A method for detecting synthetic cannabinoids based on CB2R functionalized gold electrodes

Abstract

The application discloses a kind of based on CB2R functionalized gold electrode synthetic cannabinoid detection method.The application constructs a kind of high specificity, high stability biological sensitive interface by sequentially modifying SH-PEG-NTA, CuSO4 and CB2R on the surface of gold electrode.The functionalized electrode can utilize the specific binding of CB2R and synthetic cannabinoid in solution, realize the sensitive detection of synthetic cannabinoid by detecting the change of physical or electrochemical property of electrode interface before and after binding.The method has the advantages of good selectivity, strong anti-interference ability, etc.The prepared functionalized electrode can be used as a sensitive element, and provides an innovative technical solution for developing portable synthetic cannabinoid detection equipment.

Description

Technical Field

[0001] This invention relates to the field of biosensing technology, and in particular to a method for detecting synthetic cannabinoids based on CB2R functionalized gold electrodes. Background Technology

[0002] Synthetic cannabinoids are a class of artificially synthesized novel psychoactive substances with similar pharmacological and physiological effects to Δ9-tetrahydrocannabinol (THC). These substances primarily produce hallucinogenic effects by acting on endocannabinoid receptors CB1R and CB2R in the human body. Compared to natural cannabis, they exhibit similar or even higher receptor affinity and potency, and their structures can be modified rapidly and are diverse. Unlike the relatively stable chemical structures of traditional drugs, synthetic cannabinoids can achieve rapid product iteration through the rapid replacement of specific functional groups. Specifically, the chemical structure of synthetic cannabinoids includes four key pharmacophores: a core skeleton, a linker group, a head, and a tail. Illegal organizations can modularly replace, modify, or rearrange these functional groups—such as introducing fluorine atoms, changing carbon chain length, or replacing the cyclic core—potentially creating new synthetic cannabinoids while maintaining or even enhancing receptor affinity. Studies have shown that the rates of poisoning and hospitalization caused by synthetic cannabinoids are higher than those caused by natural cannabis. Abuse of synthetic cannabinoids can lead to severe cardiovascular toxicity, acute kidney injury, mental confusion, and even death, becoming a serious public health problem and social security threat worldwide.

[0003] Currently, the detection of synthetic cannabinoids typically employs capillary electrophoresis, gas chromatography-mass spectrometry (GC-MS), and liquid chromatography-mass spectrometry (LC-MS). While these methods offer high accuracy and analysis speed, they often rely on expensive, large-scale instruments, complex sample pretreatment processes, and specialized operators, resulting in long detection cycles and failing to meet the needs of rapid on-site screening and real-time monitoring. In contrast, biosensor technology based on the principle of bioaffinity utilizes the specific binding of biorecognition elements to target molecules for detection. With its advantages of high specificity, fast response speed, ease of operation, and ease of integration, it shows great promise for application in the field of synthetic cannabinoid detection.

[0004] Receptor protein-based biosensors, as a class of highly sensitive analytical devices, rely on the efficient construction of biorecognition elements on the sensor surface. In existing technologies, researchers typically use membrane simulation systems such as nanodisks and nanovesicles, or cell surface display techniques, to immobilize receptors on the sensor surface for detection. However, these traditional construction methods often struggle to precisely control the spatial orientation of receptor proteins at the interface, resulting in a disordered spatial arrangement of receptor molecules. Recognition sites are thus masked or obstructed due to random distribution, limiting the sensor's ability to capture trace targets. To achieve the detection of lower concentrations of biomarkers, it is necessary to develop an electrode modification method that enables the directional immobilization of receptors, guiding them to assemble in an orderly manner in a specific posture. This ensures that recognition sites are fully exposed to the detection environment, fundamentally solving the problem of masked active sites and significantly improving the sensor's detection sensitivity and signal stability. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by proposing a method for detecting synthetic cannabinoids based on CB2R functionalized gold electrodes.

[0006] The objective of this invention is achieved through the following technical solution: a method for detecting synthetic cannabinoids based on a CB2R functionalized gold electrode, comprising:

[0007] To activate the gold electrode, SH-PEG-NTA was used to modify it to form a self-assembled monolayer; after rinsing with buffer, CuSO4 solution was used for Cu... 2+ Chelation, making Cu 2+ The CB2R was fully chelated with the NTA group at the end of SH-PEG-NTA. After rinsing with buffer, the CB2R was oriented and immobilized onto the gold electrode surface using a CB2R solution with His-tag.

[0008] Synthetic cannabinoids were detected using a solidified CB2R functionalized gold electrode.

[0009] Furthermore, the activation of the gold electrode includes: washing the surface of the gold electrode sequentially with NaOH and HCl solutions, immersing it in piranha solution for 1 minute to activate the surface, rinsing it with ultrapure water, and then drying it.

[0010] Furthermore, the process of using SH-PEG-NTA modification to form a self-assembled monolayer includes: adding 100 μL of 1 mg / mL SH-PEG-NTA solution to the pretreated gold electrode surface, incubating for 30 minutes at room temperature and in the dark, and rinsing with PBS buffer to remove physically adsorbed molecules.

[0011] Furthermore, the CuSO4 solution is used for Cu... 2+Chelation involves adding 100 μL of a 10 mg / mL CuSO4 solution to the modified electrode surface and incubating at room temperature for 10 minutes to allow Cu to chelate. 2+ The Cu groups at the ends of SH-PEG-NTA undergo a chelation reaction, followed by rinsing with PBS buffer to remove unchelated Cu. 2+ .

[0012] Furthermore, the method of directionally immobilizing CB2R onto the gold electrode surface using a His-tag-containing CB2R solution includes: adding 100 μL of a 1 μg / mL His-tag-containing CB2R solution to the chelated electrode surface and incubating at room temperature for 30 minutes; and rinsing thoroughly with PBS buffer to remove unbound CB2R.

[0013] Furthermore, the detection of synthetic cannabinoids using the cured CB2R functionalized gold electrode includes:

[0014] The test liquid was dropped onto the surface of the gold electrode and incubated at room temperature for 30 minutes. After rinsing with PBS buffer, the changes in the electrochemical signal of the electrode were detected. The biomolecular complex formed on the electrode surface increased the interfacial steric hindrance, which hindered the electron transfer of the redox probe. The higher the concentration of synthetic cannabinoids, the lower the corresponding electrode peak current and the greater the redox peak potential difference.

[0015] Furthermore, the SH-PEG-NTA solution is SH-PEG-NTA molecules dissolved in PBS buffer, and the CB2R solution is CB2R recombinant protein with His-Tag dissolved in PBS buffer.

[0016] Furthermore, the method also includes characterizing the modification of the gold electrode at different stages using cyclic voltammetry to ensure that the modification at each stage is complete; this includes employing a three-electrode system, with the gold electrode as the working electrode, the platinum wire electrode as the counter electrode, and the saturated calomel electrode as the reference electrode, at 2 mM The probe molecule solution is the electrolyte. Cyclic voltammetry is performed using an electrochemical workstation. The electrodes described above are connected to the electrochemical workstation, and the cyclic voltammetry test program is started.

[0017] The beneficial effects of this invention are:

[0018] This invention proposes for the first time a gold electrode modification method based on CB2R combined with metal chelation technology. The introduced SH-PEG-NTA can form a stable self-assembled monolayer on the gold electrode surface via gold-sulfur bonds. The antifouling properties of the PEG segments effectively reduce the non-specific adsorption of biomolecules. The terminal NTA... The metal chelation bridging strategy enables the directional and orderly immobilization of CB2R on the gold electrode surface, ensuring full exposure of the active sites of CB2R and improving its recognition efficiency for targets. The metal chelation effect is robust and highly specific, and the constructed biosensitive interface is uniform and stable, guaranteeing high reproducibility and reliability of the detection results. Directly utilizing the high affinity and specificity between synthetic cannabinoids and their natural target CB2R for recognition fundamentally avoids the potential cross-reaction of antibodies in traditional immunoassays, enabling accurate detection of synthetic cannabinoids even in complex biological matrices (such as saliva and urine).

[0019] This method for constructing functionalized electrodes is versatile. By simply replacing different types of His-Tag receptor proteins, it can be adapted to the detection of various abused substances or disease biomarkers. It provides a core platform technology for the development of serialized and modular portable biosensors. Furthermore, this functionalized electrode can be easily integrated into portable electrochemical or surface acoustic wave devices as a core sensing element, with a wide range of applications and promising prospects. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the modification method for CB2R functionalized gold electrodes according to the present invention.

[0021] Figure 2 This is a schematic diagram of the chelation connection after the gold electrode surface of the present invention is sequentially modified with SH-PEG-NTA, CuSO4 and CB2R.

[0022] Figure 3 This is a cyclic voltammetry characterization curve of the gold electrode surface of the present invention, which is sequentially modified with SH-PEG-NTA, CuSO4, and CB2R, and the synthetic cannabinoids are detected.

[0023] Figure 4 This is a cyclic voltammetric characterization curve of different concentrations of synthetic cannabinoids after the gold electrode surface of the present invention was sequentially modified with SH-PEG-NTA, CuSO4, and CB2R. Detailed Implementation

[0024] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0025] To better understand the technical solution of this application, the embodiments of this application will be described in detail below with reference to the accompanying drawings.

[0026] It should be understood that the described embodiments are merely some, not all, of the embodiments in this application. All other embodiments obtained by those skilled in the art based on the embodiments in this application without inventive effort are within the scope of protection of this application.

[0027] Example 1:

[0028] A method for detecting synthetic cannabinoids based on CB2R functionalized gold electrodes, combined with Figure 1 and Figure 2 The explanation includes:

[0029] Figure 1 This outlines the overall modification process for CB2R-based functionalized gold electrodes. Figure 2 Cu 2+ A schematic diagram of the chelation process.

[0030] The method for modifying the CB2R corresponding to the synthetic cannabinoid to be tested includes:

[0031] (1) Pretreatment of gold electrode: The gold electrode was soaked in 1 M NaOH solution for 30 minutes and 1 M HCl solution for 5 minutes. Then, the gold electrode was soaked in a freshly prepared piranha solution with concentrated H2SO4 and 30% H2O2 in a volume ratio of 3:1 for 1 minute to activate the surface of the gold electrode. After rinsing with a large amount of ultrapure water, it was dried for later use.

[0032] (2) SH-PEG-NTA modification: 100 μL of 1 mg / mL SH-PEG-NTA solution was added to the pretreated gold electrode surface and incubated at room temperature in the dark for 30 minutes to promote the subsequent targeted immobilization of the receptor. A self-assembled SH-PEG-NTA monolayer was formed on the gold electrode surface through gold-sulfur bonding. The electrode was thoroughly rinsed with PBS buffer to remove physically adsorbed molecules.

[0033] (3) Cu 2+ Chelation: 100 μL of 10 mg / mL CuSO4 solution was added to the electrode surface obtained in step (2), and incubated at room temperature for 10 minutes to allow Cu to chelate. 2+ The Cu groups at the ends of SH-PEG-NTA undergo a complete chelation reaction. Rinse thoroughly with PBS buffer to remove unchelated Cu. 2+ ,like Figure 2 As shown;

[0034] (4) CB2R directional immobilization: 100 μL of a 1 μg / mL His-tag CB2R solution was added to the electrode surface obtained in step (3), and incubated at room temperature for 30 minutes. The electrode was thoroughly rinsed with PBS buffer to remove unbound CB2R. The His-tag was then used to fix Cu. 2+ The coordination between them orients and fixes CB2R onto the surface of the gold electrode in an orderly manner, forming a CB2R-functionalized gold electrode, such as... Figure 2 As shown.

[0035] (5) Detection of synthetic cannabinoids: Using the CB2R-functionalized gold electrode as the sensing interface, 100 μg / mL of EDMB-PINACA was added to the surface of the gold electrode and incubated at room temperature for 30 minutes. The electrode was then thoroughly rinsed with PBS buffer to remove unbound EDMB-PINACA. CB2R specifically binds to EDMB-PINACA. The biomolecular complex formed on the electrode surface increases interfacial steric hindrance, hindering electron transfer of the redox probe. By detecting the electrochemical changes caused by this binding event, the detection of synthetic cannabinoids was achieved, such as... Figure 3 As shown.

[0036] The SH-PEG-NTA solution is SH-PEG-NTA molecules dissolved in PBS buffer.

[0037] The CB2R solution is a recombinant CB2R protein with His-Tag dissolved in PBS buffer.

[0038] The synthetic cannabinoids are synthetic cannabinoid molecules dissolved in methanol solution, and then diluted with PBS buffer to the corresponding concentration. The synthetic cannabinoids include, but are not limited to, EDMB-PINACA, ADB-4en-PINACA, 5F-CUMYL-PINACA, and 5F-ADB-PINACA.

[0039] Example 2:

[0040] Characterization of gold electrode modification using cyclic voltammetry

[0041] A three-electrode system was adopted, with a gold electrode as the working electrode, a platinum wire electrode as the counter electrode, and a saturated calomel electrode as the reference electrode. (2 mM) The probe molecule solution was used as the electrolyte, and cyclic voltammetry was performed using an electrochemical workstation. The electrode was connected to the electrochemical workstation, and the cyclic voltammetry test program was started. Specific experimental parameters were set as follows: the scan potential window was set to -0.4 V to 1.0 V, the scan rate was set to 100 mV / s, the settling time was 2 s, and the sampling interval was 1 mV. The test was conducted at room temperature. After the cyclic voltammetry curve stabilized, the data were recorded and saved for subsequent analysis. Each modification in Example 1 was characterized sequentially using cyclic voltammetry, and the results are as follows: Figure 3 As shown.

[0042] like Figure 3 As shown, in the In the detection system using a redox probe, the bare gold electrode exhibits a pair of clear and symmetrical redox peaks, with the highest peak current and a small redox potential difference, indicating that the bare gold electrode possesses good conductivity and a fast electron transport rate. When the gold electrode surface is modified with SH-PEG-NTA, the SH-PEG-NTA molecules self-assemble through gold-sulfur bonds to form a dense insulating organic monolayer, hindering the diffusion of probe molecules to the electrode surface. This leads to a significant decrease in the redox peak current and an increase in the peak potential difference. Subsequently, after incubation with CuSO4 solution, Cu... 2+ It undergoes a specific chelation reaction with the surface NTA groups, forming a metal-organic complex interface. However, due to the Cu ion... 2+ Its conductivity causes the peak current to increase rather than decrease, and the redox peak potential difference to decrease. At this point, the current value and potential difference fall between those of a bare gold electrode and a gold electrode modified with SH-PEG-NTA. This occurs when His-tag reacts with Cu... 2+ After the CB2R protein was directionally immobilized on the electrode surface through coordination, the insulating properties of the biomolecule created a significant steric hindrance effect on the electrode surface, greatly hindering the electron transfer process. This resulted in a further decrease in the redox peak current and a flattening of the curve. Finally, the electrode modified with SH-PEG-NTA, CuSO4, and CB2R was used to detect the synthetic cannabinoid EDMB-PINACA. Here, CB2R specifically binds to EDMB-PINACA, causing further changes in the peak current and the redox peak potential difference.

[0043] In summary, as the modification steps proceed step by step, the peak current and the redox peak potential difference on the electrode surface show corresponding changes, confirming that each functional layer has been successfully and stably constructed on the electrode surface, thus proving the feasibility of the modification method described in this invention.

[0044] Example 3:

[0045] Characterization of CB2R functionalized gold electrode for detecting different concentrations of synthetic cannabinoids using cyclic voltammetry

[0046] The CB2R functionalized gold electrode obtained in Example 1 was characterized using cyclic voltammetry at concentrations of 10 μg / mL, 100 μg / mL, and 200 μg / mL of EDMB-PINACA, as described in Example 2. The results are as follows: Figure 4 As shown.

[0047] like Figure 4As shown, the CB2R functionalized gold electrode obtained in Example 1 exhibited different changes in response to different concentrations of EDMB-PINACA. The lowest concentration of EDMB-PINACA (10 μg / mL) showed the highest peak current and the smallest redox peak potential difference. The highest concentration of EDMB-PINACA (200 μg / mL) showed the lowest peak current and the largest redox peak potential difference. The highest concentration of EDMB-PINACA (100 μg / mL) fell between the two, with the peak current and redox peak potential difference also falling between the two.

[0048] In summary, the CB2R functionalized gold electrode obtained in Example 1 can distinguish between different concentrations of synthetic cannabinoids, demonstrating the feasibility of this invention for practical detection. Other embodiments of this application will readily conceive of by those skilled in the art upon consideration of the specification and practice of the disclosure herein. This application is intended to cover any variations, uses, or adaptations of this application that follow the general principles of this application and include common knowledge or customary techniques in the art not disclosed herein. The specification and examples are to be considered exemplary only, and the true scope and spirit of this application are indicated by the claims.

[0049] It should be understood that the foregoing general description and the following detailed description are exemplary and explanatory only, and are not intended to limit this application. This application is not limited to the precise structures described above and shown in the accompanying drawings, and various modifications and changes can be made without departing from its scope. The scope of this application is limited only by the appended claims.

Claims

1. A method for detecting synthetic cannabinoids based on a CB2R functionalized gold electrode, characterized in that, include: The gold electrode was activated, and SH-PEG-NTA modification was used to form a self-assembled monolayer. After rinsing with buffer solution, CuSO4 solution was used for Cu... 2+ Chelation, making Cu 2+ The CB2R was fully chelated with the NTA group at the end of SH-PEG-NTA. After rinsing with buffer, the CB2R was oriented and immobilized onto the gold electrode surface using a CB2R solution with His-tag. Synthetic cannabinoids were detected using a solidified CB2R functionalized gold electrode.

2. The method for detecting synthetic cannabinoids based on a CB2R functionalized gold electrode according to claim 1, characterized in that, The activation of the gold electrode includes: washing the surface of the gold electrode sequentially with NaOH and HCl solutions, then immersing it in piranha solution for 1 minute to activate the surface, rinsing it with ultrapure water, and then drying it.

3. The method for detecting synthetic cannabinoids based on a CB2R functionalized gold electrode according to claim 1, characterized in that, The process of using SH-PEG-NTA to modify and form a self-assembled monolayer includes: adding 100 μL of 1 mg / mL SH-PEG-NTA solution to the pretreated gold electrode surface, incubating for 30 minutes at room temperature and in the dark, and rinsing with PBS buffer to remove physically adsorbed molecules.

4. The method for detecting synthetic cannabinoids based on a CB2R functionalized gold electrode according to claim 1, characterized in that, The CuSO4 solution was used for Cu 2+ Chelation involves adding 100 μL of a 10 mg / mL CuSO4 solution to the modified electrode surface and incubating at room temperature for 10 minutes to allow Cu to chelate. 2+ The Cu groups at the ends of SH-PEG-NTA undergo a chelation reaction, followed by rinsing with PBS buffer to remove unchelated Cu. 2+ .

5. The method for detecting synthetic cannabinoids based on a CB2R functionalized gold electrode according to claim 1, characterized in that, The method of directionally immobilizing CB2R onto the gold electrode surface using a His-tag-containing CB2R solution includes: adding 100 μL of a 1 μg / mL His-tag-containing CB2R solution to the chelated electrode surface and incubating at room temperature for 30 minutes; and rinsing thoroughly with PBS buffer to remove unbound CB2R.

6. The method for detecting synthetic cannabinoids based on a CB2R functionalized gold electrode according to claim 1, characterized in that, The detection of synthetic cannabinoids using the cured CB2R functionalized gold electrode includes: The test liquid was dropped onto the surface of the gold electrode and incubated at room temperature for 30 minutes. After rinsing with PBS buffer, the changes in the electrochemical signal of the electrode were detected. The biomolecular complex formed on the electrode surface increased the interfacial steric hindrance, which hindered the electron transfer of the redox probe. The higher the concentration of synthetic cannabinoids, the lower the corresponding electrode peak current and the greater the redox peak potential difference.

7. The method for detecting synthetic cannabinoids based on a CB2R functionalized gold electrode according to claim 1, characterized in that, The SH-PEG-NTA solution is SH-PEG-NTA molecules dissolved in PBS buffer, and the CB2R solution is CB2R recombinant protein with His-Tag dissolved in PBS buffer.

8. The method for detecting synthetic cannabinoids based on a CB2R functionalized gold electrode according to claim 1, characterized in that, The method also includes characterizing the modification of the gold electrode at different stages using cyclic voltammetry to ensure that the modification at each stage is completed; this includes using a three-electrode system with a gold electrode as the working electrode, a platinum wire electrode as the counter electrode, and a saturated calomel electrode as the reference electrode, at 2 mM The probe molecule solution is the electrolyte. Cyclic voltammetry is performed using an electrochemical workstation. The electrodes described above are connected to the electrochemical workstation, and the cyclic voltammetry test program is started.

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