A method for preparing a nanoplasma metasurface biosensor and its application in detecting new psychoactive substances.

Abstract

This invention discloses a method for preparing a nanoplasmic metasurface biosensor and its application in the detection of new psychoactive substances, belonging to the field of environmental monitoring technology. The invention forms a nanoplasmic metasurface by fabricating a nanopore array and a plasmonic-active metal layer on the sensor surface. The metal nanostructure generates a strong local electromagnetic field enhancement effect under photoexcitation. Subsequently, a stable, non-specific adsorption-resistant biological interface is formed through modification with a carboxyl / methoxy mixed thiol-polyethylene glycol self-assembled monolayer. After activation with N-hydroxysuccinimide / 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, the surface carboxyl groups are converted into active esters, facilitating covalent coupling of specific monoclonal antibodies against the target substance. Finally, antibody coupling allows the sensor to capture the target analyte through antigen-antibody specific binding, causing a change in the interfacial refractive index, which in turn leads to a shift in the plasmonic resonance peak, achieving optical signal conversion.

Description

Technical Field

[0001] This invention belongs to the field of environmental monitoring technology, and in particular relates to a method for preparing a nanoplasma metasurface biosensor and its application in the detection of new psychoactive substances. Background Technology

[0002] The advancement of global industrialization and urbanization has inadvertently facilitated the proliferation of unregulated new psychoactive substances (NPS). These compounds exist in aquatic systems at trace concentrations ranging from nanograms to micrograms per liter, persisting due to their structural stability and resistance to conventional water treatment processes. NPS primarily originate from urban wastewater, certain industrial emissions, and recreational waste, entering water bodies through sewage discharge, surface runoff, and groundwater seepage. Their lipophilic and pseudo-persistent properties raise significant concerns about bioaccumulation and potential long-term human health effects—even at low exposure levels, they can lead to neurotoxicity and metabolic disorders. Therefore, developing effective strategies for monitoring emerging aquatic pollutants has become an urgent task for safeguarding public health and environmental safety.

[0003] Existing methods for detecting new psychoactive substances in water primarily rely on centralized laboratory instruments, such as liquid chromatography-tandem mass spectrometry (LC-MS / MS), which offers high sensitivity and specificity for a wide range of analytes. Ancillary techniques such as gas chromatography, advanced spectroscopy, and immunoassay provide additional pathways for identification and quantification. However, these traditional methods typically require complex sample pretreatment, sophisticated equipment, and highly trained technicians, lacking the rapid responsiveness, portability, and cost-effectiveness needed for widespread deployment, thus limiting their application in actual field wastewater monitoring. This technological gap not only delays the timely identification of emerging drug threats but, more importantly, hinders the assessment and mitigation of public health risks posed by long-term, low-dose environmental exposure to these bioactive compounds. Therefore, a rapid and low-cost method for on-site environmental monitoring of new psychoactive substances is urgently needed to address these technical challenges. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention proposes a method for preparing a nanoplasma metasurface biosensor and its application in detecting new psychoactive substances.

[0005] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a method for preparing a nanoplasma metasurface biosensor, comprising the following steps: (1) A nanopore array and a plasma-active metal layer were sequentially fabricated on a substrate to obtain a nano-plasma metasurface sensor; (2) The nanoplasma metasurface sensor is incubated in a self-assembled monolayer solution, and then surface activated using a mixed solution of N-hydroxysuccinimide solution and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide solution to obtain the activated nanoplasma metasurface sensor; the self-assembled monolayer solution contains carboxyl-terminated thiol-polyethylene glycol and methoxy-terminated thiol-polyethylene glycol; (3) The activated nanoplasma metasurface sensor is coupled with a specific monoclonal antibody of the target novel psychoactive substance to obtain the nanoplasma metasurface biosensor.

[0006] Further, in step (1), the plasma-active metal layer includes a titanium adhesion layer and a gold film; the thickness of the titanium adhesion layer is 10 nm, and the thickness of the gold film is 100 nm.

[0007] Further, in step (1), the fabrication method of the nano-plasma metasurface sensor includes: spin-coating photoresist on a silicon wafer, defining a nanopore array pattern using deep ultraviolet lithography, then transferring the defined nanopore array pattern onto the silicon wafer to form a nanopore array; removing the photoresist from the silicon wafer and cleaning it, then sequentially depositing a titanium adhesion layer and a gold film on the patterned silicon wafer surface to form a plasma-active metal layer, thereby obtaining the nano-plasma metasurface sensor.

[0008] Further, in step (2), the molar ratio of carboxyl-terminated thiol-polyethylene glycol and methoxy-terminated thiol-polyethylene glycol in the self-assembled monolayer solution is 1:3; the total concentration of carboxyl-terminated thiol-polyethylene glycol and methoxy-terminated thiol-polyethylene glycol in the self-assembled monolayer solution is 10 mmol / L.

[0009] Further, in step (2), the concentration of N-hydroxysuccinimide in the mixed solution of N-hydroxysuccinimide solution and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide solution is 0.4 mol / L, and the concentration of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide solution is 0.1 mol / L.

[0010] Furthermore, in step (2), the surface activation temperature is 0~35℃ and the time is 0.5~2h.

[0011] Further, in step (3), the target novel psychoactive substance includes one or more of morphine, methamphetamine, ketamine and etomidate; the specific monoclonal antibody of the target novel psychoactive substance includes one or more of morphine monoclonal antibody, methamphetamine monoclonal antibody, ketamine monoclonal antibody and etomidate monoclonal antibody.

[0012] This invention provides a nanoplasma metasurface biosensor prepared according to the preparation method described above.

[0013] This invention provides a microfluidic chip, including the nanoplasma metasurface biosensor described in the above technical solution.

[0014] The present invention also provides an application of the nanoplasma metasurface biosensor or microfluidic chip as described above in the detection of new psychoactive substances.

[0015] Compared with the prior art, the present invention has the following advantages and technical effects: This invention creates a nanoplasma metasurface by fabricating a nanopore array and a plasmonic-active metal layer on the sensor surface. The metal nanostructure generates a strong local electromagnetic field enhancement effect under photoexcitation. Subsequently, a stable, non-specific adsorption-resistant biological interface is formed through modification with a self-assembled monolayer of carboxyl / methoxy mixed thiols and polyethylene glycol. After activation with N-hydroxysuccinimide / 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, the surface carboxyl groups are converted into active esters, facilitating covalent coupling of specific monoclonal antibodies to the target substance. Finally, antibody coupling allows the sensor to capture the target analyte through antigen-antibody specific binding, causing a change in the interfacial refractive index, which in turn leads to a shift in the plasmonic resonance peak, achieving optical signal conversion.

[0016] The sensor provided by this invention combines high sensitivity and high specificity. The nanoplasma metasurface significantly enhances the interaction between light and matter, transforming minute changes in refractive index into significant signal responses, with detection limits reaching trace levels. The self-assembled layer effectively suppresses biological contamination, improves the signal-to-noise ratio and stability, and antibody conjugation maintains its biological activity, ensuring accurate identification of target psychoactive substances. The overall method has a high degree of operational integration and is suitable for rapid, real-time detection. Attached Figure Description

[0017] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings: Figure 1 The images shown are physical images and scanning electron microscope images of the four-channel microfluidic chip prepared in Example 2, where A is a physical image and B is a scanning electron microscope image. Figure 2The specificity of different functionalized nanoplasma metasurface biosensors prepared in Example 1 to the same antigen is shown in the figures. A represents the specificity of the nanoplasma biosensor functionalized with antimorphine antibody, B represents the specificity of the nanoplasma biosensor functionalized with antimethamphetamine antibody, C represents the specificity of the nanoplasma biosensor functionalized with antiketamine antibody, and D represents the specificity of the nanoplasma biosensor functionalized with antietomidate antibody (the same applies below). Figure 3 The specificity of different functionalized nanoplasma metasurface biosensors prepared in Example 1 for different antigens; Figure 4 Real-time binding kinetics were monitored after injecting novel target psychoactive substances into the nanoplasma metasurface biosensors prepared in Example 1 with different functionalized nanoplasma metasurface biosensors. Figure 5 Calibration curves after injecting corresponding novel psychoactive substances into the nanoplasma metasurface biosensors prepared in Example 1 with different functionalized nanoplasma metasurface biosensors; Figure 6 This section assesses the robustness of the nanoplasma metasurface biosensor prepared in Example 1 under environmentally relevant interferences. A represents the stability of the new psychoactive substance detection signal within a certain temperature range (no significant change, ns); B represents the performance of the nanoplasma metasurface biosensor under different pH conditions; C represents the effect of water salinity on the detection response; and D represents the effect of cation type on the sensing signal (compared to the control). P<0.0001), E represents the effect of anion type on the sensing signal (compared to the control). P<0.0001), F represents the comparison of the recovery rate of new psychoactive substances with and without membrane filtration in the presence of suspended solids (compared to unfiltered). P<0.0001), G represents the reusability test results of the nanoplasma metasurface biosensor under a complex mixture of anions, cations, suspended solids and dissolved organic matter. Detailed Implementation

[0018] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0020] This invention provides a method for preparing a nanoplasma metasurface biosensor, comprising the following steps: (1) A nanopore array and a plasma-active metal layer were sequentially fabricated on a substrate to obtain a nano-plasma metasurface sensor; (2) The nanoplasma metasurface sensor is incubated in a self-assembled monolayer solution, and then surface activated using a mixed solution of N-hydroxysuccinimide solution and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide solution to obtain the activated nanoplasma metasurface sensor; the self-assembled monolayer solution contains carboxyl-terminated thiol-polyethylene glycol and methoxy-terminated thiol-polyethylene glycol; (3) The activated nanoplasma metasurface sensor is coupled with a specific monoclonal antibody of the target novel psychoactive substance to obtain the nanoplasma metasurface biosensor.

[0021] In a preferred embodiment, step (1) of the fabrication method of the nanoplasma metasurface sensor includes: spin-coating photoresist on a silicon wafer, defining a nanopore array pattern using deep ultraviolet lithography (DUV), and then transferring the defined nanopore array pattern onto the silicon wafer to form a nanopore array; removing the photoresist from the silicon wafer and cleaning it, and then sequentially depositing a titanium adhesion layer and a gold film on the patterned silicon wafer surface to form a plasma-active metal layer, thereby obtaining the nanoplasma metasurface sensor; the method for transferring the defined nanopore array pattern onto the silicon wafer is reactive ion etching (RIE); and the method for depositing the titanium adhesion layer and the gold film is electron beam evaporation.

[0022] In a preferred embodiment, in step (1), the pore diameter of the nanopore array is 200 nm and the period is 500 nm. The nanopore array in this invention supports long-range surface plasmon resonance and is sensitive to novel psychoactive substances captured by a plasmonic active metal layer.

[0023] In a preferred embodiment, in step (1), the plasma-active metal layer includes a titanium adhesion layer and a gold film; the thickness of the titanium adhesion layer is 5-10 nm, and the thickness of the gold film is 30-200 nm.

[0024] In a preferred embodiment, step (1), after obtaining the nanoplasma metasurface sensor, further includes cutting the sensor into 2×2mm pieces. 2 A single sensor for biosensing analysis.

[0025] In a preferred embodiment, in step (2), the molar ratio of carboxyl-terminated thiol-polyethylene glycol and methoxy-terminated thiol-polyethylene glycol in the self-assembled monolayer solution is 1:3; the total concentration of carboxyl-terminated thiol-polyethylene glycol and methoxy-terminated thiol-polyethylene glycol in the self-assembled monolayer solution is 10 mmol / L; and the solvent of the self-assembled monolayer solution is phosphate buffered saline (PBS). The carboxyl-terminated thiol-polyethylene glycol (HS-PEG-COOH) in this invention is the core modifying molecule for sensor surface functionalization. Its functions include: 1) its thiol group (-SH) can form a stable Au-S covalent bond with the sensor substrate (such as gold electrodes or gold nanomaterials), achieving molecular anchoring on the substrate surface; 2) its carboxyl group (-COOH) is an active functional group, which can covalently couple with the specific recognition element of the target analyte (such as monoclonal antibody) through an amidation reaction, constructing a specific recognition site for the sensor, which is the basis for achieving target analyte detection; 3) its hydrophilic PEG segments can form a hydration layer on the substrate surface, repelling the adsorption of non-target substances and reducing background signal; at the same time, the flexible PEG chain can regulate the distance between the recognition element and the substrate, avoiding conformational distortion of the recognition element and ensuring its binding efficiency with the target analyte. In this invention, the methoxy-terminated thiol-polyethylene glycol (HS-PEG-OCH3) co-modifies the sensor surface with HS-PEG-COOH, achieving the following effects: First, it dilutes the density of active carboxyl groups, avoiding steric hindrance caused by excessive crowding of recognition elements and optimizing the distribution of recognition sites; second, its methoxy-terminus without active functional groups can further thicken the hydration layer on the substrate surface, enhancing its resistance to non-specific adsorption; third, it can fill the modification gaps of HS-PEG-COOH, reducing direct contact between the substrate and the target system, and improving the stability and lifespan of the sensor.

[0026] If the methoxy-terminated thiol-polyethylene glycol is replaced with a short-chain alkyl thiol (such as dodecyl thiol), the lack of hydrophilic segments and active functional groups leads to a surge in non-specific adsorption on the sensor surface, making it impossible to couple the recognition element and directly resulting in a loss of detection capability. Furthermore, the substrate is easily corroded, shortening its lifespan. If the carboxyl-terminated thiol-polyethylene glycol is replaced with polypropylene glycol thiol, its weaker hydrophilicity compared to PEG reduces its resistance to non-specific adsorption, increasing the background signal. Additionally, the insufficient flexibility of the polymer chain reduces the binding efficiency of the recognition element, decreasing detection sensitivity. Using only HS-PEG-COOH as a single modification results in excessively high active carboxyl group density, leading to crowded recognition elements, reduced target binding efficiency, and poor uniformity of the hydration layer, thus decreasing the signal-to-noise ratio. Replacing the functional group end groups (e.g., replacing -COOH with -NH2) alters the surface charge distribution, causing a shift in the binding force of the recognition element, requiring re-optimization of coupling conditions. Replacing -OCH3 with -OH increases the background signal due to the weak reactivity of the hydroxyl group.

[0027] In a preferred embodiment, in step (2), the incubation temperature is room temperature and the incubation time is 2 hours.

[0028] In a preferred embodiment, step (2) further includes a washing step after the incubation is completed; the washing reagent is phosphate buffer (PBS).

[0029] In a preferred embodiment, in step (2), the concentration of N-hydroxysuccinimide in the mixed solution of N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is 0.4 mol / L, and the concentration of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is 0.1 mol / L. This invention uses a mixed solution of N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to activate the carboxyl groups on the sensor surface via a carbodiimide chemical method, in order to couple a target-specific antibody.

[0030] In a preferred embodiment, in step (2), the surface activation temperature is 0~35℃ and the time is 0.5~2h.

[0031] In a preferred embodiment, in step (3), the target novel psychoactive substance includes one or more of morphine, methamphetamine, ketamine, and etomidate; the specific monoclonal antibody of the target novel psychoactive substance includes one or more of morphine monoclonal antibody, methamphetamine monoclonal antibody, ketamine monoclonal antibody, and etomidate monoclonal antibody; and the amount of the specific monoclonal antibody of the target novel psychoactive substance is as follows.

[0032] In a preferred embodiment, in step (3), the amount of the specific monoclonal antibody against the target novel psychoactive substance is 6-20 ng / L. This invention enables the nanoplasma metasurface biosensor to specifically recognize novel psychoactive substances by coupling a specific monoclonal antibody against the target novel psychoactive substance to the surface of the activated nanoplasma metasurface sensor.

[0033] In a preferred embodiment, step (3) further includes a washing step after the coupling is completed; the washing reagent is phosphate buffer (PBS), which removes unbound antibodies by washing.

[0034] This invention provides a nanoplasma metasurface biosensor prepared according to the preparation method described above.

[0035] This invention provides a microfluidic chip, including the nanoplasma metasurface biosensor described in the above technical solution.

[0036] In a preferred embodiment, the microfluidic chip is a four-channel microfluidic chip; the fabrication method of the four-channel microfluidic chip includes: spin-coating SU-8 negative photoresist (SU8-2025, Microchem) on a silicon wafer at a speed of 2000 r / min for 30 s, then baking it sequentially at 65°C for 2 min and at 95°C for 5 min to obtain an SU-8 mold; exposing a photomask defining four parallel channel patterns to ultraviolet light to expose the SU-8 mold to ultraviolet light, followed by baking, and then developing it under stirring; the developed SU-8 mold is then exposed to trichlorosilane vapor in a desiccator for 15 min for silanization. A silanized SU-8 mold was obtained. A polydimethylsiloxane (PDMS) precursor mixture (composed of PDMS and crosslinking agent ethylenediamine, with a mass ratio of PDMS to crosslinking agent of 10:1) was cast onto the silanized SU-8 mold, degassed, and cured at 65°C for 4 hours. The cured PDMS layer was then peeled off from the SU-8 mold, and an inlet and outlet for each channel were created using a 1.1 mm biopsy punch. Finally, the PDMS layer was aligned and permanently bonded to four nanoplasma metasurface biosensors to obtain the four-channel microfluidic chip.

[0037] The present invention also provides an application of the nanoplasma metasurface biosensor or microfluidic chip as described above in the detection of new psychoactive substances.

[0038] In this embodiment of the invention, room temperature refers to "25±2℃".

[0039] Unless otherwise specified, all raw materials used in the embodiments of this invention were purchased through commercial channels.

[0040] Example 1 A method for fabricating a nanoplasma metasurface biosensor, comprising the following specific steps: (1) A layer of photoresist was spin-coated on a standard 8-inch silicon wafer. A nanopore array pattern was defined using deep ultraviolet (DUV) lithography. Then, the defined nanopore array pattern was transferred to the silicon wafer by reactive ion etching (RIE) to form a nanopore array with a hole diameter of 200 nm and a period of 500 nm. After removing the photoresist and cleaning, a titanium adhesion layer with a thickness of 10 nm was uniformly deposited on the patterned silicon wafer surface by electron beam evaporation. Subsequently, a gold film with a thickness of 100 nm was deposited to form a plasma-active metal layer. After that, the silicon wafer was cut into 2×2 mm pieces. 2 From a single sensor, multiple nanoplasma metasurface sensors are obtained; (2) The nanoplasma metasurface sensor obtained in step (1) was placed in a self-assembled monolayer solution and incubated at room temperature for 2 h. The self-assembled monolayer solution contained carboxyl-terminated thiol-polyethylene glycol and methoxy-terminated thiol-polyethylene glycol in a molar ratio of 1:3, with a total concentration of 10 mmol / L. The solvent was phosphate buffer. After incubation, the nanoplasma metasurface sensor was washed with phosphate buffer (PBS). Then, the incubated nanoplasma metasurface sensor was placed in a mixed solution of N-hydroxysuccinimide solution and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide solution and incubated at 0~35℃. Surface activation was performed, and after 0.5 h, the activated nano-plasma metasurface sensor was obtained. In the mixed solution of N-hydroxysuccinimide solution and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide solution, the concentration of N-hydroxysuccinimide was 0.4 mol / L, the concentration of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide solution was 0.1 mol / L, the solvent of N-hydroxysuccinimide solution was morpholine ethanesulfonic acid (MES) buffer, and the solvent of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide solution was morpholine ethanesulfonic acid (MES) buffer. (3) The activated nanoplasmonic metasurface sensor was coupled with specific monoclonal antibodies (morphine monoclonal antibody, etomidate monoclonal antibody, ketamine monoclonal antibody and methamphetamine monoclonal antibody) of the target novel psychoactive substances (morphine, methamphetamine, ketamine and etomidate). The amount of specific monoclonal antibody of the target novel psychoactive substances was 10 ng / L. Each antibody was coupled to the corresponding sensor. Then, the unbound antibody was thoroughly washed with phosphate buffer (PBS) to remove the unbound antibody and obtain different functionalized nanoplasmonic metasurface biosensors, namely, antimorphine antibody functionalized nanoplasmonic metasensor, antimethamphetamine antibody functionalized nanoplasmonic metasensor, antiketamine antibody functionalized nanoplasmonic metasensor and antietomidate antibody functionalized nanoplasmonic metasensor.

[0041] Example 2 A method for fabricating a four-channel microfluidic chip, comprising the following steps: SU-8 negative photoresist (SU8-2025, Microchem) was spin-coated onto a silicon wafer at 2000 rpm for 30 seconds, followed by baking at 65°C for 2 minutes and then at 95°C for 5 minutes to obtain an SU-8 mold. A photomask defining four parallel channel patterns was exposed to ultraviolet light to expose the SU-8 mold, which was then baked and developed with stirring. The developed SU-8 mold was then exposed to trichlorosilane vapor in a desiccator for 15 minutes for silanization to obtain a silanized SU-8 mold. Polydimethylsiloxane (PD) was then applied... The precursor mixture of polydimethylsiloxane (PDMS) (composed of polydimethylsiloxane and ethylenediamine in a mass ratio of 10:1) was cast onto a silanized SU-8 mold, degassed, and cured at 65°C for 4 hours. The cured PDMS layer was then peeled off from the SU-8 mold, and an inlet and outlet for each channel were created using a 1.1 mm biopsy punch. Finally, the PDMS layer was aligned and permanently bonded to four nanoplasma metasurface biosensors prepared in Example 1 to obtain the four-channel microfluidic chip.

[0042] Figure 1 The images show a physical photograph and a scanning electron microscope (SEM) image of the four-channel microfluidic chip prepared in Example 2, where A is the physical photograph and B is the SEM image. Figure 1 As can be seen from Part A, a single water sample can be simultaneously analyzed by four nanoplasma metasurface biosensors on a four-channel microfluidic chip. From... Figure 1 As can be seen from Part B, a periodic array of nanopore structures is formed on the surface of the nanoplasma metasurface biosensor. The pore diameter is 200 nm and the period is 500 nm. These periodic nanostructures support long-range surface plasmon resonance and are sensitive to novel psychoactive substances captured on the gold surface.

[0043] To systematically evaluate the detection performance of the nanoplasma biosensors, four different functionalized nanoplasma metasurface biosensors obtained in Example 1 and a non-specific antibody-functionalized metasurface sensor obtained in Comparative Example 1 were exposed to different types of novel psychoactive substances (morphine, methamphetamine, ketamine, and etomidate) for testing. The results are shown in [Figure 1]. Figure 2-5 .

[0044] Figure 2The specificity of different functionalized nanoplasma metasurface biosensors prepared in Example 1 for the same antigen is shown, where A represents the specificity of the nanoplasma biosensor functionalized with anti-morphine antibody, B represents the specificity of the nanoplasma biosensor functionalized with anti-metamphenicol antibody, C represents the specificity of the nanoplasma biosensor functionalized with anti-ketamine antibody, and D represents the specificity of the nanoplasma biosensor functionalized with anti-etomidate antibody. Figure 2 As can be seen from Part A, when using antimorphine antibody-functionalized nanoplasma biosensors to detect different types of new psychoactive substances, a significant signal response was observed only in the presence of morphine, while the response from non-target new psychoactive substances was minimal. From... Figure 2 As can be seen from the BD section, a similar trend of high specificity was also observed in cross-reactivity tests performed on other functionalized nanoplasma biosensors.

[0045] Figure 3 The different functionalized nanoplasma metasurface biosensors prepared in Example 1 demonstrate their specificity for different antigens. Specifically, A is a nanoplasma biosensor functionalized with an anti-morphine antibody, B is a nanoplasma biosensor functionalized with an anti-metamphenicol antibody, C is a nanoplasma biosensor functionalized with an anti-ketamine antibody, and D is a nanoplasma biosensor functionalized with an anti-etomidate antibody. Figure 3 As shown in Part A, when using a metasurface sensor functionalized with a non-specific antibody to analyze morphine, the induced signal is negligible. In contrast, the nanoplasma biosensor functionalized with the corresponding anti-morphine antibody exhibits a strong and statistically significant response (P < 0.0001). Figure 3 As can be seen from the BD section, the other three new psychoactive substance biomarkers all consistently exhibit antibody-dependent specificity, meaning that only nanoplasma biosensors functionalized with their corresponding antibodies can produce a significant response to the target analyte.

[0046] Figure 4 This study describes the real-time binding kinetics of different functionalized nanoplasma metasurface biosensors prepared in Example 1 after injecting corresponding novel psychoactive substances. In this biosensor, A is an anti-morphine antibody-functionalized nanoplasma biosensor, B is an anti-metamine antibody-functionalized nanoplasma biosensor, C is an anti-ketamine antibody-functionalized nanoplasma biosensor, and D is an anti-etomidate antibody-functionalized nanoplasma biosensor. The concentrations of the target novel psychoactive substances were 10 ng / L, 25 ng / L, and 50 ng / L, respectively. Figure 4 As can be seen from Part A, different morphine concentrations lead to different final wavelength shifts. Figure 4The BD portion of the study also demonstrated concentration-dependent behavior for methamphetamine (Met), ketamine (Ket), and etomidate (Eto).

[0047] Figure 5 Calibration curves were generated for the nanoplasma metasurface biosensors prepared in Example 1 after injecting corresponding novel psychoactive substances. In this model, A represents a nanoplasma biosensor functionalized with an anti-morphine antibody, B with an anti-metamine antibody, C with an anti-ketamine antibody, and D with an anti-etomidate antibody. Data are expressed as mean ± standard deviation (n=5). The dashed line represents linear fitting, indicating the dynamic detection range and sensitivity of each novel psychoactive substance. Figure 5 As shown in Part A, the target concentration range detected by the morphine-specific metasurface sensor is 10 ng / L to 100 ng / L, and the resonant wavelength shift increases proportionally with concentration. Similarly, the detection of the other three biomarkers also showed a strong concentration-dependent linear relationship (see...). Figure 5 (BD part in the text).

[0048] Figures 2-5 The results show that the nanoplasma metasurface biosensor provided by this invention achieves multiple, specific, concentration-dependent and high-sensitivity detection of target new psychoactive substances in water samples, confirming its practicality in reliable environmental monitoring.

[0049] To evaluate the adaptability of the nanoplasma metasurface biosensors in complex real-world aquatic environments, the detection performance of the four nanoplasma metasurface biosensors prepared in Example 1 for their respective target biomarkers was tested within a temperature range of 0-40℃. The results are shown in [Figure 1]. Figure 6 Part A of the report; considering the variable pH of natural water, the stability of the metasurface sensor under different pH conditions was further evaluated, and the results are shown in [see section A]. Figure 6 Part B of the report evaluated the impact of water salinity on sensor performance, and the results are shown in [see section B]. Figure 6 Part C of the sample; 5 mmol of different cations (Ca) were added to the sample to be tested. 2+ Mg 2+ Al 3 + Fe 3+ Fe 2+ K + Ca 2+ +Mg 2+ +Al 3+ +Fe 3+ +Fe 2+ +K +The effect of cation type on the sensing signal was tested, and the results are shown in [the table below]. Figure 6 Part D of the sample; 5 mmol of different anions (CO3) were added to the sample to be tested. 2- PO4 3- NO3 - SO4 2- CO3 2- +PO4 3- +NO3 - +SO4 2- The effect of cation type on the sensing signal was tested, and the results are shown in [the table below]. Figure 6 Part E of the report; considering the presence of suspended solids in environmental water, the detection performance of samples containing added particulate matter (Microspheres SiO2, CaCO3, Microspheres SiO2+CaCO3) was compared under conditions with and without a filter membrane. The results are shown in [see section E]. Figure 6 The F part; The effect of a complex mixture of anions, cations, suspended solids, and dissolved organic matter on the sensing signal was tested in the sample. The results are shown in […]. Figure 6 The G part in the text.

[0050] from Figure 6 As can be seen from Part A, all metasurface sensors maintained effective detection performance, producing consistent responses to their targets at different temperatures, confirming their reliable function under various thermal conditions. From Figure 6 As can be seen from Part B, all four nanoplasma metasurface biosensors exhibited stable detection performance under different pH conditions. From... Figure 6 As can be seen from Part C, even at different salt concentrations, the nanoplasma metasurface biosensor can reliably distinguish samples containing novel psychoactive substances from blank controls. From... Figure 6 As shown in section D, there was no significant difference (ns) in the response of the nanoplasma metasurface biosensor between samples with and without added cations. A similar trend was observed in various anion assays (see...). Figure 6 Part E of the middle section). From Figure 6 As can be seen from section F, although no significant difference was observed between filtered and unfiltered samples relative to the control without added solids, using a filter generally increases the total amount of new psychoactive substances captured. Figure 6 As can be seen from the G part, even in the presence of a complex mixture of various interfering substances, the resonant wavelength shift response of the nanoplasma metasurface biosensor to morphine remains highly consistent across multiple measurements. Figure 6 The results show that the nanoplasma metasurface biosensor prepared in this invention has high specificity and significant antifouling properties, and shows great potential for reliable detection of new psychoactive substances in actual aquatic environments.

[0051] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for fabricating a nanoplasma metasurface biosensor, characterized in that, Includes the following steps: (1) A nanopore array and a plasma-active metal layer were sequentially fabricated on a substrate to obtain a nano-plasma metasurface sensor; (2) The nanoplasma metasurface sensor is incubated in a self-assembled monolayer solution, and then surface activated using a mixed solution of N-hydroxysuccinimide solution and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide solution to obtain the activated nanoplasma metasurface sensor; the self-assembled monolayer solution contains carboxyl-terminated thiol-polyethylene glycol and methoxy-terminated thiol-polyethylene glycol; (3) The activated nanoplasma metasurface sensor is coupled with a specific monoclonal antibody of the target novel psychoactive substance to obtain the nanoplasma metasurface biosensor.

2. The method for preparing the nanoplasma metasurface biosensor according to claim 1, characterized in that, In step (1), the plasma-active metal layer includes a titanium adhesion layer and a gold film; the thickness of the titanium adhesion layer is 10 nm, and the thickness of the gold film is 100 nm.

3. The method for preparing the nanoplasma metasurface biosensor according to claim 1, characterized in that, In step (1), the fabrication method of the nanoplasma metasurface sensor includes: spin-coating photoresist on a silicon wafer, defining a nanopore array pattern using deep ultraviolet lithography, then transferring the defined nanopore array pattern onto the silicon wafer to form a nanopore array; removing the photoresist from the silicon wafer and cleaning it, then sequentially depositing a titanium adhesion layer and a gold film on the patterned silicon wafer surface to form a plasma active metal layer, thereby obtaining the nanoplasma metasurface sensor.

4. The method for preparing the nanoplasma metasurface biosensor according to claim 1, characterized in that, In step (2), the molar ratio of carboxyl-terminated thiol-polyethylene glycol and methoxy-terminated thiol-polyethylene glycol in the self-assembled monolayer solution is 1:3; the total concentration of carboxyl-terminated thiol-polyethylene glycol and methoxy-terminated thiol-polyethylene glycol in the self-assembled monolayer solution is 10 mmol / L.

5. The method for preparing the nanoplasma metasurface biosensor according to claim 1, characterized in that, In step (2), the concentration of N-hydroxysuccinimide in the mixed solution of N-hydroxysuccinimide solution and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide solution is 0.4 mol / L, and the concentration of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide solution is 0.1 mol / L.

6. The method for preparing the nanoplasma metasurface biosensor according to claim 1, characterized in that, In step (2), the surface activation temperature is 0-35℃ and the time is 0.5-2h.

7. The method for preparing the nanoplasma metasurface biosensor according to claim 1, characterized in that, In step (3), the target novel psychoactive substance includes one or more of morphine, methamphetamine, ketamine and etomidate; the specific monoclonal antibody of the target novel psychoactive substance includes one or more of morphine monoclonal antibody, methamphetamine monoclonal antibody, ketamine monoclonal antibody and etomidate monoclonal antibody.

8. A nanoplasma metasurface biosensor prepared by the preparation method according to any one of claims 1-7.

9. A microfluidic chip, characterized in that, Including the nanoplasma metasurface biosensor as described in claim 8.

10. The application of a nanoplasma metasurface biosensor as described in claim 8 or a microfluidic chip as described in claim 9 in the detection of new psychoactive substances.

Images