively evaluate the distribution density of hotspot regions in the nano-interstic gaps and the electromagnetic field enhancement factor (EF) of surface-enhanced Raman scattering. Test Principle: Based on a combination of finite element simulation and experimental verification, the EF is calculated by measuring the Raman signal enhancement factor of the nitrobenzenethiophenol (4-NTP) probe molecule. Experimental Method: Scanning electron microscopy (SEM) was used to count the number of hotspots <10 nm per unit area (at least 5 fields of view, 20 μm per field of view). 2 The hotspot density was calculated. The substrate was immersed in 4-NTP ethanol solution (1 mmol / L) for 2 h, rinsed with ethanol, and dried. A Raman spectrometer (excitation wavelength 785 nm, power 5 mW, integration time 10 s) was used to collect data at a depth of 1336 cm⁻¹. -1 The enhancement factor of the characteristic peak signal is calculated using EF = (ISERS / NSERS) / (Iref / Nref). Key parameters: nanometer gap threshold 10 nm, statistical area ≥100 μm. 2 4-NTP concentration: 1 mmol / L; Raman excitation wavelength: 785 nm. Data processing: Hotspot density is expressed as hotspots / μm. 2 EF calculation takes the mean ± standard deviation of at least 10 points, n≥3.

[0217] Experiment 3: Test of Selective Adsorption and Enrichment Capacity of Imprinted Pores

[0218] Test Object: Imprinted MOF composite membrane. Test Objective: To evaluate the selective recognition and enrichment ability of the imprinted channels for target impurity I (E / Z configuration 4-trimethylammonium-2-butenoic acid inner salt). Test Principle: The adsorption capacity, adsorption rate, and selectivity coefficient of the imprinted channels were quantitatively analyzed through adsorption kinetics and isothermal adsorption experiments. Experimental Method: A standard solution of impurity I (concentration gradient 0.1-10 μg / mL, deionized water) was prepared. The Raman substrate was immersed in the solution and incubated at 25℃ for different times (5-120 min). The residual concentration of impurity I in the solution was determined by high-performance liquid chromatography (HPLC), and the adsorption capacity was calculated. For the selectivity experiment, a mixed solution of impurity I and the structural analog L-carnitine (molar ratio 1:1) was used to determine the ratio of the adsorption amounts of the two on the substrate surface after adsorption. Key Parameters: Temperature 25℃, initial concentration 0.1-10 μg / mL, adsorption time 5-120 min, pH 3.5. Data processing: Adsorption capacity qe = (C0 - Ce)V / m, selectivity coefficient α = (qe,I / Ce,I) / (qe,L / Ce,L), n≥3.

[0219] Experiment 4: Non-specific adsorption and background interference test in complex aqueous matrix

[0220] Test Subject: Performance of Raman substrate in a simulated L-carnitine oral solution matrix. Test Objective: To evaluate the anti-interference ability and non-specific adsorption inhibition effect of the imprinted channels in a complex matrix. Test Principle: By comparing the detection signal intensity and signal-to-noise ratio of impurity I in a pure water system and a simulated oral solution matrix (containing excipients such as DL-malic acid, sodium saccharin, and methylparaben), the degree of matrix interference is assessed. Experimental Method: A simulated oral solution matrix was prepared (according to the formulation ratio in the example). Impurity I was added to a final concentration of 0.5-5 μg / mL. The Raman substrate was immersed in the solution and incubated for 30 min. After gentle rinsing with deionized water and drying, Raman spectra were acquired (785 nm excitation, 5 mW, 10 s integration time). The characteristic peak intensity and baseline noise of impurity I were recorded. A parallel control experiment was conducted using a pure water system. Key Parameters: The matrix composition was consistent with the oral solution; impurity I concentration was 0.5-5 μg / mL; incubation time was 30 min; and rinsing conditions were gentle to avoid membrane damage. Data processing: Signal-to-noise ratio S / N = signal strength / baseline noise standard deviation, matrix effect = (matrix signal - pure water signal) / pure water signal × 100%, n≥3.

[0221] Experiment 5: Testing the anchoring strength of Au-S and Au-OP bonds and the stability of gold nanoislands

[0222] Test Subject: MOF-Au composite film with dual anchoring points. Test Objective: To quantitatively evaluate the anchoring strength of Au-S and Au-OP bonds and their contribution to the long-term retention of gold nanoislands. Test Principle: X-ray photoelectron spectroscopy (XPS) was used to characterize the binding energies and chemical states of Au 4f, S 2p, and P 2p, combined with a cyclic rinsing experiment to determine the gold loss rate and assess the stability of the anchoring bonds. Experimental Method: XPS was used to analyze the peak positions and peak areas of Au 4f7 / 2 (binding energy approximately 84 eV), S 2p3 / 2 (approximately 162 eV corresponding to Au-S bonds), and P 2p3 / 2 (approximately 133 eV corresponding to Au-OP bonds) on the surface of the composite film, and the relative contents of Au-S and Au-OP bonds were calculated. Stability tests were performed using a pH 3.5 simulated oral solution for 20 cyclic rinsing cycles (5 min each time, flow rate 5 mL / min, 30 min intervals). The gold ion concentration in the rinsing solution was determined by inductively coupled plasma optical emission spectrometry (ICP-OES), and the cumulative gold loss rate was calculated. Key parameter: XPS vacuum degree ≤10. -7 Pa, rinsing pH 3.5, flow rate 5 mL / min, 20 cycles. Data processing: Gold loss rate = (gold loss mass / initial gold mass) × 100%, n≥3.

[0223] Experiment 6: Long-term repeatability and batch-to-batch consistency test of Raman signals

[0224] Test Subject: Performance stability of Raman substrates after long-term storage and multiple uses. Test Objective: To evaluate the signal repeatability, batch-to-batch consistency, and long-term accessibility of the plasmon-active surface of the substrate. Test Principle: Repeatability and consistency are assessed by calculating the relative standard deviation (RSD) through multiple tests on the same substrate and parallel tests on substrates from different batches. Experimental Method: Three substrates from the same batch were selected, and Raman detection (785 nm, 5 mW, 10 s) was performed on days 1, 7, 14, 30, and 60 using a standard impurity I solution (2 μg / mL). The intensity of the characteristic peak was recorded, and the time stability RSD was calculated. Five substrates from each of three different batches were selected, and parallel tests were performed using the same concentration of impurity I solution. The batch-to-batch RSD was calculated. After each test, the substrate was regenerated by rinsing with ethanol, and the number of reusable substrates was evaluated (ending when the signal intensity decayed to 80% of the initial value). Key Parameters: Storage conditions: 4℃, protected from light and sealed; impurity I concentration: 2 μg / mL; regeneration conditions: ethanol sonication for 5 min. Data processing: RSD = (standard deviation / mean) × 100%, with requirements of intra-batch RSD ≤ 10%, inter-batch RSD ≤ 15%, and n ≥ 3.

[0225] Figure 1XPS full-spectrum superposition images were obtained to deeply analyze the Au-S and Au-OP bonding states of gold nanoislands, aiming to enhance the film stability and gold nanoisland retention capacity through dual anchoring synergistic enhancement. Fixed parameters included a zirconium-based imprinted MOF-supported gold nanoisland composite film as the substrate material, XPS testing energy range of 0 to 1200 eV, and consistent acquisition conditions and data processing procedures for all samples. Variations included sample type, from Example 1 to Comparative Example 7 (no anchoring modification) to Comparative Example 8 (Au-S single anchoring). Example 1 showed both S and P related characteristic signals on top of the Au signal, while Comparative Example 7 lacked both S and P signals, and Comparative Example 8 lacked a P signal. This indicates that the introduction of dual anchoring sites provides a chemical environment for sulfur and phosphorus sites associated with gold, providing a prerequisite for subsequent high-resolution spectroscopy confirmation of the Au-S and Au-OP bonding states, thus supporting the establishment of a chemical retention mechanism for gold nanoislands.

[0226] Figure 2 To illustrate the synergistic enhancement of film stability and gold nanoisland retention through dual-anchoring, X-ray photoelectron spectroscopy was used to deeply analyze the Au 4f high-resolution peak fitting of Au-S and Au-OP bonding states. The parameters were fixed at the Au 4f spectral range of 78 to 92 eV, with consistent background subtraction and peak fitting models, and consistent spin-orbit splitting and area ratio constraints. The sample type varied from Example 1 (no anchoring modification) to Comparative Example 7 (Au-S single anchoring). The presence of the Au 4f main peak in all three examples indicates the widespread presence of gold nanostructures. However, Example 1 and Comparative Example 8 show peak shapes closer to the distribution of gold species in a stable chemical environment. Comparative Example 7 exhibits more unstable peak shapes and a trend of increasing fitting residuals. Combined with subsequent S 2p and P 2p results, this can be attributed to the interfacial chemical stabilization effect of the anchoring points, supporting the belief that dual anchoring helps improve the consistency and reproducibility of the gold-support interface.

[0227] Figure 3 To illustrate the synergistic enhancement of film stability and gold nanoisland retention through dual-anchoring, X-ray photoelectron spectroscopy (XPS) analysis of the S 2p high-resolution peak fitting of Au-S and Au-OP bonding states, a high-resolution peak fitting comparison was obtained. The parameters were fixed at the S 2p spectral range of 158 to 170 eV, employing consistent bimodal spin-orbit splitting constraints and peak shape parameters while maintaining consistent background subtraction. The parameters varied from sample type (Example 1 to Comparative Example 7, unanchored, to Comparative Example 8, Au-S single-anchored). Example 1 and Comparative Example 8 exhibited an S 2p 3 / 2 chemical state component consistent with the Au-S bond around approximately 162 eV, while Comparative Example 7 did not show a corresponding peak component. This indicates that the sulfide sites can form stable interfacial bonds with gold, significantly different from the unanchored system, providing direct chemical evidence for anchoring enhancement.

[0228] Figure 4X-ray photoelectron spectroscopy (XPS) analysis of the P 2p high-resolution spectra of Au-S and Au-OP bonding states to enhance film stability and gold nanoisland retention through dual-anchoring synergistic enhancement is presented as a comparison of peak fitting. The parameters were fixed at the P 2p spectral range of 128 to 138 eV, with consistent double-peak splitting constraints and peak shape parameters, and consistent background subtraction. The parameters varied from sample type (Example 1 to Comparative Example 7, no anchoring modification) to Comparative Example 8, which only had Au-S single anchoring. Only Example 1 showed a P 2p 3 / 2 chemical state component consistent with the Au-OP bond near approximately 133 eV, while Comparative Examples 7 and 8 lacked this peak component. This indicates that the introduction of phosphorus sites creates an additional interfacial bonding channel between gold and phosphate groups, forming the chemical basis for dual-anchoring synergistic retention with Au-S, thereby improving the resistance of gold nanoislands to detachment in the liquid phase.

[0229] Figure 5 The graph shows the relationship between the morphology and distribution stability of gold nanoislands before and after cyclic rinsing, and the number of rinsing cycles, to illustrate the synergistic enhancement of film stability and gold nanoisland retention capacity through dual anchoring. Fixed parameters were: rinsing medium pH 3.5 (simulating oral solution) and flow rate 5 mL / min. -1 Each rinse lasted 5 minutes, and the number of islands per unit area was obtained and the retention rate was calculated under the same microscopic statistical area and threshold criteria. The parameters varied as follows: sample type: Example 1 and Comparative Example 7 without anchoring modification, and Comparative Example 8 with only Au-S single anchoring; and the number of rinses increased from 0 to 10 to 20. The retention rate of Example 1 approached a stable plateau with increasing number of rinses, while Comparative Examples 7 and 8 showed a significant decrease. This indicates that the dual-anchoring system can better maintain the fixation of gold nanoislands and the continuity of hot spot structure under cyclic shearing and solution scouring, thus providing a structural basis for long-term signal consistency.

[0230] Figure 6 This image shows the particle size difference distribution curves of gold nanoislands before and after cyclic rinsing to demonstrate the synergistic enhancement of film stability and gold nanoisland retention capacity through dual anchoring. Fixed parameters included a minimum of 200 gold nanoislands per group, consistent particle size definition, statistical caliber, and distribution estimation method. Variations included sample types (Example 1 and Comparative Example 7 without anchoring modification and Comparative Example 8 with only Au-S anchoring) and rinsing cycles ranging from 0 to 20. Example 1 showed minimal changes in peak position and broadening before and after rinsing, while Comparative Examples 7 and 8 exhibited overall distribution drift and increased broadening. This indicates that unanchored or single-anchored systems are more prone to aggregation or selective loss of effective particles under rinsing stress, leading to changes in apparent particle size statistics. This supports the view that dual anchoring can better stabilize the particle size and distribution consistency of gold nanoislands.

[0231] Figure 7The image shows the nano-gap difference distribution curves of the gold nano-island morphology and distribution stability before and after cyclic rinsing to illustrate the synergistic enhancement of film stability and gold nano-island retention capacity through dual anchoring. Fixed parameters included gap defined as the minimum surface distance between adjacent gold nano-islands, with at least 200 statistical points per group and a consistent distribution estimation method. Variation parameters included sample type (Example 1 and Comparative Example 7 without anchoring modification) and Comparative Example 8 with only Au-S single anchoring, and rinsing cycles ranging from 0 to 20. In Example 1, the gap distribution peak remained in the small gap region with minimal change in distribution width before and after rinsing. In contrast, the gap distribution in Comparative Examples 7 and 8 shifted towards larger gaps and broadened significantly. This indicates that dual anchoring is more beneficial for maintaining a high-density nano-gap hotspot structure under liquid-phase rinsing conditions, thereby supporting more stable electromagnetic field coupling enhancement.

[0232] Figure 8 The cumulative particle size distribution of gold nanoislands in Example 1, Comparative Examples 8 and 7 is shown. The cumulative distribution Q was calculated based on the same particle size statistics and compared between 0 and 20 washes. The basic parameters are particle size in nanometers on the horizontal axis and cumulative distribution Q from 0 to 1 on the vertical axis, with solid lines representing 0 washes and dashed lines representing 20 washes. The samples are stacked separately. The variable parameters are sample type and the degree of rightward shift of the overall distribution after washing. The results show that the two cumulative curves of Example 1 are close, while the cumulative curves of Comparative Examples 8 and 7 shift significantly to the right after washing. This proves that Example 1 can keep most particles within the target particle size range, thus explaining the correctness and consistency of its performance retention.

[0233] Figure 9 The cumulative distribution curves of the gold nanoisland morphology and distribution stability before and after cyclic rinsing are used to illustrate the synergistic enhancement of film stability and gold nanoisland retention capacity through dual anchoring. The fixed parameter is that the cumulative distribution is calculated from the original gap statistics according to a consistent rule and remains monotonically increasing. The varying parameters are: sample type (Example 1 and Comparative Example 7 without anchoring modification) and Comparative Example 8 with only Au-S single anchoring, and rinsing cycles from 0 to 20. The cumulative distribution curve of Example 1 still maintains a high proportion falling within the less than 10 nm range after 20 rinsing cycles, while the cumulative proportion in the same range for Comparative Examples 7 and 8 decreases significantly. This indicates that dual anchoring can achieve higher structural fidelity across the overall hotspot-related gap population, thus providing statistical support for signal retention and reusability.

[0234] Figure 10 To demonstrate the selective identification and high enrichment capacity of nitrogen adsorption-desorption isotherms in complex matrices with robustness against interference, a superimposed plot of nitrogen adsorption-desorption isotherms with pore size distribution was created. Fixed parameters included consistent nitrogen adsorption-desorption test temperature and degassing conditions, and the use of consistent isotherm acquisition pressure points and adsorption unit conversion methods. Variations included sample type (Example 1 and Comparative Example 1) and specific surface area (420 m²). 2The adsorption capacity of Example 1 was below the lower limit, while that of Comparative Example 2 (pore size 2.4 nm) was above the upper limit. Example 1 exhibited a higher adsorption capacity and a more typical micropore-dominated isothermal characteristic, while the overall adsorption capacity of Comparative Example 1 was limited, and Comparative Example 2 showed a more significant tail contribution in the high relative pressure range. This indicates that Example 1 can better meet the requirements of sufficient adsorption sites and reasonable pore accessibility in terms of specific surface area and pore structure, providing a structural basis for selective enrichment.

[0235] Figure 11 This is a comparison of the nitrogen adsorption-desorption isotherm and the DFT pore size distribution curves, which are superimposed to demonstrate the selective identification and high enrichment capacity of imprinted pores in complex matrices, exhibiting strong resistance to interference. The pore size distribution is calculated using the same type of DFT model and a consistent data processing workflow, with the following parameters varying: sample type (Example 1 and Comparative Example 1), specific surface area (420 m²). 2 The pore size distribution of Example 1 is below the lower limit, while that of Comparative Example 2 (pore size 2.4 nm) is above the upper limit. The main peak of the pore size distribution in Example 1 is located at approximately 1.2 nm and is concentrated within the 0.5 to 2.0 nm window, while the main peak of the distribution in Comparative Example 2 shifts to a larger pore size region and exceeds the upper limit of the window. The overall distribution intensity of Comparative Example 1 is lower, indicating that the pore size of Example 1 is closer to the target molecule size matching condition and also has sufficient pore volume distribution contribution, thus supporting its selective recognition and enrichment ability to be superior to samples outside the boundary.

[0236] Figure 12 Raman spectral overlays were used to verify the selective identification and high enrichment capacity of imprinted channels in complex matrices, demonstrating their anti-interference properties. The parameters were fixed at an excitation wavelength of 785 nm, a laser power of 5 mW, an integration time of 10 s, and the same incubation and gentle rinsing procedures. The variable parameters were: sample type (Example 1 and Comparative Example 2), pore size of 2.4 nm (higher than the upper limit), and testing in a mixed solution of equimolar impurity I and L-carnitine. Example 1 was tested at approximately 1580 cm⁻¹. -1 The characteristic peak of impurity I in the vicinity is relatively more prominent at approximately 1420 cm⁻¹. -1 The contribution of the L-carnitine peak is relatively lower in Comparative Example 2, while the contrast between impurity I and the background peak decreases. This indicates that the imprinted channels within the aperture window can preferentially enrich impurity I and suppress non-specific adsorption background, thereby improving the readability of fingerprint recognition in the mixed system.

[0237] As can be seen from the performance of the examples and comparative examples in Tables 1-2, Examples 1-4 are significantly superior to the comparative examples in multiple dimensions, including composite film adhesion, wash-resistant stability, nano-gap hotspot density, electromagnetic field enhancement capability, imprinted pore adsorption and enrichment performance, resistance to matrix interference, gold nano-island anchoring stability, and long-term signal repeatability. Comparative Example 1 suffers from insufficient adsorption capacity and decreased hotspot density due to its low specific surface area; Comparative Example 2 suffers from reduced selectivity and increased non-specific adsorption due to its excessively large pore size; Comparative Examples 3 and 4 suffer from weakened electromagnetic field enhancement due to deviations in the particle size or gaps of the gold nano-islands from the optimal range; Comparative Example 5 suffers from insufficient gold content, which directly limits the Raman signal intensity; Comparative Example 6 suffers from reduced effective detection area and decreased adhesion due to insufficient coverage; and Comparative Examples 7 and 8 suffer from significant gold nano-island loss during cyclic washing, drastic deterioration of film structure stability, and a sharp decrease in the number of reusable applications due to the lack of dual-anchoring point modification. Example 4 showed the best performance in several key indicators, verifying that the parameter combination close to the technical solution boundary can still maintain excellent performance, and proving the wide applicability and process robustness of the technical solution of the present invention.

[0238] Table 1 Performance comparison data between the examples and comparative examples (Part 1)

[0239]

[0240] Table 2 Performance comparison data of the examples and comparative examples (Part 2)

[0241]

[0242] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that any equivalent structural transformations made under the concept of the present invention and using the contents of the specification and drawings of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A levocarnitine-imprinted MOF-Au Raman substrate, characterized in that, Includes a substrate and a composite film loaded on the surface of the substrate; The substrate is selected from either a silicon dioxide substrate or a silicon substrate; The composite film comprises an imprinted MOF and gold nano islands anchored to the imprinted MOF via both Au-S and Au-OP bonds. The imprinted MOF contains zirconium and terephthalic acid ligands, and has imprinted channels formed using template molecules as templates and obtained by elution to remove the template molecules; The imprinted MOF has a specific surface area of ​​500-1500 m² / g and a pore size of 0.5-2.0 nm. The gold nano islands have a particle size of 10-40 nm and the nano gaps between adjacent gold nano islands are less than 10 nm. The mass fraction of gold in the composite film is 20-60 wt% based on the dry basis of the composite film, and the area coverage of the composite film on the substrate surface is 40-80%.

2. The Raman substrate according to claim 1, characterized in that, The imprinted MOF was prepared under the following conditions: A1) Raw material preparation: Zirconium tetrachloride, terephthalic acid, template molecule, glacial acetic acid and N,N-dimethylformamide are provided, wherein the molar ratio of zirconium tetrachloride to terephthalic acid is 0.40–0.60:1, the molar ratio of template molecule to terephthalic acid is 0.05–0.60:1, the molar ratio of glacial acetic acid to zirconium tetrachloride is 10–60:1, and N,N-dimethylformamide is added to make the molar concentration of zirconium tetrachloride in the mixed system 0.02–0.20 mol / L; A2) Solvent-thermal reaction: The mixture obtained in A1) is placed in a closed reaction vessel and reacted at 90-120 ℃ for 6-18 h; A3) Post-treatment and template elution: The solid obtained in A2) was washed sequentially with N,N-dimethylformamide and ethanol, 2-6 times with N,N-dimethylformamide and 2-6 times with ethanol. The solid-liquid ratio for each wash was 1:5-1:50 g / mL and the washing time was 5-30 min. Then, the template molecules were removed by elution with a mixed solvent of ethanol and deionized water, with a volume ratio of ethanol to deionized water of 30:70-70:

30. The elution was repeated 3-10 times, with a solid-liquid ratio of 1:5-1:50 g / mL and the elution time was 10-60 min. Finally, the solid was dried at 40-120 °C for 4-12 h. A4) Endpoint Criteria and Quality Control: When the specific surface area of ​​the obtained MOF is 500-1500 m² 2 When the pore size is 0.5-2.0 nm, the pore size is calculated using the DFT method based on nitrogen adsorption-desorption data to obtain the imprinted MOF.

3. The Raman substrate according to claim 1, characterized in that, The imprinted MOF in the composite film is a dual-anchor positioning point imprinted MOF, which is prepared under the following conditions: B1) Raw material supply: Providing imprinted MOF, 3-mercaptopropionic acid, phosphoric acid, ethanol, and deionized water; B2) Introduction of sulfide sites: The imprinted MOF is dispersed in a mixed solvent of ethanol and deionized water, wherein the volume ratio of ethanol to deionized water is 10:90-90:10, and the mass concentration of the imprinted MOF in the mixed solvent is 1-50 mg / mL. The dispersion is achieved by ultrasonic treatment, wherein the ultrasonic power is 100-800 W and the ultrasonic time is 1-30 min. Then, 3-mercaptopropionic acid is added to a concentration of 0.01-0.20 mol / L, and the reaction is carried out at 25-70 ℃ for 1-8 h. B3) Phosphorus site introduction: Phosphoric acid is added to the dispersion system obtained in B2) to make the concentration of phosphoric acid in the system 0.005-0.10 mol / L, and then the reaction is carried out at 25-70 °C for 1-8 h; B4) Post-treatment: The obtained solid was washed with ethanol and deionized water 2-6 times and 2-6 times with deionized water. The solid-liquid ratio of each wash was 1:5-1:50 g / mL and the washing time was 5-30 min. Then it was dried at 40-120℃ for 4-12 h. B5) Quality control: When the mass fraction of sulfur in the obtained solid is 0.10-2.00 wt% and the mass fraction of phosphorus is 0.05-1.50 wt%, the double anchor positioning point imprint MOF is obtained.

4. The Raman substrate according to claim 1, characterized in that, The imprinted MOF in the composite film is formed from an imprinted MOF-Au composite intermediate, which is prepared under the following conditions: C1) Raw material supply: Provide dual-anchor positioning point imprint MOF, tetrachloroauric acid, L-ascorbic acid, ethanol and deionized water; C2) Adsorption: The dual-anchor positioning point imprinted MOF is dispersed in a mixed solvent of ethanol and deionized water, wherein the volume ratio of ethanol to deionized water is 10:90-90:10, and the mass concentration of the dual-anchor positioning point imprinted MOF in the mixed solvent is 1-50 mg / mL. Tetrachloroauric acid is then added to adjust the gold ion concentration to Au. 3+ The concentration was calculated to be 0.2–5.0 mmol / L, and the addition method was dropwise addition at a rate of 0.05–5 mL / min, followed by adsorption at 5–40 °C for 5–60 min; C3) Reduction and nucleation: L-ascorbic acid is added to the system obtained in C2) to make the concentration of L-ascorbic acid 0.5-20 mmol / L, and then the reaction is carried out at 5-40 ℃ for 5-60 min to obtain the imprinted MOF-Au complex intermediate; C4) Endpoint Criteria and Quality Control: When the mass fraction of gold in the obtained composite intermediate is 20-60 wt%, and the particle size of the gold nano islands is 10-40 nm, and the nano-gap between adjacent gold nano islands is less than 10 nm, it is judged to be qualified.

5. The Raman substrate according to claim 1, characterized in that, The composite film is constructed on the surface of the substrate through the following steps: D1) Substrate provision: providing the silicon dioxide substrate or the silicon substrate; D2) Coupling treatment: The substrate is immersed in an ethanol solution of 3-aminopropyltriethoxysilane, wherein the volume fraction of 3-aminopropyltriethoxysilane is 0.5-5.0 vol%, and the solvent of the ethanol solution is ethanol with a volume fraction of 95.0-99.5 vol%. The reaction is then carried out for 5-60 min and rinsed with ethanol. Finally, the substrate is dried at 60-120 °C for 10-60 min. D3) Film Formation: The imprinted MOF-Au composite intermediate is dispersed in a mixed solvent of ethanol and deionized water to form a dispersion, wherein the volume ratio of ethanol to deionized water is 10:90-90:10, and the mass concentration of the dispersion is 0.5-10 mg / mL. The dispersion is achieved by ultrasonic treatment with an ultrasonic power of 100-800 W and an ultrasonic time of 1-30 min. Subsequently, a composite film is formed on the substrate surface by dip coating or spin coating. The dip coating speed is 0.5-10 mm / s and then dried at 25-60℃ for 10-120 min, or the spin coating speed is 500-4000 rpm and the spin coating time is 10-60 s and then dried at 25-60℃ for 10-120 min. D4) Endpoint criterion: When the area coverage of the composite film is 40-80%, the Raman substrate is obtained.

6. The Raman substrate according to claim 1, characterized in that, The thickness of the composite membrane is 50-500 nm, and the thickness is obtained by measuring the cross-sectional scanning electron microscope image of the composite membrane and taking the average value of at least 5 different positions.

7. A method for preparing a levocarnitine-imprinted MOF-Au Raman substrate as described in any one of claims 1-6, characterized in that, Includes the following steps: S1) Preparation of imprinted MOF: Using zirconium tetrachloride and terephthalic acid as raw materials, in the presence of template molecules, a solvothermal reaction is carried out in a mixed solvent system of N,N-dimethylformamide and glacial acetic acid to obtain imprinted MOF; S2) Preparation of MOF with dual anchor points: The surface of the imprinted MOF was treated with 3-mercaptopropionic acid and phosphoric acid to obtain MOF with dual anchor points. S3) Preparation of composite intermediate: Tetrachloroauric acid is adsorbed onto the surface of the double anchoring site imprinted MOF and reduced with L-ascorbic acid to form gold nano islands on the surface of the double anchoring site imprinted MOF, thus obtaining the imprinted MOF–Au composite intermediate. S4) Film formation and substrate construction: The imprinted MOF-Au composite intermediate is coated on the surface of a silicon dioxide substrate or a silicon substrate, and then dried to obtain a Raman substrate.

8. The preparation method according to claim 7, characterized in that, In step S1), the molar ratio of zirconium tetrachloride to terephthalic acid is 0.40–0.60:1, the molar ratio of template molecule to terephthalic acid is 0.05–0.60:1, the molar ratio of glacial acetic acid to zirconium tetrachloride is 10–60:1, and N,N-dimethylformamide is added to make the molar concentration of zirconium tetrachloride in the mixed system 0.02–0.20 mol / L.

9. The preparation method according to claim 7, characterized in that, In step S2), the concentration of 3-mercaptopropionic acid is 0.01-0.20 mol / L, the concentration of phosphoric acid is 0.005-0.10 mol / L, and the mass concentration of the imprinted MOF in the mixed solvent of ethanol and deionized water is 1-50 mg / mL, and the volume ratio of ethanol to deionized water is 10:90-90:

10. In step S3), the gold ion concentration is expressed as Au. 3+ The concentration of L-ascorbic acid is 0.5-20 mmol / L, and the mass concentration of the dual-anchored positioning point imprinted MOF in the mixed solvent of ethanol and deionized water is 1-50 mg / mL. The volume ratio of ethanol to deionized water is 10:90–90:

10. Tetrachloroauric acid is added dropwise at a rate of 0.05-5 mL / min.

10. The application of the L-carnitine imprinted MOF-Au Raman substrate as described in any one of claims 1-6 in the rapid detection of impurity I in L-carnitine oral solution, characterized in that, Impurity I is (E)-4-(trimethylammonium)-2-butenoic acid inner salt and / or its (Z)-isomer; by contacting the L-carnitine oral solution sample to be tested with the imprinted MOF-Au Raman substrate and acquiring the surface-enhanced Raman spectral signal, the qualitative identification or quantitative detection of impurity I can be achieved for product pre-inspection in the production process, thereby reducing or replacing the complex sample pretreatment required by high-performance liquid chromatography and shortening the detection time.

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