A flexible SERS substrate and its preparation method and application

Abstract

This invention belongs to the field of drug detection technology, and discloses a flexible SERS substrate, its preparation method, and its application. The preparation method includes: Step S1, preparing PVP-coated AgNPs based on Citrate-Ag colloid; Step S2, using PDMS as a flexible substrate, transferring the AgNPs sol to the PDMS surface to form a PDMS@Citrate-Ag flexible SERS substrate. The flexible SERS substrate is the substrate obtained by the above method. It also includes a method for detecting narcotic and psychotropic drugs based on the flexible SERS substrate, comprising the following steps: Step S1, contacting the narcotic and psychotropic drug to be tested with the flexible SERS substrate and performing SERS measurement using a Raman spectrometer; Step S2, introducing a dSERS strategy to perform digital quantitative analysis of the target narcotic and psychotropic drug. The flexible substrate of this invention can meet the needs of rapid on-site detection of narcotic and psychotropic drugs in various scenarios, and the dSERS strategy can solve the problem of false negatives caused by low content, cumulative abuse, and interference from complex matrices. The combination of the two is beneficial to promoting the scientific rigor and reliability of drug detection in drug control work.

Description

Technical Field

[0001] This invention relates to the field of drug detection technology, specifically to a flexible SERS substrate, its preparation method, and its application. Background Technology

[0002] The latest drug situation report in my country shows that the drug abuse situation is severe and complex, with the problem of overlapping abuse of new types of drugs and substitutes continuing to spread, and the problem of substitution abuse of addictive substances such as narcotic and psychotropic drugs being prominent. In drug control work, narcotic and psychotropic drugs seized on site are in different dosage forms (powder injections, injections, tablets), illegally added to different carriers (face masks, alcohol, tobacco oil, tobacco leaves, etc.), and there are also cases of overlapping abuse of different drugs.

[0003] Surface-enhanced Raman spectroscopy (SERS) can detect the characteristic vibrational information of target molecules, enabling rapid, sensitive, and specific trace molecule detection, complex sample analysis, and real-time monitoring, making it one of the mainstream methods for rapid drug detection. Traditional rigid SERS substrates, due to their fixed detection platform shape, cannot meet the detection needs on complex shapes and irregular surfaces, limiting their application range. In recent years, flexible SERS substrates have demonstrated superior flexibility and plasticity, offering significant advantages in non-destructive and in-situ detection, making them particularly suitable for addressing the needs of rapid on-site detection of drugs and psychotropic substances in various complex scenarios in drug enforcement work.

[0004] Although flexible SERS substrates have many advantages, they still have some drawbacks, mainly including: 1. High Detection Difficulty: In drug enforcement operations, the different dosage forms, carriers, and instances of combined abuse of narcotic and psychotropic drugs seized on-site lead to overlapping and overlapping signals from different target substances. Trace amounts of these signals are difficult to separate from the matrix background, resulting in false negatives. Furthermore, traditional quantification methods using averaged SERS intensity may fail to capture positive signals due to a small number of samplings or because they are masked by the background after averaging, leading to false negatives. False negatives in drug and psychotropic drug testing can allow drug users to escape legal supervision, posing a threat to public safety.

[0005] 2. Limitations of SERS performance: The performance of SERS depends on the interaction between the target molecule and the plasma hotspot on the substrate. Recent SERS studies have revealed significant signal fluctuations in the target molecule-metal interaction, making stable SERS quantification a persistent challenge.

[0006] To address the aforementioned shortcomings, Brolo et al. proposed a pixel-based digital surface-enhanced Raman spectroscopy (dSERS) strategy. The dSERS strategy solves the false negative problem caused by traditional average SERS intensity quantification methods by significantly reducing the minimum detection concentration through large-area SERS sampling and single-pixel-level digital visualization. Building on this, Ye's team proposed a single-molecule recognition technology based on digital colloidal Raman spectroscopy, achieving stable and reliable quantitative detection of ultra-low concentration targets, further improving the performance of the dSERS strategy. However, this research is only suitable for liquid detection in practical applications and is difficult to meet the needs of rapid on-site testing in various complex situations in drug enforcement work. Summary of the Invention

[0007] The purpose of this invention is to provide a flexible SERS substrate, its preparation method, and its application, which at least solves one of the technical problems mentioned in the background art.

[0008] To achieve the above objectives, this invention proposes a method for preparing a flexible SERS substrate, specifically including the following steps: Step S1: Based on Citrate-Ag colloid, prepare PVP-coated AgNPs; Step S2: Using PDMS as a flexible substrate, AgNPs sol is transferred to the PDMS surface to form a PDMS@Citrate-Ag flexible SERS substrate.

[0009] Further, in step S1, the method for preparing Citrate-Ag colloid is as follows: heating AgNO3 solution to boiling, adding sodium citrate solution dropwise, and boiling again to obtain Citrate-Ag colloid.

[0010] Further, in step S1, the method for preparing PVP-coated AgNPs is as follows: after centrifuging and washing the Citrate-Ag colloid, it is redispersed in a PVP ethanol solution to obtain PVP-coated AgNPs. Further, in step S2, dichloromethane is mixed with PVP-coated AgNPs, then n-hexane is added, the upper n-hexane layer is removed, and the stickier side of the PDMS film is brought into contact with the monolayer AgNPs to transfer the AgNPs sol to the PDMS surface.

[0011] Furthermore, in step S2, after forming the PDMS@Citrate-Ag flexible SERS substrate, it is washed sequentially with ultrapure water and anhydrous ethanol, dried, and stored in a dry container.

[0012] This invention also proposes a flexible SERS substrate prepared using the above-described method. This substrate combines the flexibility of PDMS with the SERS-enhancing properties of AgNPs, making it suitable for rapid detection in complex field environments.

[0013] This invention also proposes a method for detecting narcotic and psychotropic drugs based on a flexible SERS substrate, specifically including the following steps: Step S1: The narcotic drug to be tested is brought into contact with a flexible SERS substrate, and SERS measurement is performed using a Raman spectrometer; the flexible SERS substrate is a flexible SERS substrate obtained by the above preparation method.

[0014] Step S2 introduces the dSERS strategy to perform digital quantitative analysis of the target narcotic and psychotropic drugs. In this step, digital quantitative analysis of the target narcotic and psychotropic drugs is performed through stepwise dilution, large-area SERS sampling, intensity threshold setting, and digital probability calculation.

[0015] Furthermore, the steps of the dSERS strategy include: (a) Mix the narcotic and psychotropic drug to be tested with Hya-Ag sol evenly, incubate with a flexible SERS substrate, and then dry; (b) Perform large-area SERS sampling within the designated area to collect multiple spectral data; (c) Set an intensity threshold, convert the spectral data into digital “0” and “1” signals, and perform quantitative analysis by statistically analyzing the probability of occurrence of the “1” signal.

[0016] Further, in step (a), the preparation method of Hya-Ag sol is as follows: AgNO3 is added to a mixture of Hya·HCl and NaOH, and after stirring, Hya-Ag is obtained.

[0017] Furthermore, the narcotic drugs include one or more of heroin, cocaine, or etomidate.

[0018] The beneficial effects of this invention are as follows: 1. In the preparation of flexible SERS substrate, AgNPs are used as reinforcing materials and PDMS is used as substrate to prepare flexible SERS substrate, which can meet the needs of rapid detection of drugs and psychotropic substances in various complex on-site environments.

[0019] 2. When using a flexible SERS substrate to detect narcotic and psychotropic drugs, this invention introduces a digital surface-enhanced Raman spectroscopy strategy. Through stepwise dilution, large-area SERS sampling, intensity threshold setting, and digital probability calculation, it successfully solves the problem of false negatives in the detection of narcotic and psychotropic drugs in judicial practice caused by low content, superimposed abuse, and interference from complex matrices.

[0020] 3. The flexible SERS substrate combined with the dSERS strategy of the present invention can not only meet the needs of rapid on-site testing, but also effectively solve the problem of false negatives in testing, providing a universal new solution for drug and psychotropic drug testing in drug control practice. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the present invention will be further described below in conjunction with the accompanying drawings and embodiments. The drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort: Figure 1 This is a flowchart of the overall process of preparing and testing flexible SERS substrates.

[0022] Figure 2 This is a process diagram of the flexible SERS substrate preparation process in Example 1.

[0023] Figure 3 This is a diagram illustrating the performance of a flexible SERS substrate.

[0024] Figure 4 It is a Raman spectrum of narcotic and psychotropic drugs.

[0025] Figure 5 This is a concentration gradient SERS spectrum of narcotic and psychotropic drugs.

[0026] Figure 6 This is a schematic diagram of the principle of digital quantitative analysis.

[0027] Figure 7 This is a spectral data graph for quantitative detection of narcotic and psychotropic drugs.

[0028] Figure 8 This is a digital quantitative chart of narcotic and psychotropic drugs.

[0029] Figure 9 It is a digital quantitative analysis chart of the superimposed abuse of narcotic and psychotropic drugs and complex matrices. Detailed Implementation

[0030] The embodiments described are provided to better illustrate the present invention, but are not intended to limit the scope of the invention to the embodiments described. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0031] In this invention, the sources of the raw materials and instruments are as follows: Silver nitrate (AgNO3, 99.8%) was purchased from Guangdong Guanghua Science & Technology Co., Ltd. (China); Sodium citrate (99%), hydroxylamine hydrochloride (Hya·HCl, 98.5%), sodium hydroxide (NaOH, 98%), crystal violet (CV, 90%), tetrabutylammonium nitrate (TBA NO3) - 98%), dichloromethane (CH2Cl2, 99.9%), n-hexane (C6H 14 Anhydrous ethanol (C2H6O, ≥99.7%) was purchased from Shanghai McLean Co., Ltd. (China).

[0032] The polydimethylsiloxane (PDMS) film was purchased from Hefei Keliao New Material Technology Co., Ltd.

[0033] The heroin and cocaine came from the Narcotics Control Division of the Chongqing Municipal Public Security Bureau.

[0034] Etomidate was obtained from the Chongqing Institute for Food and Drug Control.

[0035] The experimental water used in the sample synthesis process was ultrapure water (RES-10-B type pure water machine from Chongqing Kerun Water Treatment Equipment Co., Ltd., China), BK-360B ultrasonic cleaner (Jinan Barker Ultrasonic Technology Co., Ltd., China), and ESJ200-4B analytical balance (Shenyang Longteng Electronics Co., Ltd., China).

[0036] The ultraviolet-visible spectra were measured using a UV-2450 ultraviolet-visible spectrophotometer (Shimadzu Corporation, Japan).

[0037] Scanning electron micrographs were obtained using a Hitachi S-4800 cold field emission scanning electron microscope (Hitachi, Japan).

[0038] Transmission electron microscopy images were obtained using a Tecnai G2 F20 field emission transmission electron microscope (FEI Corporation, USA).

[0039] Raman spectra were measured using a DXR3 Raman spectrometer (Thermo Fisher Scientific, USA).

[0040] Example 1 A method for preparing a flexible SERS substrate specifically includes the following steps: Step S1: Prepare PVP-coated AgNPs S1.1, Based on Citrate-Ag colloid, prepare Citrate-Ag colloid. Dissolve 18 mg of silver nitrate (AgNO3) in 100 ml of ultrapure water, heat to boiling, then add 2 ml of 1% sodium citrate solution and continue boiling for 1 hour to obtain yellow-green silver citrate colloid (Citrate-Ag).

[0041] S1.2, Preparation of PVP-coated AgNPs. The Citrate-Ag colloid prepared in step S1.1 was placed in a centrifuge tube and centrifuged at 10,000 rpm. After removing the supernatant, the precipitate was redispersed in 1 mL of 0.001 wt% polyvinylpyrrolidone (PVP) ethanol solution. By vigorous stirring, PVP-coated AgNPs were obtained.

[0042] Step S2: Forming a PDMS@Citrate-Ag flexible SERS substrate S2.1, mix 200 μL of dichloromethane with 2 mL of water in a 10 mL centrifuge tube, then add 3.8 mL of water, shake vigorously for 1 min, and let stand for 30 s. Then, slowly add 800 μL of n-hexane along the container wall. Due to the Marangoni force, AgNPs are pulled to the upper water / oil interface to form a monolayer.

[0043] In this step, after adding n-hexane, an oil / water / oil three-phase system of n-hexane / water / dichloromethane is formed, which is then self-assembled on the surface of Citrate-Ag in step S1.2 through oil / water / oil.

[0044] S2.2 After removing the top layer of n-hexane, the stickier side of the pretreated polydimethylsiloxane (PDMS) film is brought into contact with the monolayer AgNPs to transfer the AgNPs sol to the PDMS surface. After drying in a nitrogen atmosphere, it is washed twice with ultrapure water and anhydrous ethanol, and then further dried and stored in a clean desiccator to form a PDMS@Citrate-Ag flexible SERS substrate.

[0045] Example 2 The flexible SERS substrate in this embodiment was prepared using the preparation method of Example 1. The overall flowchart of the flexible SERS substrate preparation and detection process is shown below. Figure 1 As shown.

[0046] A method for detecting narcotic and psychotropic drugs based on the above-mentioned flexible SERS substrate specifically includes the following steps: Step S1: On-site sampling and SERS measurement The sample of the narcotic and psychotropic drugs to be tested was brought into contact with a flexible SERS substrate. SERS measurements were performed using a Raman spectrometer with a 50×0.75NA microscope objective, with excitation and exposure at 785 nm for 0.1 s at 30 mW laser power. Spectra were collected through a 50 μm slit. A grating of 400 lines / mm was used, providing a range of 350–1800 cm⁻¹. -1 Spectral range.

[0047] In this step, the preparation method for the samples of the narcotic and psychotropic drugs to be tested (including heroin, cocaine, or etomidate) is as follows: a stock solution containing 1 mg / mL crystal violet, heroin, cocaine, and etomidate is prepared using anhydrous ethanol as a solvent. Crystal violet is used as a Raman reporter molecule to analyze the SERS performance of the flexible substrate. Different concentrations of narcotic and psychotropic drugs are prepared from the stock solution through stepwise dilution. Ethanol is a volatile solvent and will evaporate before measurement, thus not affecting the Raman spectrum of the target molecules.

[0048] For rapid on-site testing, for solid samples, a small amount of ethanol can be sprayed onto the surface before attaching a flexible SERS substrate; for liquid samples, a flexible SERS substrate membrane can be used to directly dip into the sample solution, and after drying, SERS measurements can be performed.

[0049] Step S2: SERS detection S2.1, Mix the sample of the narcotic and psychotropic drug to be tested with Hya-Ag sol at a volume ratio of 1:9, and then homogenize by ultrasonication.

[0050] In this step, the preparation method of Hya-Ag sol is as follows: 10 ml of AgNO3 (10 mM) is rapidly added to a mixture of 90 ml of Hya·HCl (1.67 mM) and NaOH (3.33 mM), and the mixture is stirred vigorously to obtain a gray-green silver hydroxylamine hydrochloride sol (Hya-Ag). S2.2, the mixed solution was incubated with a flexible SERS substrate at room temperature for 2 hours, and then dried under nitrogen.

[0051] S2.3, large-area SERS sampling was performed in an area of ​​approximately 20 μm × 20 μm, and 20 × 20 spectral data were collected using a Raman spectrometer.

[0052] Step S3: Digital Quantitative Analysis S3.1 employs the dSERS strategy and sets an intensity threshold. The spectral data of each sampling point is compared with the threshold and converted into a digital "0" (negative) or "1" (positive) signal. By statistically analyzing the probability of the "1" signal and combining it with a pre-established quantitative relationship model, digital quantitative analysis of the target narcotic and psychotropic drugs is performed.

[0053] In this step, each site within the acquired 20μm × 20μm region is considered a pixel. The maximum intensity (Is) within the window containing the target molecule's characteristic Raman peaks (feature window) is used to determine whether the target molecule is detected in that pixel. When Is is greater than a threshold calculated based on the "noise window," the pixel is designated as a positive "1," otherwise a negative "0." Quantification is ultimately performed using the probability of "1" and "0" occurrences. The feature window should be selected on the unique band of the target molecule and far from other peaks present in the background. When selecting an appropriate threshold, factors such as the confidence level of the positive peak of the target molecule's characteristic peak under the same measurement parameters and false positives in the absence of the target molecule should be considered.

[0054] The detection method in this embodiment can achieve highly sensitive and rapid on-site detection of narcotic and psychotropic drugs, and effectively solves the problem of false negatives caused by low content, cumulative abuse, and interference from complex matrices.

[0055] I. Process Inspection and Result Data Analysis This analysis mainly focuses on the data and results from the preparation and testing of flexible SERS substrates, and includes the following parts.

[0056] 1. Material Characterization Figure 2 This is a process diagram illustrating the fabrication of the flexible SERS substrate in Example 1, wherein... Figure 2 (a) shows the surface self-assembly process of oil / water / oil with Citrate-Ag in Example 1; (b) shows the transfer process of the monolayer AgNPs thin film in Example 1; (c) shows the SEM (scanning electron microscope) and TEM (transmission electron microscope) characterization images of PDMS@Citrate-Ag in Example 1. Figure 2 In the image, the scale bar is 1 μm, and the scale bar in the upper right corner is 100 nm.

[0057] Figure 2 In the process shown in (a), the driving force for the transfer and compression of AgNPs in the three-phase system is the Marangoni force, which originates from the surface tension gradient difference between the upper and lower water / oil interfaces (n-hexane / water / dichloromethane). When the surface tension of the upper oil / water interface (n-hexane / water; γ=51.1 mN / m) is higher than that of the water / lower oil interface (dichloromethane / water; α=28.3 mN / m), the resultant force pulls the AgNPs towards the upper water / oil interface. During this process, the Marangoni force can overcome the electrostatic repulsion between AgNPs, thereby producing a high-density monolayer AgNPs film. Compared with the traditional two-phase self-assembly method, the self-assembly effect of the three-phase system of this invention is not affected by the AgNPs concentration and has a self-healing function, facilitating large-scale transfer.

[0058] Figure 2 (b) In the process shown, hexane is removed as much as possible without damaging the monolayer film. Then, the stickier side of the PDMS film is directly contacted with the monolayer AgNPs to transfer the AgNPs to the surface of the PDMS film. After drying, washing and drying are performed to obtain PDMS@Citrate-Ag.

[0059] Figure 2 In (c), the SEM and TEM images of PDMS@Citrate-Ag show that AgNPs are uniformly and densely distributed on the PDMS film. Compared to the traditional peeling and transfer method, which involves first transferring to a silicon wafer, then pressing PDMS onto the silicon wafer surface and peeling it off to obtain PDMS@Citrate-Ag, the flexible SERS substrate obtained by the direct contact and drying method of this invention is more uniform. Compared to the nanoparticles prepared by self-assembling three-phase systems CTAC or CTAB in the prior art, the nanofilm obtained by preparing Citrate-Ag with citrate in this invention has insufficient density, but it already possesses certain SERS properties and can basically meet the needs of preliminary on-site screening. To improve its enhancement performance, this invention combines the flexible SERS substrate with Hya-Ag to provide more and better hotspots for the target analyte.

[0060] 2. Performance Investigation of Flexible SERS Substrates Figure 3 These are performance analysis diagrams for flexible SERS substrates, in which... Figure 3 (a) SERS spectra of CV (1 μg / mL) detected on different SERS substrates; Figure 3 (b) is the SERS spectrum of CV at different concentrations detected by PDMS@Ag; Figure 3 (c) is a physical image of the PDMS@Ag flexible substrate; Figure 3 (d) shows 30 random spectra of PDMS@Ag at different sites at 1176 cm⁻¹. -1 422cm -1 Scatter plot of SERS intensity.

[0061] Figure 3In (a), crystal violet (CV) was used as the Raman reporter molecule to investigate the enhancement performance of PDMS@Citrate-Ag+Hya-Ag (PDMS@Ag), PDMS@Citrate-Ag, and Hya-Ag. Clear CV molecular fingerprint peaks were generated on all three substrates. Hya-Ag produced the lowest SERS signal due to its smallest particle size; PDMS@Ag produced the highest SERS signal, possibly because the combination of Citrate-Ag and Hya-Ag provided more and stronger SERS hotspots for CV molecules, or perhaps due to the newly generated intermetallic charge tunneling enhancement effect between Citrate-Ag, Hya-Ag, and CV molecules. The study showed that although Citrate-Ag is larger than Hya-Ag, its residual citrate layer may create steric hindrance, hindering the target from approaching the metal surface to some extent. Therefore, the invention employs a method of combining PDMS@Citrate-Ag with Hya-Ag for SERS detection.

[0062] according to Figure 3 The results in (b) show that PDMS@Ag can detect at least 1 ng / mL CV, according to the EF formula EF=I SERS / C SERS ×C RS / I RS Where I represents 1176cm -1 The peak intensity at [value], where C represents the concentration of the analyte. When CSERS and CRS are each set to 1×10 [value], [the value is indicated by the peak intensity at [value], and C represents the concentration of the analyte]. -5 At 1 mg / mL, the calculated EF is approximately 3.14 × 10⁻⁶. 7 This indicates that the substrate has good sensitivity.

[0063] according to Figure 3 The results in (c) show that the prepared PDMS@Ag flexible substrate can not only be placed on a smooth carrier surface for conventional SERS sampling, such as... Figure 3 (c) Ⅱ can also be assembled onto rubber gloves for flexible application of target solutions, such as... Figure 3 (c) Points III and IV demonstrate that PDMS@Ag possesses good adaptability. Furthermore, due to the flexibility of PDMS, bending is unavoidable in practical applications. This paper simulates real deformation by bending PDMS@Ag at different angles, such as... Figure 3 (c) I. The bent PDMS@Ag still exhibits the bright silver color of the AgNPs film. Random SERS sampling is shown below. Figure 3 As shown in (d). According to Figure 3 The results in (d) show that at 1176cm -1 and 422cm -1The RSDs at the two locations were 6.25% and 7.09%, respectively, indicating that PDMS@Ag has good stability.

[0064] 3. SERS detection of narcotic and psychotropic drugs PDMS@Ag was used for SERS measurements of heroin, cocaine, and etomidate. Figure 4 These are Raman spectra of narcotic and psychotropic drugs, in which... Figure 4 (a) ~ Figure 4 (c) are the Raman spectra of Heroin, Cocaine and Etomidate, respectively.

[0065] according to Figure 4 (a) ~ Figure 4 (c) shows that the Raman spectra of different narcotic and psychotropic drugs are significantly different.

[0066] exist Figure 4 In (a), 444cm -1 531cm -1 590cm -1 628cm -1 and 1242cm -1 The peak value at that point indicates a moderate but detectable signal, confirming the presence of heroin.

[0067] exist Figure 4 In (b), 848cm -1 886cm -1 1104cm -1 1036cm -1 1279cm -1 1453cm -1 1605cm -1 and 1712cm -1 The characteristic peak at 848 cm⁻¹ is the vibrational peak of cocaine. The cocaine solid exhibits a peak at 848 cm⁻¹ due to the C-T stretching vibration of the tropane ring. -1 874cm -1 898cm -1 The three characteristic peaks generated at the point; after SERS enhancement, i.e. (PDMS@Ag+Cocaine), only 848 cm⁻¹ was observed. -1 886cm -1 Two characteristic peaks, which can be attributed to adsorption-induced vibrational mode coupling between Cocaine molecules and AgNPs, resulting in a peak at 874 cm⁻¹. -1 and 898cm -1 The energy difference between two similar vibrational modes decreases, and the polarizability tensor changes, causing them to merge into a single 886 cm⁻¹. -1 Single peak.

[0068] exist Figure 4 In (c), 618cm -1 785cm -1 985cm -1 1003cm -1 1196cm -1 1351cm -1 and 1723cm -1 The new characteristic peak at this location is the vibrational peak of etomidate.

[0069] 3. Digital quantitative analysis Quantitative analysis was performed on three types of narcotic and psychotropic drugs, and the concentration gradient SERS spectra of the drugs are shown below. Figure 5 As shown, where, Figure 5 (a) is the SERS spectrum of the Heronin concentration gradient; Figure 5 (b) is the SERS spectrum of the Cocaine concentration gradient; Figure 5 (c) is the SERS spectrum of the Etomidate concentration gradient; Figure 5 (d) is the SERS spectrum of the "0" and "1" phenomenon after removing the background.

[0070] according to Figure 5 The results from (a) to 5 (d) show that Heroin, Cocaine, and Etomidate were at concentrations of 100 ng / mL and 10 ng / mL (approximately 10 ng / mL), respectively. -7 M and 10 -8 The Raman signal drops sharply at concentrations M and below. In fact, when SERS sampling is performed on target molecule concentrations at and below this level, the number of target molecules is too small, resulting in a significantly reduced Raman signal. Figure 5 The "0" and "1" phenomenon shown in (d) indicates that some sites can detect the characteristic fingerprint peaks of the target analyte, while others cannot detect a valid spectral signal. Red represents a positive spectrum "1" with a recognizable characteristic fingerprint peak, and black represents a negative spectrum "0" where no target signal was detected, similar to the observation in the blank control group (no target analyte added, BG) under the same conditions. The concentration of different target analytes produces this phenomenon differently, and the probability of "0" and "1" varies with different concentrations of the same target analyte. At lower target analyte concentrations, the probability of "0" is higher and the probability of "1" is lower, making traditional SERS detection methods prone to false negatives.

[0071] exist Figure 5 In (a), within the concentration range of 1 μg / mL to 100 μg / mL, Heroin at 628 cm⁻¹ -1The peak intensity at the concentration is not linear; the SERS intensity at 1 μg / mL is actually higher than that at 10 μg / mL. This can be attributed to the limited adsorption sites on the AgNP surface. When the heroin concentration is high, intermolecular competition for adsorption may lead to the ineffective occupation of some sites (such as multilayer adsorption or disordered stacking), thus reducing the enhancement efficiency. At low concentrations, monolayer adsorption is more ordered, the polarizability changes more significantly, and the signal may be enhanced. In summary, traditional detection methods are prone to false negatives at low concentrations and nonlinear quantitative challenges at high concentrations. Based on this, this invention introduces a digital surface-enhanced Raman spectroscopy (dSERS) strategy, which uses stepwise dilution, large-area SERS sampling, intensity threshold setting, and digital probability calculation to perform quantitative analysis of the target analyte. Specifically, as follows... Figure 6 As shown.

[0072] Figure 6 This is a schematic diagram of the principle of digital quantitative analysis, in which... Figure 6 (a) is a simulation diagram of the spatial distribution between narcotic and psychotropic drug molecules and AgNPs; Figure 6 (b) is a schematic diagram of dSERS sampling; Figure 6 (c) Schematic diagram of digital heat map description.

[0073] according to Figure 6 (a) It can be seen that, at low concentrations, there are theoretically four possible distribution states between the target molecule and AgNPs: the target molecule adsorbed between three nanoparticles (best enhancement effect), between two nanoparticles (good enhancement effect), on a single nanoparticle (extremely low enhancement effect), and not adsorbed on any nanoparticle. The SERS signals detected for these four distribution states are as follows: Figure 6 (b) The first two states exhibit obvious Raman peaks of the target molecule, resulting in a positive detection ("1"). The latter two states lack Raman peaks of the target molecule, resulting in a negative detection ("0"). Since the actually synthesized AgNPs are not perfectly smooth spheres, the enhancement effects of different nanoparticles and their interstices vary, leading to different Raman signal intensities. Specifically... Figure 6 (c) refers to the SERS signal fluctuation phenomenon.

[0074] Figure 6(c) is a 20×20 thermal image representing 400 independent SERS samples, where the color of each pixel represents the intensity of the target analyte's characteristic peak. After SERS sampling, by setting an intensity threshold, the complex Raman intensity thermal image can be converted into a digital "0" and "1" thermal image. A higher threshold reduces false positives and increases false negatives, while a lower threshold increases false positives and reduces false negatives; increasing the number of measurements reduces the probability of false negatives. Therefore, by adjusting the intensity threshold and the number of measurements in dSERS, the false positives and false negatives can be effectively reduced. Simultaneously, during SERS sampling, the independence of each sample should be ensured, and the scan step size should be greater than the excitation volume to avoid overlap of adjacent detection sites.

[0075] This invention investigates a region of approximately 20 μm × 20 μm using Raman spectroscopy, collecting a total of 20 × 20 spectra at an excitation wavelength of 785 nm, resulting in each pixel being approximately 1 μm × 1 μm in size. While increasing the number of SERS spectra collected in a given region theoretically improves the resolution per pixel and thus enhances detection accuracy, this is limited by the inherent spatial resolution of Raman microscopy. In this invention, a confocal Raman microscope with a 50 × objective (NA = 0.75) is used for SERS spectrum collection at an excitation wavelength of 785 nm. Based on r = 0.61λ / NA, this provides a theoretical spatial resolution of approximately 638 nm, where r is the spatial resolution, λ is the excitation wavelength, and NA is the numerical aperture of the objective. However, the practical resolution of Raman microscopy is typically lower than the theoretical diffraction limit due to factors such as optical aberrations, noise, and sample characteristics that degrade signal quality. Furthermore, environmental vibrations, focusing stability, and detector limitations further reduce the effective resolution in practical applications. Therefore, it is reasonable to perform 20×20 SERS samplings in a 20μm×20μm region.

[0076] Figure 7 This is a spectral data graph of quantitative detection of narcotic and psychotropic drugs, in which... Figure 7 (a) Spectral data of the blank control group and Heroin. Figure 7 (b) Spectral data of Cocaine and Etomidate. Figure 7 Each set of data contains 100 spectra.

[0077] This invention requires the selection of a feature window before setting the intensity threshold. According to... Figure 7 Therefore, the feature window should be selected in the unique band of the target molecule and far from the background peaks present in the blank control group, while the noise window should be selected far from the fingerprint peaks belonging to the target molecule and the background peaks. The threshold setting needs to comprehensively consider factors such as the confidence level of positive peaks and false positives caused by background interference. The threshold formula is:

[0078] In the above formula, The maximum Raman intensities of Heroin, Cocaine, and Etomidate at their respective feature windows are ( , ) represents the Raman mean and standard deviation of the noise window. This is a constant. That is, as long as the Raman intensity of the target molecule at the feature window is greater than or equal to the average value at the corresponding position in the noise window plus... An intensity that is more than one standard deviation is marked as "1", and vice versa, it is marked as "0". The specific parameter values ​​of the feature window, noise window, and intensity threshold of this invention are shown in Table 1.

[0079] Table 1: Parameters for positive assays of different narcotic and psychotropic drugs

[0080] Figure 8 This is a digital quantitative chart of narcotic and psychotropic drugs, in which... Figure 8 (a) is a graph showing the logarithmic relationship between the positive signal probability (digital count) and concentration of three narcotic and psychotropic drugs. Figure 8 (b) ~ Figure 8 (d) are log-log diagrams showing the relationship between the probability of positive signals and concentration of three narcotic and psychotropic drugs (i.e., Heroin, Cocaine, and Etomidate).

[0081] exist Figure 8 In (a), based on the standard of a positive signal "1", from 0.01 ng / mL to 100 ng / mL, the percentage of dSERS counts of positive signals (the ratio of the number of positive signals to the total sample volume) showed a monotonic change with the logarithmic concentration of the narcotic and psychotropic drug molecules. Each measurement was repeated three times to generate an error bar. Etomidate exhibited a lower detection range compared to Heronin and Cocaine, possibly due to its stronger affinity for AgNPs, smaller molecular weight (approximately 244.3 g / mol) compared to Heronin (approximately 369.4 g / mol) and Cocaine (approximately 303.4 g / mol), larger molar mass at the same concentration, and its smaller size facilitates entry into the "hot spot" regions between silver nanoparticles, enhancing the electromagnetic coupling effect.

[0082] According to classical Gibbs adsorption-binding thermodynamics, the relationship between the probability and concentration of a positive signal is expected to conform to the Freundlich equation, exhibiting a linear relationship on a log-log scale. Figure 8 (b) ~ Figure 8(d) When plotted on a log-log scale, the concentration dependence of the percentage of positive dSERS counts for the three narcotic and psychotropic drugs can be expressed using Rd. 2 The linear relationship of 0.975, 0.969, and 0.996 provides a good description and is consistent with the low analyte concentration adsorption event phenomenon in classical Gibbs thermodynamics. The repeatability of this monotonic correspondence allows for the conversion of measured percentages into actual concentrations. For higher concentrations outside this range, stepwise dilution of the sample is necessary, which precisely addresses this issue. Figure 5 (a) Nonlinear problems in quantification at medium to high concentrations.

[0083] II. Application of Simulated Crime Scenes In practical drug control work, it has been found that drug users tend to mix and abuse multiple drugs and psychotropic substances to enhance the pleasure of use, which makes drug detection difficult. Based on this, this invention addresses the problem of mixed abuse of psychotropic substances and digital quantification in complex matrices, specifically as follows: Figure 9 As shown, where, Figure 9 (a) Schematic diagram of adding Heroin and Etomidate to alcoholic beverages; Figure 9 (b) is a diagram of the SERS signal; Figure 9 (c) SERS signals after background removal at different concentrations (Ⅰ); Figure 9 (d) are digital quantitative heatmaps (II~III) after background removal at different concentrations.

[0084] This invention simulates samples seized by law enforcement agencies, adding heron and ethyl acetate to alcoholic beverages to examine the practical application of this technique. Specifically, as follows... Figure 9 As shown in (a). Figure 9 In (b), I~III represent single SERS signals and mixed SERS signals of two different targets. According to... Figure 9 The results in (b) show that the concentration of Heronin in the mixed signal is 5 times higher than that of Etomidate. However, due to the stronger affinity of Etomidate for AgNPs, the overall peak shape after superposition is closer to that of Etomidate, as shown in the figure below. Figure 9 As shown in Ⅲ in (b), this phenomenon can easily lead people to misjudge it as a single target object of Etomidate. Figure 9 (b) Ⅳ is the SERS signal of the wine mixed with Heroin and Etomidate, which caused the target signal to overlap and the target signal to be difficult to separate from the matrix background.

[0085] To address the aforementioned challenges, this invention involves serially diluting a beverage containing heroin and ethyl acetate, and then performing dSERS analysis following the steps described in Part I. As the concentration of the target analyte decreases, the SERS signal changes from a predominantly mixed signal to a combination of mixed and single signals, as shown below. Figure 9 (a) and the proportion of mixed signals further decreases, with single signals becoming the dominant signal, as shown in the following cases. Figure 9 (d) Statistically, a single signal is most likely generated by a single molecule, indicating that the PDMS@Ag proposed in this invention may achieve single-molecule detection capability. This single-molecule detection capability can distinguish between two narcotic and psychotropic drugs that are abused in combination, solving the problem of misidentification as a single component of ethyl ester at high concentrations. Calculations show that... Figure 9 (c) II~III and Figure 9 In (d), the probabilities of positive signals II to III are 40.75%, 41.75%, 11.00%, and 9.75%, respectively, compared with... Figure 8 The quantitative relationship is consistent with that in the present invention, further verifying the accuracy and universality of the method.

[0086] III. Conclusion To address the challenge of false negatives caused by complex factors in drug and psychotropic substance testing, this invention focuses on heroin, cocaine, and etomidate, which are heavily abused in society. It prepares a flexible PDMS@Ag SERS substrate suitable for multi-scenario detection and, for the first time, introduces a digital surface-enhanced Raman spectroscopy (dSERS) strategy, achieving ultrasensitive detection of 0.1 ng / mL heroin, cocaine, and 0.01 ng / mL etomidate, while also solving the nonlinearity problem in quantification at high concentrations. Furthermore, PDMS@Ag demonstrates statistically significant single-molecule detection potential in the combined detection of heroin and etomidate abuse, successfully overcoming the challenges of target signal overlap and difficulty in separating the target signal from the matrix background.

[0087] The flexible SERS substrate of this invention can meet the needs of rapid detection of drugs and psychotropic substances in various complex environments. The dSERS strategy can effectively avoid false negatives caused by the traditional average SERS intensity quantification method, which fails to capture positive signals due to insufficient sampling or is masked by the background after averaging. The combination of the two is particularly suitable for the detection of drugs and psychotropic substances. However, it is worth noting that dSERS-related research involves the collection, processing, and analysis of a large amount of data, resulting in a huge workload. At the same time, most existing dSERS studies use a single feature window as the basis for judging positive and negative results, which may increase the probability of false positives caused by the background and inevitably encounter the problem of overlapping feature windows of multiple targets. Selecting multiple feature windows as the basis for judgment can solve these problems, but this will inevitably further increase the workload. Therefore, the selection of feature windows should be comprehensively considered according to actual needs, and it is also necessary to introduce technologies such as machine learning to assist.

[0088] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for preparing a flexible SERS substrate, characterized in that, Specifically, the following steps are included: Step S1: Based on Citrate-Ag colloid, prepare PVP-coated AgNPs; Step S2: Using PDMS as a flexible substrate, AgNPs sol is transferred to the PDMS surface to form a PDMS@Citrate-Ag flexible SERS substrate.

2. The method for preparing a SERS substrate according to claim 1, characterized in that, In step S1, the Citrate-Ag colloid is prepared by heating AgNO3 solution to boiling, adding sodium citrate solution dropwise, and boiling again to obtain Citrate-Ag colloid.

3. The method for preparing a SERS substrate according to claim 1 or 2, characterized in that, In step S1, the preparation method of PVP-coated AgNPs is as follows: Citrate-Ag colloid is centrifuged, washed, and then redispersed in PVP ethanol solution to obtain PVP-coated AgNPs.

4. The method for preparing a SERS substrate according to claim 3, characterized in that, In step S2, dichloromethane is mixed with PVP-coated AgNPs, then n-hexane is added. After removing the upper layer of n-hexane, the stickier side of the PDMS film is brought into contact with the monolayer AgNPs, and the AgNPs sol is transferred to the PDMS surface.

5. The method for preparing a SERS substrate according to claim 1, 2, or 4, characterized in that, In step S2, after forming the PDMS@Citrate-Ag flexible SERS substrate, it is washed sequentially with ultrapure water and anhydrous ethanol, dried, and stored in a dry container.

6. A flexible SERS substrate obtained by the preparation method according to any one of claims 1 to 5.

7. A method for detecting narcotic and psychotropic drugs based on a flexible SERS substrate, characterized in that, Specifically, the following steps are included: Step S1: The narcotic drug to be tested is brought into contact with a flexible SERS substrate, and SERS measurement is performed using a Raman spectrometer; wherein, the flexible SERS substrate is a flexible SERS substrate obtained by the preparation method of any one of claims 1 to 5 or a flexible SERS substrate as described in claim 6; Step S2: Introduce the dSERS strategy to perform digital quantitative analysis of the target narcotic and psychotropic drugs.

8. The method for detecting narcotic and psychotropic drugs based on a flexible SERS substrate according to claim 7, characterized in that, The steps of the dSERS strategy include: (a) Mix the narcotic and psychotropic drug to be tested with Hya-Ag sol evenly, incubate with a flexible SERS substrate, and then dry; (b) Perform large-area SERS sampling within the designated area to collect multiple spectral data; (c) Set an intensity threshold, convert the spectral data into digital "0" and "1" signals, and perform quantitative analysis by statistically analyzing the probability of the occurrence of "1" signals.

9. The method for detecting narcotic and psychotropic drugs based on a flexible SERS substrate according to claim 8, characterized in that, In step (a), the preparation method of Hya-Ag sol is as follows: AgNO3 is added to a mixture of Hya·HCl and NaOH, and after stirring, Hya-Ag is obtained.

10. The method for detecting narcotic and psychotropic drugs based on a flexible SERS substrate according to any one of claims 7 to 9, characterized in that, The narcotic drugs include one or more of heroin, cocaine, or etomidate.

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