A novel MOF material-based SERS substrate for quantitative detection of fentanyl

Abstract

The application discloses a novel SERS substrate based on MOF material NH2-MIL-101(Fe) with high sensitivity and good stability and a preparation and application thereof. The NH2-MIL-101 has high adsorption and can be used for adsorbing fentanyl, and the amino group on the structure guides the in-situ growth of gold nanoparticles to form hot spots, thereby significantly enhancing the Raman signal of fentanyl. The novel Raman substrate constructed can be successfully used for detecting trace fentanyl in water samples, blood plasma and urine. Meanwhile, with deuterated fentanyl as an internal standard, quantitative detection of fentanyl can be realized.

Description

Technical Field

[0001] This invention belongs to the field of nanodetection and designs a novel Raman substrate for the detection of fentanyl in complex biological environments. Background Technology

[0002] Fentanyl, chemically known as N-[l-(2-phenylethyl)-4-piperidinyl]-N-phenylpropionamide, has the molecular formula C1. 22 H 28 N2O, with a molecular weight of 336, is an opioid receptor agonist and a potent narcotic analgesic, with pharmacological effects similar to morphine. Animal studies have shown that its analgesic potency is approximately 80 times that of morphine. In 2017, fentanyl surpassed heroin as the leading cause of drug-related deaths in the United States. Subsequently, the problem of fentanyl-related drug abuse has attracted attention in many countries.

[0003] Surface-enhanced Raman scattering (SERS) is an ultrasensitive spectroscopic analysis technique that provides analyte fingerprints. The enhancement effects of SERS are mainly categorized into electromagnetic enhancement (EM) and chemical enhancement (CM). Traditional SERS substrates, such as AuNPs and AgNPs, suffer from unstable Raman signals due to the random distribution of gaps caused by the uncontrolled aggregation of nanoparticles. Therefore, how to construct controllable aggregation of the enhancement substrate to obtain stable hotspots and accurate Raman enhanced signals has always been a research hotspot in the field of SERS.

[0004] Metal-organic frameworks (MOFs) are highly ordered porous materials with advantages such as tunable pore structure, large porosity, high surface area, and ease of functionalization. They have been widely used in gas storage and separation, sensing, enzyme immobilization, and removal of organic pollutants. The porous structure formed by MOFs through van der Waals forces, electrostatics, and π-π interactions can protect nanoparticles from aggregation while simultaneously adsorbing and enriching analytes, providing an effective means for the separation, analysis, and detection of target substances.

[0005] Currently, the main methods for detecting fentanyl include ion mobility chromatography, GC / MS, and LC / MS. However, these instruments are cumbersome to operate and therefore difficult to apply in complex field environments. Portable Raman spectrometers, due to their portability, ease of operation, and ability to perform rapid and non-destructive analysis, are used for rapid on-site analysis. Summary of the Invention

[0006] Based on this, the present invention aims to synthesize a novel SERS substrate based on MOF material, and characterize the substrate through a series of experiments including SEM, TEM, FT-IR, XPS, and XRD. A highly sensitive and stable method for the quantitative detection of fentanyl is established, exhibiting a low detection limit and strong anti-interference ability. The method's ability to detect trace amounts of fentanyl in water samples, plasma, and urine is verified. Simultaneously, the detection performance of this method in the main compounds of heroin and morphine is investigated.

[0007] The SERS substrate involves co-incubating NH2-MIL-101 with chloroauric acid and growing AuNPs in situ on the material under the action of a reducing agent to synthesize NH2-MIL-101 / AuNPs.

[0008] Furthermore, the effects of different chloroauric acid concentrations on the growth of gold nanoparticles were screened.

[0009] Furthermore, the effects of different reaction times on the growth of gold nanoparticles were screened.

[0010] This invention also discloses a method for preparing NH2-MIL-101 / AuNPs, comprising the following steps:

[0011] First, NH2-MIL-101 was synthesized using a solvothermal method. Then, an aqueous solution of chloroauric acid was added, and the reaction was carried out in an ice bath. AuNPs were then grown in situ under the action of a reducing agent.

[0012] The microstructure, lattice, and physical and chemical properties of the material were characterized by UV-Vis, infrared spectroscopy, scanning electron microscopy, transmission electron microscopy, XRD, XPS, and nitrogen adsorption analysis.

[0013] The present invention also discloses the above-mentioned process for quantitative detection of fentanyl content using a Raman substrate:

[0014] (1) Centrifuge NH2-MIL-101 / AuNPs particles at room temperature, discard the supernatant, and obtain concentrated NH2-MIL-101 / AuNPs particles.

[0015] (2) Add fentanyl to the particle solution obtained in step (1) and perform Raman detection.

[0016] (3) Investigate the effect of the type and concentration of agglomerating agent on the fentanyl signal.

[0017] (4) Investigate the stability of the substrate under different salt solutions and pH values.

[0018] (5) Measurement of fentanyl in water, plasma and urine samples using deuterated fentanyl as an internal standard.

[0019] (6) Detection of fentanyl in heroin and morphine main compounds.

[0020] (7) The detection parameters of the SERS instrument in the above steps are: excitation wavelength 785nm, laser intensity 90mW, integration time 10s, microscope objective magnification 20x. When the field of view spot is concentrated to the brightest point and is circular, Raman spectroscopy scanning is performed using BWSpec3.27 software.

[0021] The advantages and beneficial effects of this invention are as follows:

[0022] The NH2-MIL-101 used in this invention possesses porosity and high porosity, which can protect AuNPs and generate controllable hot spots. Simultaneously, its large specific surface area and the presence of organic ligands allow analytes to be adsorbed near the SERS active sites, thereby significantly enhancing the Raman intensity of the analytes. The excellent SERS activity of this SERS substrate was tested by detecting the signal of the Raman reporter molecule R6G. Furthermore, it enables the quantitative detection of fentanyl, demonstrating promising application prospects in practical systems. Attached Figure Description

[0023] Figure 1 The image shows the SEM characterization of NH2-MIL-101 (left) and the TEM characterization of NH2-MIL-101 (right).

[0024] Figure 2 TEM characterization of NH2-MIL-101 / AuNPs with different amounts of chloroauric acid. Figure 2 A (0.04%) Figure 2 B (0.06%) Figure 2 C (0.08%) Figure 2 D (0.10%) and Figure 2 E(0.12%) (w / v)

[0025] Figure 3 The UV-Vis absorption spectra of the NH2-MIL-101 / AuNPs composite material were characterized.

[0026] Figure 4 X-ray diffraction (XRD) characterization of NH2-MIL-101 / AuNPs composite material.

[0027] Figure 5 Fourier transform infrared (FT-IR) characterization of NH2-MIL-101 / AuNPs composite material.

[0028] Figure 6 X-ray photoelectron spectroscopy (XPS) characterization of NH2-MIL-101 / AuNPs composite material.

[0029] Figure 7 Characterized as 60nm AuNPs, Figure 7 A stands for UV-vis. Figure 7 B stands for TEM. Figure 7 C represents the particle size distribution.

[0030] Figure 8 The effect of chloroauric acid concentration on the SERS enhancement effect of the substrate.

[0031] Figure 9 The effect of incubation time on the enhancement of substrate SERS.

[0032] Figure 10 To investigate the agglomerating agent and its concentration. Figure 10 A represents the effect of different agglomerating agents on the fentanyl signal. Figure 10 B is an investigation into the optimal concentration of NaI as an agglomerating agent.

[0033] Figure 11 A comparison of the SERS enhancement effects of AuNPs and NH2-MIL-101 / AuNPs on R6G.

[0034] Figure 12 A comparison of the SERS enhancement effects of AuNPs and NH2-MIL-101 / AuNPs on fentanyl.

[0035] Figure 13 The SERS spectrum of fentanyl homologues.

[0036] Figure 14 To investigate the stability of SERS substrates, the salt resistance of the substrates was examined. Figure 14 A) Resistant to acids and alkalis ( Figure 14 B) In-batch stability Figure 14 C) and anti-aggregation effect ( Figure 14 D).

[0037] Figure 15 For trace detection of fentanyl in heroin and morphine main compounds.

[0038] Figure 16 Deuterated fentanyl was used as an internal standard for quantitative determination of fentanyl content in water samples.

[0039] Figure 17 Linear standard curve of fentanyl in water sample when deuterated fentanyl is used as internal standard.

[0040] Figure 18 Linear standard curve of fentanyl in plasma when deuterated fentanyl is used as an internal standard.

[0041] Figure 19 Linear standard curve of fentanyl in urine when deuterated fentanyl is used as an internal standard.

[0042] Figure 20 This is for the verification of batch-to-batch stability of the substrate. Specific implementation methods:

[0043] The preparation method and application of the substrate of the present invention will be further described below with reference to the accompanying drawings and examples.

[0044] Example 1:

[0045] The SERS substrate was named NH2-MIL-101 / AuNPs, and the preparation method is as follows:

[0046] (1) Synthesis of NH2-MIL-101: Ferric chloride hexahydrate (0.845 g), 2-aminoterephthalic acid (0.54 g, 3 mmol), and N,N-dimethylformamide (15 mL) were added to a 50 mL Erlenmeyer flask and sonicated for 15 min to completely dissolve the ferric chloride hexahydrate. The solution was then transferred to a 50 mL high-pressure reactor with a polytetrafluoroethylene substrate and heated at 110 °C for 20 h. After cooling to room temperature, the crude product was washed with DMF and hot ethanol (60 °C) to remove unreacted 2-aminoterephthalic acid from the pores of NH2-MIL-101. The product was collected after centrifugation at 11000 rpm for 10 min. The synthesized nanoparticles were dried in an oven at 70 °C for 12 h to obtain aminated NH2-MIL-101(Fe).

[0047] (2) Synthesis of NH2-MIL-101 / AuNPs: 30 mg of NH2-MIL-101 was suspended in 30 mL of chloroauric acid solutions of different concentrations (0.04%, 0.06%, 0.08%, 0.10%, and 0.12% (w / v)), sonicated for 15 min, and stirred at 0 °C for 1 h. 300 μL (10, 15, 20, 25, 30 mM) of freshly prepared sodium borohydride solution was added, and stirring was continued for 1 h in an ice bath. The solution color turned purple-red. The synthesized NH2-MIL-101 / AuNPs nanoparticles were washed three times by centrifugation with ultrapure water, redissolved in 30 mL of ultrapure water, and stored at room temperature for later use.

[0048] (3) Investigation of the synthesis time of NH2-MIL-101 / AuNPs: To investigate the effect of the incubation time of the material with chloroauric acid on the growth of AuNPs, 30 mg of NH2-MIL-101 was suspended in 30 mL of 0.08% (w / v) chloroauric acid solution and reacted for 0.5 h, 1 h, 1.5 h, 2 h, 4 h, 6 h, 8 h, 12 h, and 24 h, respectively. Freshly prepared NaBH4 solution was then added to the system. The synthesized NH2-MIL-101 / AuNPs nanoparticles were washed three times by centrifugation with ultrapure water, and the product was stored at room temperature for later use.

[0049] Example 2:

[0050] In this embodiment, UV-Vis, infrared spectroscopy, scanning electron microscopy, transmission electron microscopy, XRD, and XPS were used to characterize the microstructure, lattice, and physical and chemical properties of the material.

[0051] Electron microscopy characterization

[0052] The morphology and size of the samples were observed using a JSM-7800F scanning electron microscope, and the surface microstructure of NH2-MIL-101 was obtained. As shown in Figure 1A, the microstructure of a single sample was analyzed using a cryo-TEM (Talos L120C G2) transmission electron microscope. A small amount of sample was dissolved in ultrapure water, ultrasonically dispersed, and then dropped onto a 200-mesh copper grid coated with a carbon film. The operating voltage was 120 kV, and after vacuum drying, TEM testing was performed. The microstructure of NH2-MIL-101 was obtained, as shown in Figure 1A. Figure 1 As shown in Figure B. TEM images of NH2-MIL-101 / AuNPs prepared by varying the amount of chloroauric acid added are shown below. Figure 2 As shown.

[0053] Microstructure observations of NH2-MIL-101(Fe) using SEM and TEM revealed that the synthesized MIL-101(Fe) exhibited uniform size distribution, a smooth surface, and high crystallinity. Transmission electron microscopy of a single NH2-MIL-101(Fe) particle confirmed that the synthesized material possessed a regular octahedral structure.

[0054] By changing the concentration of added chloroauric acid, AuNPs are reduced inside the material, and the particle size of AuNPs gradually increases with the increase of chloroauric acid concentration. When the concentration of chloroauric acid exceeds 0.10% (w / v), AuNPs tend to aggregate and agglomerate on the outside of NH2-MIL-101.

[0055] Ultraviolet-visible absorption spectroscopy characterization:

[0056] The UV absorption spectra of NH2-MIL-101(Fe) and NH2-MIL-101 / AuNPs are as follows: Figure 3 As shown.

[0057] NH₂-MIL-101 without AuNPs loading exhibits no UV absorption peak. However, after AuNPs are intercalated into NH₂-MIL-101, a new peak appears near 550 nm, which is typical of AuNPs' surface plasmon resonance absorption. This demonstrates the successful intercalation of AuNPs within NH₂-MIL-101.

[0058] Powder X-ray diffraction (XRD) characterization

[0059] The crystal structure of the nanomaterials was determined using a Rigaku Smart Lab 3kW instrument to analyze the material composition, sample structure, and morphology. The tube voltage was 40 kV, the tube current was 40 mA, the scan rate was 8° / min, the scan step size was 0.01, and the 2θ range was 3°–80°. The XRD patterns of NH₂-MIL-101(Fe) and NH₂-MIL-101 / AuNPs are shown below. Figure 4 As shown.

[0060] The XRD patterns of NH2-MIL-101 and NH2-MIL-101 / AuNPs, with NH2-MIL-101(Fe) diffraction patterns collected at 2θ = 8.86°, 10.25°, 16.23°, and 18.58°, showed good agreement with the reported diffraction patterns, indicating successful preparation of NH2-MIL-101(Fe). No peaks of NH2-MIL-101 were observed in NH2-MIL-101(Fe) / Au, possibly due to the high content of AuNPs, which masked their characteristic peaks. The NH2-MIL-101(Fe) / Au exhibited good diffraction peaks at 2θ = 39.02°, 45.60°, 65.48°, and 78.61°, which correspond to the Au(111), Au(200), Au(220), and Au(311) crystal planes, respectively, demonstrating the formation of highly crystalline AuNP nanoparticles on NH2-MIL-101.

[0061] Fourier Infrared Characterization

[0062] The functional groups of the nanomaterials were analyzed using VERTEX 70 infrared spectroscopy. After drying at 60℃, the samples were mixed with potassium bromide and pressed into a semi-transparent film. A blank potassium bromide film was used as a blank. The wavenumber range was 4000 cm⁻¹. -1 -450cm -1 The FT-IR spectra of NH2-MIL-101(Fe) and NH2-MIL-101 / AuNPs are shown below. Figure 5 As shown.

[0063] FT-IR spectra of NH2-MIL-101 and NH2-MIL-101 / AuNPs, at 3455.41 cm⁻¹ -1 and 3317.58cm -1 A peak of NH stretching vibration of the amino group on the benzene ring appears at 1658 cm⁻¹. -1 and 1384.9cm -1 These correspond to the asymmetric stretching vibrations of OCO, 1258.08 cm. -1 A CN stretching vibration peak was observed. 766.34 cm⁻¹ -1 The peak at 578.74 cm⁻¹ is a characteristic peak of the benzene ring. -1The peak position is for the Fe-O stretching vibration. After loading AuNPs, the peak position remained unchanged. This demonstrates that loading gold nanoparticles within the framework does not affect the original material skeleton.

[0064] Multifunctional electronic spectroscopy characterization

[0065] Using the Axis Ultra DLD to measure the X-ray photoelectron spectroscopy (XPS) and corresponding high-resolution energy dispersive spectroscopy (HDED) of materials, precise analysis of the elemental composition, atomic valence states, and other information of nanomaterials can be achieved. The sample is fixed on a sample holder, any loose powder is blown away, and then placed into the instrument for testing. The spectrum is corrected using Avantage software with the binding energy at C1s (284.4 eV) as the reference. XPS values ​​for NH2-MIL-101 and NH2-MIL-101 / AuNPs are shown below. Figure 6 As shown in Figure A, the peak fitting of the Au element is as follows: Figure 6 As shown in B.

[0066] XPS spectra of Fe and Au in NH2-MIL-101 / AuNPs, with Fe2p at 725.46 eV and 711.55 eV respectively. 1 / 2 and Fe 2p 3 / 2 The peaks at 88.01 eV and 84.36 eV showed gold signals, corresponding to Au 4f, respectively. 5 / 2 and Au4f 7 / 2 The peaks confirmed the presence of Au in NH2-MIL-101 / AuNPs. XPS spectra of NH2-MIL-101 and NH2-MIL-101 / AuNPs confirmed the presence of Fe, O, N, and C.

[0067] Example 3:

[0068] In this embodiment, 60nm AuNPs were prepared from 20nm AuNPs using a seed growth method as a control group. The steps are as follows:

[0069] Synthesis of 20nm AuNPs: 100mL of 0.01% (w / v) HAuCl4 solution was added to a 250mL three-necked flask. The solution was heated to boiling under uniform stirring, and then 2.7mL of 1% (w / v) sodium citrate solution was quickly added. After reacting for 10min, heating was stopped, and stirring was continued for 6h until the system cooled to room temperature. The 20nm AuNPs were then collected, and the obtained AuNPs seeds were stored at 4℃ for later use.

[0070] Synthesis of 60nm AuNPs: 100mL of ultrapure water, 1mL of 20nm AuNPs, and 800μL of 1% (w / v) HAuCl4 solution were added to a 250mL three-necked flask. The mixture was stirred vigorously for 1min. Then, 200μL of 1% (w / v) sodium citrate solution and 100μL of 30mM L6H6O2 solution were quickly added as reducing agents in the reaction system. The two reducing agents were added every 10min. The reaction was stopped after five cycles. The obtained 60nm AuNPs were stored at room temperature.

[0071] 60nm AuNPs UV-Vis Figure 7 As shown in A, TEM is as follows Figure 7 As shown in Figure B, the particle size distribution is as follows: Figure 7 As shown in C.

[0072] The maximum absorption peak of the 60nm AuNPs in the UV-Vis spectrum is about 533nm. Transmission electron microscopy proved that the synthesized AuNPs have a uniform morphology. The average particle size distribution of the nanoparticles was measured to be about 60nm using a particle size analyzer, which proved the successful synthesis of 60nm gold seeds.

[0073] Example 4:

[0074] This embodiment illustrates the effect of varying the amount of chloroauric acid added on enhancing the fentanyl signal. Figure 8 As shown.

[0075] The SERS enhancement effect of MOF / AuNPs synthesized by varying the amount of chloroauric acid added on FTNs was investigated. It was found that the enhancement effect of the material on FTNs was most significant when the concentration of chloroauric acid was 0.08% (w / v).

[0076] Example 5:

[0077] This example illustrates the effect of chloroauric acid incubation time on the enhancement of fentanyl signal. Figure 9 As shown.

[0078] To verify the effect of the incubation time of the material with chloroauric acid on the enhancement of fentanyl SERS signal, the incubation time of chloroauric acid with the material had a weak effect on the SERS enhancement of FTNs when the concentration of chloroauric acid was 0.08% (w / v).

[0079] Example 6:

[0080] In this embodiment, the types and concentrations of the most potent agglomerating agents with enhanced effects are as follows: Figure 10 As shown in A and 10B.

[0081] Accurately weigh the powders and dissolve them in ultrapure water to prepare 2M solutions. Take 180 μL of 500 μg / L fentanyl aqueous solution and add 20 μL of 2M solution to each solution. After mixing, add NH2-MIL-101 / AuNPs particles and transfer to a 96-well plate for SERS detection.

[0082] Take 180 μL of 500 μg / L fentanyl aqueous solution, add NH2-MIL-101 / AuNPs particles, mix well, then add 20 μL of NaI aqueous solution with concentrations of 0.5, 1, 2, 3, and 4 M respectively, mix well again, and then use portable Raman spectroscopy for SERS detection.

[0083] The results proved that I - and Br - The SERS enhancement effect on FTNs is strong. Comparison of NaI and KI shows that NaI exhibits better repeatability in detecting fentanyl signals. Therefore, NaI was selected as the agglomerating agent for fentanyl detection. The introduction of I... -1 Subsequently, it can be rapidly adsorbed onto the Au surface while reducing background interference such as fluorescence. The fentanyl molecule has two tertiary amine structures, which allows fentanyl to approach the negatively charged Au surface more quickly. Furthermore, the concentration of NaI was optimized; the SERS response was strongest at a NaI concentration of 200 mM.

[0084] Example 7:

[0085] In this embodiment, under optimal screening conditions, 60nm AuNPs were used as a control to verify the enhancement effect of the substrate. Raman reporter molecule R6G was used as a model molecule, such as... Figure 11 As shown.

[0086] Using 60nm AuNPs as a positive control and NH2-MIL-101 as a negative control, and with NH2-MIL-101 / AuNPs as a substrate, 500ppb of Rhodamine 6B molecules were detected. NH2-MIL-101 alone without AuNPs loading did not detect the R6G signal peak. The R6G Raman signal intensity was lower when using 60nm AuNPs, while the signal intensity was stronger when using NH2-MIL-101 / AuNPs as a substrate. This may be because the porosity of NH2-MIL-101 concentrates the analyte in the Raman hotspot region, thereby enhancing the detection of R6G molecules.

[0087] Example 8:

[0088] In this embodiment, under optimal screening conditions, 60nm AuNPs were used as a control to verify the enhancing effect of the substrate on fentanyl. Figure 12 As shown.

[0089] Fentanyl induced by inorganic salts 1238cm-1 and 1355cm -1 Skeletal vibrations attributed to piperidine, 1000 cm -1 Bending vibrations attributable to CCCs on the phenethyl and aniline rings, 1024 cm⁻¹ -1 It belongs to the C-C stretching vibration, 1166 cm⁻¹ belongs to the C-C stretching vibration on the phenylethyl ring, 1583 cm⁻¹ -1 This is attributed to the CC stretching vibration on the aniline ring. Compared to 60 nm AuNPs alone, this substrate provides a more significant SERS enhancement for FTNs.

[0090] Example 9:

[0091] This example shows the SERS spectra of fentanyl and its homologues 3-methylfentanyl, sufentanyl, norfentanyl, carfentanyl, and 4-fluorobutyrylfentanyl, as shown below. Figure 13 As shown.

[0092] SERS spectra of fentanyl and its homologues were analyzed at 1000 cm⁻¹. -1 The presence of fentanyl-like characteristic fingerprints at the substrate demonstrates that the substrate can rapidly detect fentanyl and its homologues.

[0093] Example 9:

[0094] This embodiment investigated the salt resistance of the substrate. Figure 14 A) Resistant to acids and alkalis ( Figure 14 B) In-batch stability Figure 14 C) and anti-aggregation effect ( Figure 14 D).

[0095] Salt tolerance: NaCl salt solutions of fentanyl at different concentrations were prepared and tested using this substrate.

[0096] Acid and alkali resistance investigation: Fourteen 500 μg / L fentanyl standard solutions were prepared with pH values ​​of 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0, and 14.0. 180 μL of the fentanyl standard solutions at different pH values ​​were added to concentrated NH2-MIL-101, followed by 20 μL of NaI solution. After thorough mixing, the solutions were transferred to 96-well plates for SERS detection.

[0097] Intra-batch stability: Intra-batch stability tests were conducted using the same substrate.

[0098] Anti-aggregation effect: 60nm AuNPs were prepared by seed growth method. 1 mL of the solution was centrifuged at 6000 rpm for 10 min, and 180 μL of 500 μg / L fentanyl solution was added. After mixing thoroughly, 20 μL of 2M NaI solution was added, and the mixture was transferred to a 96-well plate for measurement. SERS signals were detected at different time points. 200 μL of NH2-MIL-101 / AuNPs solution was centrifuged and SERS was detected under the same conditions as the 60nm AuNPs.

[0099] The material remained stable in NaCl solution for 30 minutes, indicating its applicability to complex biomonitoring. Since AuNPs tend to aggregate in acidic and alkaline solutions, affecting the SERS signal of the analyte, the material was investigated for detecting fentanyl in aqueous solution at pH 4–12. It was found that SERS stability was enhanced under alkaline conditions at pH 8–10. The same batch of material was used to repeatedly detect the fentanyl signal ten times at 1000 cm⁻¹. -1 The intra-batch recovery rate of the material was calculated from the signal, yielding an RSD of 5.39%, demonstrating the material's stability. Compared to bare AuNPs alone, NH2-MIL-101 / AuNPs stabilized the fentanyl signal for 36 hours. This may be attributed to the aggregation of AuNPs in the salt solution after the addition of the agglomerating agent, which weakens the SERS signal of fentanyl. In contrast, the substrate, protected by the MOF shell, prevents AuNPs from agglomerating, thus maintaining signal stability.

[0100] Example 10:

[0101] Selective SERS detection of fentanyl in heroin and morphine samples, such as Figure 15 As shown.

[0102] The detection of trace amounts of fentanyl, primarily heroin and morphine, was demonstrated. While the presence of heroin inhibits the detection of FTNs signals, 0.5% fentanyl could still be detected in heroin. In the detection of morphine-based FTNs, fentanyl levels as low as 0.05% could be detected. These results prove that this novel, post-synthesized SERS substrate can achieve trace detection of fentanyl.

[0103] Example 11:

[0104] Deuterated fentanyl was chosen as the internal standard. Figure 16 As shown, the linear standard curve of fentanyl in the water sample is as follows: Figure 17 As shown.

[0105] Add a certain concentration of fentanyl and deuterated fentanyl to a blank water sample to prepare fentanyl solutions with different concentration gradients, with a final concentration of 100 ng / mL deuterated fentanyl as the internal standard. Take 180 μL of the analyte and concentrate NH2-MIL-101 / AuNPs.

[0106] In quantitative analysis of samples, uncontrollable factors such as background, laser fluctuations, and substrate physicochemical properties make quantitative detection difficult. This invention selects deuterated fentanyl as an internal standard and establishes a SERS method for the detection of fentanyl using the internal standard method. Deuterated fentanyl at 958 cm⁻¹... -1 The characteristic peak at 1000 cm⁻¹ is similar to that of fentanyl. -1 Using the ratio of characteristic peak intensity at a given location as a relative intensity can effectively avoid interference from various factors such as the environment.

[0107] The assay was used to detect fentanyl levels in water samples. The linear range was 10 ng / mL to 200 ng / mL, and the correlation coefficient (R) was [value missing]. 2 The RSD was 0.9802, the recovery rate ranged from 75.65% to 110.82, and all RSD values ​​were less than 15%.

[0108] Example 12:

[0109] Linearity standard curve for fentanyl detection in plasma samples as follows Figure 18 As shown.

[0110] Take FTNs spiked samples from healthy human plasma, add bovine serum albumin (BSA), add 20 μL of 2M NaI solution to NH2-MIL-101 / AuNPs particles, mix well, add 180 μL of plasma containing internal standard, mix, and transfer to a 96-well plate for measurement.

[0111] Plasma consists of proteins, lipids, inorganic substances, salts, amino acids, and a large amount of water. Bovine albumin (BSA) is added to avoid interference from large molecules in the plasma. The linear range is 20 ng / mL-400 ng / mL, and the correlation coefficient (R) is [missing value]. 2 The value was 0.9488. The recovery rate ranged from 81.55% to 109.32%, with all RSD values ​​less than 15%.

[0112] Example 11:

[0113] linearity standard curve for the detection of FTNs in urine samples as follows Figure 19 As shown.

[0114] Take 70 μL of Wistar urine, add 70 μL of 10% NaOH solution, 70 μL of 5M NaCl solution, 35 μL of 10 ppm FTNs-D5 solution, and 35 μL of 10 ppm FTNs solution to prepare a urine sample with a final concentration of 500 μg / L of FTNs, wherein the concentration of the internal standard is 500 μg / L.

[0115] The presence of a large amount of urea in urine interfered with the signal peak. Therefore, urine diluted tenfold was selected as the analyte. The strongly alkaline environment created by adding NaOH helped improve drug separation, and NaCl reduced the drug's solubility in water. The linear range was 20 ng / mL–500 ng / mL, and the correlation coefficient (R0) was [not specified]. 2 The value was 0.9437. The recovery rate ranged from 86.29% to 121.07%, with all RSD values ​​less than 15%.

[0116] Example 12:

[0117] inter-batch stability, such as Figure 20 As shown.

[0118] Three batches of materials were synthesized, loaded with AuNPs, and the batch-to-batch recovery was verified by standard curves.

[0119] To verify the accuracy of the invention, batch-to-batch accuracy tests were conducted, with recoveries ranging from 71.36% to 129.62%, demonstrating the stability of the substrate.

[0120] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for detecting trace amounts of fentanyl using a SERS-enhanced substrate for rapid fentanyl detection, characterized in that, The process for detecting trace amounts of fentanyl using this substrate is as follows: (1) Centrifuge NH2-MIL-101 / AuNPs particles at room temperature, discard the supernatant, and obtain concentrated NH2-MIL-101 / AuNPs particles; (2) Take concentrated NH2-MIL-101 / AuNPs particles, add them to the analyte solution, mix well, and then transfer them to a 96-well plate for SERS detection; The substrate comprises NH2-MIL-101 / AuNPs synthesized by co-incubating octahedral NH2-MIL-101(Fe) with chloroauric acid and growing AuNPs in situ on the material under the action of a reducing agent; and the substrate, when used in conjunction with a deuterated fentanyl internal standard, enables the quantitative detection of fentanyl content in complex biological samples.

2. The method for detecting trace amounts of fentanyl using a SERS-enhanced substrate for rapid fentanyl detection according to claim 1, characterized in that, This substrate can trap analyte molecules around AuNPs, thereby enhancing their Raman signal.

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