Electrochemiluminescence aptamer sensor for dual-mode detection of 4-CEC using electrochemiluminescence and colorimetry, its preparation method and application

Abstract

This invention belongs to the field of electrochemiluminescence detection, and relates to an electrochemiluminescence aptamer sensor for detecting synthetic cathinone 4-CEC, its preparation method, and its application. The sensor can perform dual-mode detection of 4-CEC through electrochemiluminescence and colorimetry. The electrochemiluminescence aptamer sensor is formed by loading the aptamer aptamer onto the surface of conductive glass FTO modified with the composite material PCN-224 / SIOPCs; SIOPCs are SiO2 inverse opal photonic crystals synthesized using PS microspheres as templates and tetraethyl orthosilicate as raw material. The electrochemiluminescence detection method uses a traditional three-electrode system consisting of an aptamer / PCN-224 / SIOPCs / FTO modified electrode, an Ag / AgCl electrode, and a platinum electrode. Colorimetric detection is calculated based on the ultraviolet absorption peak of Rhodamine B. The aptamer sensor in this invention exhibits high sensitivity, good selectivity, and a wide linear range for 4-CEC detection.

Description

Technical Field

[0001] This invention belongs to the field of electrochemiluminescence detection, and relates to an electrochemiluminescence aptamer sensor for dual-mode detection of synthesized cathinone 4-CEC using both electrochemiluminescence and colorimetry, as well as its preparation method and application. The sensor can perform dual-mode detection of 4-CEC through electrochemiluminescence and colorimetry. Background Technology

[0002] Synthetic cathinones (SCs) are a class of artificially synthesized drugs developed based on the cathinone structure. They belong to the category of new psychoactive substances (NPS) and, when acting on the human body, produce effects similar to amphetamine-type stimulants. A report by the United Nations Office on Drugs and Crime (UNODC) indicates that over 1,000 NPSs have been discovered, with synthetic cathinones accounting for approximately 20%, making them the second largest category of NPSs after synthetic cannabinoids. Because of their white crystalline appearance, synthetic cathinones are often illegally sold under the label of "bath salts." Their excessively strong stimulant effects can cause abusers to experience symptoms such as mania, hallucinations, and palpitations; in severe cases, they can lead to organ failure and even death. As the problem of abuse of these "designer drugs" becomes increasingly apparent, drug enforcement efforts in various countries face even greater challenges.

[0003] Currently, the main methods for detecting synthetic cathinone include chromatography-mass spectrometry (C-MS) and fluorescence spectroscopy (FL). However, these methods are limited by instruments and testing conditions. Therefore, simple, rapid, and highly sensitive detection methods have become a focus of attention. In recent years, electrochemiluminescence (ECL) has gained increasing importance in the field of trace detection due to its advantages such as low background interference, high sensitivity, and wide detection range. Currently, there are few reports on the use of a three-electrode system for the detection of synthetic cathinone 4-CEC using electrochemiluminescence methods.

[0004] Electrochemiluminescence (ECL) plays a crucial role in trace analysis due to its low background signal, high sensitivity, and wide detection range. "Signal-off" and "signal-on" are the main detection modes for ECL, but single-signal detection is highly susceptible to experimental conditions, leading to false positives or false negatives. Currently, multi-mode detection has been successfully developed for self-calibration of detection results, such as electrochemical-colorimetric, photoelectrochemical-electrochemical, and electrochemiluminescence-colorimetric methods. However, reports on three-electrode systems for detecting 4-CEC, as well as electrochemiluminescence and colorimetric methods, are still scarce.

[0005] Nucleic acid aptamers (aptamers) are single-stranded DNA or RNA molecules that possess the ability to bind to specific targets, exhibiting very high affinity and selectivity. Utilizing the differences in binding affinity between aptamers and other substances allows for analyte substitution; combining aptamers with ECLs is also a current focus in the field of bioanalytical detection. Summary of the Invention

[0006] To address the shortcomings of existing technologies in detecting synthetic cathinone 4-CEC, this invention provides an electrochemiluminescence aptamer sensor for dual-mode detection of synthetic cathinone 4-CEC using both electrochemiluminescence and colorimetry, along with its preparation method and application. The sensor enables dual-mode detection of 4-CEC through both electrochemiluminescence and colorimetry.

[0007] This invention utilizes electrostatic bonding to sequentially immobilize SIOPCs, PCN-224, and the aptor apt on the surface of conductive glass FTO, obtaining an apt / PCN-224 / SIOPCs / FTO modified electrode, which significantly improves the sensitivity and stability of electrochemiluminescence (ECL). The luminescent material PCN-224 is a metal-organic framework synthesized with Zr as the central metal ion and methyl-2-tetra(4-carboxyphenyl)porphyrin (TCPP) and benzoic acid as organic ligands. SIOPCs are SiO2 inverse opal photonic crystals synthesized using PS microspheres as templates and tetraethyl orthosilicate as raw material. SIOPCs effectively immobilize PCN-224 using their unique inverse opal cavity structure, achieving ECL signal stability. Furthermore, the photonic bandgap of the photonic crystal reflects light of the same wavelength as PCN-224, enhancing the ECL signal. The aptor apt is loaded onto the surface of the PCN-224 / SIOPCs / FTO modified electrode to fabricate an electrochemiluminescence aptor sensor. After adding the analyte to synthesize cathinone 4-CEC, the ECL signal value of the electrochemiluminescence aptamer sensor showed significant quenching, enabling the detection of 4-CEC. The addition of other interfering agents revealed that the aptamer sensor exhibited good specificity. Furthermore, by utilizing the different binding forces of aptamers to SRB and 4-CEC, colorimetric detection of 4-CEC was successfully achieved through the substitution of SRB and 4-CEC.

[0008] An electrochemiluminescence aptamer sensor for electrochemiluminescence-colorimetric dual-mode detection of the synthesis of cathinone 4-CEC is disclosed. The electrochemiluminescence aptamer sensor is formed by loading an aptamer aptamer onto the surface of a composite material PCN-224 / SIOPCs / FTO modified electrode. PCN-224 is a metal-organic framework synthesized with Zr as the central metal ion and TCPP and benzoic acid as organic ligands. SIOPCs are SiO2 inverse opal photonic crystals synthesized using PS microspheres as templates and tetraethyl orthosilicate as raw material. The aptamer aptamer is an aptamer containing the 3' base sequence 5'-GGCACTTACGACCTTAAGTGGGGTTCGGGTGGAGTTTATGGGGTCGTAAG.

[0009] Furthermore, the preparation method of the electrochemiluminescence aptamer sensor for the dual-mode electrochemiluminescence-colorimetric detection of synthesized cathinone 4-CEC includes the following steps:

[0010] (1) Preparation of PS template: The pretreated hydrophilic FTO was placed vertically in an ethanol suspension containing PS photonic crystals and placed in a constant temperature oven at 60°C for about 3 days without vibration. After the suspension was completely dried, the PS template was heated at 80°C for 1 hour to enhance compact packing. The particle size of the PS photonic crystals was 300 nm and the mass fraction of the ethanol solvent was 75%.

[0011] Furthermore, the mass concentration of the PS photonic crystal in the ethanol suspension is 0.1–0.5 wt%; preferably 0.3 wt%.

[0012] Furthermore, the pretreatment steps for the hydrophilic FTO electrode include: cutting FTO glass into 4cm×1cm thin slices, ultrasonically cleaning them sequentially with water, ethanol, and acetone to remove surface grease and impurities, and drying them at room temperature. The cleaned FTO is then immersed in a dilute alkaline H₂O₂ solution for hydrophilic treatment, followed by washing and drying to obtain the hydrophilic FTO electrode.

[0013] Furthermore, the specific requirements for the basic H2O2 dilute solution are: H2O2 mass fraction of 3wt%, pH = 12; and the hydrophilic treatment temperature condition is heating at 80℃ for 1 hour.

[0014] (2) Preparation of SIOPCS: First, silica sol was dropped onto the PS template. Then, the resulting PS template / silica composite material was left to stand in the air to allow it to fully wet, and then excess silica sol was removed. After that, it was naturally dried and then heated at 90°C for 1 hour. Next, the obtained sample was calcined at 500°C for 2 hours to etch the PS template. After being thoroughly washed and dried with deionized water, a SIOPCs / FTO modified electrode with a three-dimensional ordered macroporous structure was obtained.

[0015] Furthermore, the main components of the silica sol include: 0.1M HCl, tetraethyl orthosilicate (TEOS), and anhydrous ethanol, with a mass ratio of m:m:m = 1:1.5:1;

[0016] Furthermore, to ensure that the silica sol can fully wet the PS microspheres, the silica sol is applied in excess. The excess silica sol is removed. The preferred amount of silica sol applied is 60-100 μL, and the excess silica sol is removed by rotation at 500 rpm.

[0017] (3) Preparation of PCN-224: First, centrifugal tetra(4-carboxyphenyl)porphyrin (TCPP), ZrCl4, and benzoic acid were dissolved in DMF, and then reacted in a high-pressure reactor at 115-125℃ for 23-25 ​​h. After natural cooling, the mixture was centrifuged and washed three times each with DMF and acetone. Finally, the resulting precipitate was freeze-dried for several hours to form a good crystalline morphology. The resulting dark red powder was named PCN-224; the mass ratio of TCPP, ZrCl4, and benzoic acid was 20:60:1.

[0018] (4) PCN-224 was dispersed in DMF and ultrasonicated to make it uniformly dispersed to obtain PCN-224 dispersion; PCN-224 dispersion was drop-coated onto the surface of the prepared SIOPCs / FTO modified electrode and air-dried to obtain PCN-224 / SIOPCs / FTO electrode; apt was modified on the surface of PCN-224 / SIOPCs / FTO and incubated at room temperature to obtain electrochemiluminescence aptamer sensor apt / PCN-224 / SIOPCs / FTO.

[0019] Furthermore, the concentration of the PCN-224 dispersion is 0.5–2 mg / mL, preferably 1 mg / mL, and the modification amount is 40 μL / cm³. 2 .

[0020] Further, the modification method of aptamer: 30 μL of Tris-HCl buffer solution with an apt concentration of 3 μmol / L was drop-coated onto the surface of PCN-224 / SIOPCs / FTO and incubated for 6-8 h.

[0021] This invention also provides a method for detecting synthetic cathinone based on an electrochemiluminescence aptamer sensor, comprising the following steps:

[0022] Electrochemiluminescence detection: The three-electrode system is formed by using the electrochemiluminescence aptamer sensor (apt / PCN-224 / SIOPCs / FTO) as the working electrode, Ag / AgCl as the reference electrode, and a platinum wire electrode as the counter electrode. 4-CEC in the sample is fixed to the surface of the sensor. The detection of 4-CEC is achieved by detecting the change in the luminescence signal before and after binding of the sample.

[0023] In the electrochemiluminescence detection: using a PBS buffer solution containing K2S2O8 as the electrolyte, within the electrochemical window range of -1.8 to 0V, a photomultiplier tube voltage of 800V and a scan rate of 0.1V / s were used for cyclic voltammetry scanning, and the luminescence intensity-time curve was recorded. The difference in luminescence intensity before and after binding to 4-CEC by the electrochemiluminescence sensor was established, and the concentration of synthesized cathinone 4-CEC in the sample was calculated according to the linear regression equation. The PBS buffer solution containing K2S2O8 was prepared by using a 0.1mol / L PBS buffer solution with a pH of 7.4 to prepare a PBS buffer solution containing 0.05mol / L K2S2O8.

[0024] The colorimetric detection method uses the electrochemiluminescence aptamer sensor (apt / PCN-224 / SIOPCs / FTO) as the capture unit, as described above. First, sulfonylrhodamine B (SRB) is bound to the sample. Then, the sample is immersed in the test sample solution to allow 4-CEC to undergo a displacement reaction with SRB. The detection of 4-CEC is achieved by detecting the change in UV-Vis absorbance before and after the reaction. The UV detection wavelength is 200-800 nm.

[0025] Furthermore, the sample binding reaction time in electrochemiluminescence was 15 min, and the displacement reaction time in colorimetric testing was 30 min.

[0026] Further, the specific steps are as follows:

[0027] Step 1, Preparation of PBS buffer solution containing K2S2O8:

[0028] Prepare a PBS buffer solution containing 0.05 mol / L K2S2O8 using a 0.1 mol / L PBS buffer solution with a pH of 7.4;

[0029] Step 2, Preparation of cathinone 4-CEC standard solution and SRB solution of different concentrations:

[0030] A stock solution for synthesizing cathinone 4-CEC was prepared, and then serially diluted with Tris-HCl buffer to obtain a series of 4-CEC standard solutions with different concentrations, ranging from 1.0 × 10⁻⁶. -14 ~1.0×10 -5 g / L; prepare an SRB solution with a concentration of 1 mg / mL;

[0031] Step 3, Plotting the standard curve:

[0032] In the electrochemiluminescence detection: the above-mentioned aptamer sensor was immersed in the synthetic cathinone 4-CEC standard solutions of different concentrations prepared according to step 2 and reacted for the same time (15 min) to allow the electrochemiluminescence aptamer sensor to bind to the synthetic cathinone 4-CEC, resulting in 4-CEC / apt / PCN-224 / SIOPCs / FTO. Then, it was used as the working electrode, Ag / AgCl as the reference electrode, and a platinum electrode as the counter electrode to form a three-electrode system. The PBS buffer solution containing K2S2O8 from step 1 was used as the electrolyte. Cyclic voltammetry was performed within the electrochemical window range of -1.8 to 0 V, with a photomultiplier tube voltage of 800 V and a scan rate of 0.1 V / s. The luminescence intensity-time curve was recorded, and a linear relationship was established between the difference in luminescence intensity (ΔECL) before and after the electrochemiluminescence sensor binds to 4-CEC and the logarithm of the 4-CEC concentration in the 4-CEC standard solution. The corresponding linear regression equation was obtained.

[0033] In the colorimetric detection, the above-mentioned aptamer sensor was immersed in an SRB aqueous solution with a concentration of 1 mg / mL prepared according to step 2 and reacted for 30 min to allow the aptamer to fully bind with SRB to obtain SRB / apt / PCN-224 / SIOPCs / FTO. Then, the modified electrode was immersed in 4-CEC standard solutions of different concentrations to allow 4-CEC and SRB to fully replace each other. The replaced solution was subjected to UV-vis testing in the wavelength range of 200-800 nm and the absorbance was recorded. A linear relationship between absorbance and 4-CEC concentration was established, and the corresponding linear regression equation was obtained.

[0034] Step 4, Detection of synthetic cathinone 4-CEC in the sample

[0035] The sample was first pretreated and spiked for recovery testing. In step 3, the electrochemiluminescence aptamer sensor was immersed in the pretreated e-cigarette liquid to allow the aptamer sensor to bind to the synthesized cathinone 4-CEC. Then, it was used as the working electrode, and the luminescence intensity was detected by the method in step 3. The concentration of the synthesized cathinone 4-CEC in the sample was calculated according to the linear regression equation.

[0036] Furthermore, the pretreatment process for electronic cigarettes involves first centrifuging the electronic cigarette sample to remove insoluble impurities, then further filtering it using a vacuum filtration method to obtain a supernatant, which is then stored at 0-4℃ for later use.

[0037] The beneficial effects of this invention are:

[0038] This invention designs an electrochemiluminescence aptamer sensor based on a composite material of SiO2 inverse opal photonic crystals (SIOPCs) and zirconium-based porphyrin metal-organic frameworks (PCN-224). The two materials are combined through electrostatic interactions during synthesis, resulting in highly efficient and stable electrochemiluminescence performance. SIOPCs utilize a unique inverse opal cavity structure to effectively immobilize PCN-224, achieving ECL signal stability. Furthermore, the photonic bandgap of the photonic crystal reflects light of the same wavelength as the luminescent material PCN-224, enhancing the ECL signal. The aptamer (apt) is loaded onto the surface of a PCN-224 / SIOPCs / FTO-modified electrode to prepare the electrochemiluminescence aptamer sensor. After adding the analyte cathinone 4-CEC, the ECL signal value of the electrochemiluminescence aptamer sensor exhibits significant quenching, enabling the detection of 4-CEC. The addition of other interfering agents revealed that this aptamer sensor possesses good specificity. In addition, by utilizing the different binding forces of aptamers to SRB and 4-CEC, colorimetric detection of 4-CEC was successfully achieved through the displacement of SRB and 4-CEC. This invention offers simple operation, good selectivity, high sensitivity, and a wide detection range for detecting 4-CEC, and is of great significance for promoting the application of aptamer sensors in practical detection. Attached Figure Description

[0039] Figure 1 This is a simplified flowchart of the preparation of the electrochemiluminescence aptamer sensor and the detection of 4-CEC in this invention;

[0040] Figure 2 The image shows the ECL response of the electrochemiluminescence aptamer sensor constructed in Example 1 after binding with different concentrations of 4-CEC, where the concentrations of 4-CEC from a to g are: (a) 1.0 × 10⁻⁶ g. -12 g / L; (b) 1.0×10 -11 g / L; (c) 1.0×10 -10 g / L; (d) 1.0×10 -9 g / L; (e) 1.0 × 10 -8 g / L; (f) 1.0×10 -7 g / L; (g) 1.0×10 -6 g / L;

[0041] Figure 3 The standard curve of the difference in luminescence intensity (ΔECL) before and after the addition of 4-CEC in Example 1 versus the logarithm of the 4-CEC concentration;

[0042] Figure 4The UV-Vis absorption curves for Example 1 with different concentrations of SRB are shown below. (ag represents the concentration of 4-CEC during aptamer replacement (10, 100, 200, 400, 600, 800, 1000 ng / mL)).

[0043] Figure 5 This illustrates the linear relationship between absorbance values ​​and 4-CEC concentration in Example 1.

[0044] Figure 6 The images shown are SEM images of the PS microspheres (A), SIOPCs (B), PCN-224 (C), and PCN-224 / SIOPCs (D) prepared in Example 1.

[0045] Figure 7 ECL-time images of PCN-224 / FTO(a) and PCN-224 / SIOPCs / FTO(b). Detailed Implementation

[0046] This invention is not limited to the specific embodiments listed below. Those skilled in the art can implement this invention using various other specific embodiments based on the content disclosed herein. Any modifications or alterations made to the design structure and concept of this invention fall within the protection scope of this invention. It should be noted that, unless otherwise specified, the embodiments and features described in this invention can be combined with each other.

[0047] The present invention will be further described in detail with reference to the embodiments: In the following embodiments, apt is an aptamer containing the 5'-AGATGGGGGTTGAGGCTAAGCCGA-3' base sequence, which is fixed on the surface of the PCN-224 / SIOPCs / FTO modified electrode through electrostatic binding.

[0048] The preparation methods for 4-CEC standard solutions of different concentrations in the following examples are as follows: A 4-CEC stock solution was prepared, and then serially diluted with Tris-HCl buffer solution to obtain a series of 4-CEC standard solutions of different concentrations. In this example, the concentrations of 4-CEC in the 4-CEC standard solutions are (a) 1.0 × 10⁻⁶. -12 g / L; (b) 1.0×10 -11 g / L; (c) 1.0×10 -10 g / L; (d) 1.0×10 -9 g / L; (e) 1.0 × 10 -8 g / L; (f) 1.0×10 -7 g / L; (g) 1.0×10 -6 g / L;

[0049] Example 1:

[0050] (I) Assembling apt / PCN-224 / SIOPCs / FTO sensors

[0051] (1) Preparation of SiOPCs and PCN-224 materials:

[0052] FTO glass was cut into 4cm × 1cm slices, and then surface grease and impurities were removed sequentially using water, ethanol, and acetone via ultrasonication. Hydrophilic treatment was performed by immersing the FTO in a dilute basic H₂O₂ solution (3wt%, pH 12) and heating at 80°C for 1 hour. After washing and drying, each hydrophilic FTO was vertically placed in a vial containing a suspension of 0.3wt% PS photonic crystal (300nm) (purchased from Shanghai Yiyuan Biotechnology Co., Ltd.) in 75% ethanol (immersion area 1cm × 1cm). All vials were placed in a constant temperature oven at 60°C for approximately 3 days without vibration. After the suspension was completely dry, the template was heated at 80°C for 1 hour to enhance compact packing.

[0053] A mixed solution (m:m:m = 1:1.5:1) containing 0.1M HCl, tetraethyl orthosilicate (TEOS), and ethanol was used as a silica sol precursor. 100 μL of silica sol was first dropped onto an opal template. The silica sol readily penetrated into the voids between PS microspheres via capillary force. The resulting template / silica composite was then allowed to stand in air for 1 min, followed by rotation at 500 rpm to remove excess silica sol. The sample was then air-dried for 12 h and heated at 90 °C for 1 h. Next, the sample was calcined in a muffle furnace at 500 °C for 2 h to etch away the PS microspheres. After thorough washing and drying with deionized water, a SiOPCs substrate with a three-dimensional ordered macroporous structure was obtained.

[0054] 20 mg TCPP, 60 mg ZrCl4, and 1 mg benzoic acid were accurately weighed and dissolved in 6 mL DMF. The mixture was then reacted in a high-pressure reactor for 24 h (120 °C). After natural cooling, the mixture was centrifuged and washed three times each with DMF and acetone. Finally, the resulting precipitate was freeze-dried for several hours to form a well-defined crystalline morphology. The resulting dark red powder was named PCN-224. A portion of the prepared PCN-224 was ultrasonically dispersed in a DMF solution to form a PCN-224 dispersion with a concentration of 1 mg / mL.

[0055] (2) Preparation of an electrochemiluminescence aptamer sensor for detecting 4-CEC

[0056] The PCN-224 dispersion was drop-coated onto the prepared SIOPCs / FTO surface and allowed to air dry naturally. Then, 30 μL of Tris-HCl buffer solution containing the aptin was added and allowed to air dry naturally for 8 hours to obtain the apt / PCN-224 / SIOPCs / FTO electrochemiluminescence aptin sensor.

[0057] The modification amount of the PCN-224 dispersion was 40 μL, and the concentration of the aptamer was 3 μM.

[0058] (II) Method for detecting 4-CEC based on electrochemiluminescence aptamer sensor

[0059] (1) Plotting the standard curve

[0060] In electrochemiluminescence detection: the above-mentioned aptamer sensor was immersed in a series of synthetic cathinone 4-CEC standard solutions of different concentrations and reacted for 15 min, so that the electrochemiluminescence aptamer sensor bound the synthetic cathinone 4-CEC to obtain 4-CEC / apt / PCN-224 / SIOPCs / FTO. Then, it was used as the working electrode, Ag / AgCl as the reference electrode, and a platinum electrode as the counter electrode to form a three-electrode system. A 0.1 mol / L PBS buffer solution with a pH of 7.4 was used to prepare a solution containing 0. Using 0.5 mol / L K₂S₂O₈ PBS buffer solution as the electrolyte, cyclic voltammetry was performed within an electrochemical window of -1.8 to 0 V, with a photomultiplier tube voltage of 800 V and a scan rate of 0.1 V / s. The luminescence intensity-time curve was recorded, and a linear relationship was established between the luminescence intensity difference (ΔECL) before and after 4-CEC binding by the electrochemiluminescence sensor and the logarithm of the 4-CEC concentration in the 4-CEC standard solution. The corresponding linear regression equation was obtained: ΔECL = 19116.65 + 1507.23lg C (g / L), with a detection range of 1.0 × 10⁻⁶ g / L. -12 ~1.0×10 -6 g / L, detection limit is 2.6×10 -13 g / L;

[0061] In colorimetric detection, the aptamer sensor was immersed in an SRB aqueous solution with a concentration of 1 mg / mL and reacted for 30 min to allow the aptamer to fully bind with SRB, resulting in SRB / apt / PCN-224 / SIOPCs / FTO. The modified electrode was then immersed in a series of 4-CEC standard solutions of different concentrations for 30 min to allow for complete displacement of 4-CEC and SRB. The displaced solutions were then subjected to UV-vis testing in the wavelength range of 200-800 nm, and the absorbance was recorded. A linear relationship between absorbance and 4-CEC concentration was established, yielding the corresponding linear regression equation: Abs. = 0.065 + 0.00034c (ng / L). The detection range was 10–1000 ng / L, and the detection limit was 6.5 ng / L.

[0062] (2) Sample detection

[0063] A sample of e-liquid from a certain brand of electronic cigarettes was first centrifuged to remove insoluble impurities. Then, it was further filtered using vacuum filtration to obtain the supernatant, which was stored at 0-4℃ for later use. Electrochemiluminescence testing was performed according to the method shown in the standard curve plotting diagram, and the concentration of 4-CEC in the sample was calculated using the obtained linear regression equation. The results are listed in Table 1.

[0064] This embodiment uses PCN-224 / SIOPCs / FTO as the substrate material (the morphology of each material is as follows) Figure 6 This sensor utilizes the electrostatic interaction between PCN-224 and SIOPCs for stable bonding. SIOPCs, on the one hand, effectively immobilize PCN-224 using their unique inverse opal cavity structure, achieving ECL signal stability; on the other hand, they enhance the ECL signal by reflecting light of the same wavelength as the emitting PCN-224 through the photonic bandgap of the photonic crystal. This sensor significantly improves the electrochemiluminescence intensity of individual materials, exhibits good conductivity, stability, and selectivity.

[0065] Example 2:

[0066] The operation method in this embodiment is the same as in Example 1, except that the concentration of the PCN-224 dispersion is 0.5 mg / mL. The linear regression equation obtained by ECL detection is ΔECL=4817.75+1170.17lg C(g / L), and the detection range is 1.0×10 -12 ~1.0×10 -7 g / L, detection limit is 1.6×10 g / L. -12 g / L; the linear regression equation obtained by colorimetric detection is the same as that in Example 1, namely Abs.=0.00045c-0.01410(ng / L), the detection range is 100~1000ng / L, and the detection limit is 20ng / L.

[0067] Example 3:

[0068] The operation method in this embodiment is the same as in Example 1, except that the concentration of the PCN-224 dispersion is 2 mg / mL. The linear regression equation obtained by ECL detection is ΔECL=4312.71+1114.32lg C(g / L), and the detection range is 1.0×10 -12 ~1.0×10 -6 g / L, detection limit is 8.3×10 g / L. -13 g / L; the linear regression equation obtained by colorimetric detection is the same as that in Example 1, namely Abs.=0.000357c+0.01605(ng / L), the detection range is 100~1000ng / L, and the detection limit is 18ng / L.

[0069] Embodiment 4:

[0070] The operation method in this embodiment is the same as in Embodiment 1, except that the concentration of the PS photonic crystal is 0.1wt%. The linear regression equation obtained by ECL detection is ΔECL=3026.04+996.26lg C(g / L), and the detection range is 1.0×10 -11 ~1.0×10 -6 g / L, detection limit is 2.7×10 g / L. -12 g / L; the linear regression equation obtained by colorimetric detection is the same as that in Example 1, namely Abs.=0.00046c-0.00761(ng / L), the detection range is 100~1000ng / L, and the detection limit is 18ng / L.

[0071] Example 5:

[0072] The operation method in this embodiment is the same as in Embodiment 1, except that the concentration of the PS photonic crystal is 0.5 wt%. The linear regression equation obtained by ECL detection is ΔECL=1621.60+810.00lg C(g / L), and the detection range is 1.0×10 -10 ~1.0×10 -6 g / L, detection limit is 1.3×10 g / L. -11 g / L; the linear regression equation obtained by colorimetric detection is the same as that in Example 1, namely Abs.=0.00060c-0.00186(ng / L), the detection range is 10~1000ng / L, and the detection limit is 19ng / L.

[0073] Comparative Example 1:

[0074] (1) Fabrication of apt / SIOPCs / FTO sensor

[0075] 30 μL of 3 μM apt solution was transferred using a microsyringe and drop-coated onto the surface of the prepared SIOPCs / FTO modified electrode to obtain an apt / SIOPCs / FTO chemically modified electrode. After air drying, it was used as a sensing element for electrochemiluminescence testing. (The amount of modification and sample concentration for monomer testing remained the same as in the examples.)

[0076] (2) Plotting the standard curve

[0077] Using the apt / SIOPCs / FTO sensor obtained in step (1) as the sensing element, the above aptamer sensor was immersed in a series of synthetic cathinone 4-CEC standard solutions of different concentrations and reacted for 15 min, so that the electrochemiluminescent aptamer sensor combined with the synthetic cathinone 4-CEC to obtain 4-CEC / apt / PCN-224 / SIOPCs / FTO. Then, it was used as the working electrode, Ag / AgCl as the reference electrode, and a platinum electrode as the counter electrode to form a three-electrode system. Using PBS buffer solution containing K2S2O8 as the electrolyte, cyclic voltammetry was performed within an electrochemical window of -1.8 to 0 V, with a photomultiplier tube voltage of 800 V and a scan rate of 0.1 V / s. The luminescence intensity-time curve was recorded, and a linear relationship was established between the difference in luminescence intensity (ΔECL) before and after 4-CEC binding by the electrochemiluminescence sensor and the logarithm of the 4-CEC concentration in the 4-CEC standard solution. The corresponding linear regression equation was obtained: ΔECL = 193.32 + 47.31lg C (g / L), with a detection range of 1.0 × 10⁻⁶ g / L. -12 ~1.0×10 -7 g / L, detection limit is 6.7×10 g / L. -13 g / L;

[0078] In colorimetric detection, the aptamer sensor was immersed in an SRB aqueous solution with a concentration of 1 mg / mL and reacted for 30 min to allow the aptamer to fully bind with SRB, resulting in SRB / apt / PCN-224 / SIOPCs / FTO. The modified electrode was then immersed in a series of 4-CEC standard solutions of different concentrations for 30 min to allow for complete displacement of 4-CEC and SRB. The displaced solutions were then subjected to UV-vis testing in the wavelength range of 200-800 nm, and the absorbance was recorded. A linear relationship between absorbance and 4-CEC concentration was established, yielding the corresponding linear regression equation: Abs. = 0.1072 + 0.01412c (ng / L). The detection range was 10–1000 ng / L, and the detection limit was 6.5 ng / L.

[0079] (3) Sample detection

[0080] A sample of e-liquid from a certain brand of electronic cigarette was first centrifuged to remove insoluble impurities. Then, it was further filtered using a vacuum filtration method to obtain the supernatant, which was then stored at 0-4℃ for later use. Electrochemiluminescence testing was performed as shown in step (2), and the concentration of 4-CEC in the sample was calculated using the obtained linear regression equation. The results are listed in Table 1.

[0081] Comparative Example 2:

[0082] (1) Fabrication of apt / PCN-224 / FTO sensor

[0083] 40 μL of a 1 mg / mL DMF dispersion of PCN-224 was transferred using a microsyringe and drop-coated onto the surface of a pretreated FTO electrode (pretreatment method as in Example 1) to obtain a PCN-224 / FTO modified electrode. After air-drying, 30 μL of a 3 μM apt solution was drop-coated onto the surface of the PCN-224 / FTO modified electrode and air-dried to obtain an apt / PCN-224 / FTO sensor, which was used as the sensing element for electrochemiluminescence assays. (The amount of modification and sample concentration for monomer testing remained a single variable as in the examples.)

[0084] (2) Plotting the standard curve

[0085] Using the apt / SIOPCs / FTO sensor obtained in step (1) as the sensing element, the above aptamer sensor was immersed in a series of synthetic cathinone 4-CEC standard solutions of different concentrations and reacted for 15 min, so that the electrochemiluminescent aptamer sensor combined with the synthetic cathinone 4-CEC to obtain 4-CEC / apt / PCN-224 / SIOPCs / FTO. Then, it was used as the working electrode, Ag / AgCl as the reference electrode, and a platinum electrode as the counter electrode to form a three-electrode system. Using PBS buffer solution containing K2S2O8 as the electrolyte, cyclic voltammetry was performed within an electrochemical window of -1.8 to 0 V, with a photomultiplier tube voltage of 800 V and a scan rate of 0.1 V / s. The luminescence intensity-time curve was recorded, and a linear relationship was established between the difference in luminescence intensity (ΔECL) before and after 4-CEC binding by the electrochemiluminescence sensor and the logarithm of the 4-CEC concentration in the 4-CEC standard solution. The corresponding linear regression equation was obtained: ΔECL = 3995.43 + 1012.64lg C (g / L), with a detection range of 1.0 × 10⁻⁶. -12 ~1.0×10 -6 g / L, detection limit is 6.3×10 - 13 g / L;

[0086] In the colorimetric detection, the aptamer sensor was immersed in an SRB aqueous solution with a concentration of 1 mg / mL and reacted for 30 min to allow the aptamer to fully bind with SRB, resulting in SRB / apt / PCN-224 / SIOPCs / FTO. Then, the modified electrode was immersed in a series of 4-CEC standard solutions of different concentrations for 30 min to allow 4-CEC and SRB to fully exchange. The solutions after exchange were subjected to UV-vis testing in the wavelength range of 200-800 nm, and the absorbance was recorded. A linear relationship between absorbance and 4-CEC concentration was established, yielding the corresponding linear regression equation: Abs. = 0.1082 + 0.01421c (ng / L). The detection range was 10–1000 ng / L, and the detection limit was 7.2 ng / L.

[0087] (3) Sample detection

[0088] A sample of e-liquid from a certain brand of electronic cigarette was first centrifuged to remove insoluble impurities. Then, it was further filtered using a vacuum filtration method to obtain the supernatant, which was then stored at 0-4℃ for later use. Electrochemiluminescence testing was performed as shown in step (2), and the concentration of 4-CEC in the sample was calculated using the obtained linear regression equation. The results are listed in Table 1.

[0089] Comparative Example 3:

[0090] (1) Fabrication of PCN-224 / SIOPCs / FTO sensor

[0091] 40 μL of a 1 mg / mL DMF dispersion of PCN-224 was transferred using a microsyringe and drop-coated onto the prepared SIOPCs / FTO surface (pretreatment method as in Example 1) to obtain a PCN-224 / SIOPCs / FTO modified electrode. After air drying, the sensing element for electrochemiluminescence testing was obtained. (The amount of modification and sample concentration for monomer testing remained the same as in the example.)

[0092] (2) Plotting the standard curve

[0093] Using the apt / SIOPCs / FTO sensor obtained in step (1) as the sensing element, the above aptamer sensor was immersed in a series of synthetic cathinone 4-CEC standard solutions of different concentrations and reacted for 15 min, so that the electrochemiluminescent aptamer sensor combined with the synthetic cathinone 4-CEC to obtain 4-CEC / apt / PCN-224 / SIOPCs / FTO. Then, it was used as the working electrode, Ag / AgCl as the reference electrode, and a platinum electrode as the counter electrode to form a three-electrode system. Using PBS buffer solution containing K2S2O8 as the electrolyte, cyclic voltammetry was performed within an electrochemical window of -1.8 to 0 V, with a photomultiplier tube voltage of 800 V and a scan rate of 0.1 V / s. The luminescence intensity-time curve was recorded, and a linear relationship was established between the luminescence intensity difference (ΔECL) before and after 4-CEC binding by the electrochemiluminescence sensor and the logarithm of the 4-CEC concentration in the 4-CEC standard solution. The corresponding linear regression equation was obtained: ΔECL = 362.86 + 108.57lg C (g / L), with a detection range of 1.0 × 10⁻⁶ g / L. -9 ~1.0×10 -3 g / L, detection limit is 8.0×10 g / L. -10 g / L, the linear correlation coefficient is 0.66, which is almost non-linear;

[0094] In colorimetric detection, the aptamer sensor was immersed in an SRB aqueous solution with a concentration of 1 mg / mL and reacted for 30 min to allow the aptamer to fully bind with SRB, resulting in SRB / apt / PCN-224 / SIOPCs / FTO. Then, the modified electrode was immersed in a series of 4-CEC standard solutions of different concentrations for 30 min to allow 4-CEC and SRB to fully exchange. The solutions after exchange were subjected to UV-vis testing in the wavelength range of 200-800 nm, and the absorbance was recorded. A linear relationship between absorbance and 4-CEC concentration was established, yielding the corresponding linear regression equation. However, due to the lack of an aptamer, a corresponding linear regression equation could not be obtained.

[0095] (3) Sample detection

[0096] A sample of e-liquid from a certain brand of electronic cigarette was first centrifuged to remove insoluble impurities. Then, it was further filtered using a vacuum filtration method to obtain the supernatant, which was then stored at 0-4℃ for later use. Electrochemiluminescence testing was performed as shown in step (2), and the concentration of 4-CEC in the sample was calculated using the obtained linear regression equation. The results are listed in Table 1.

[0097] Table 1. Measurement results of a certain electronic cigarette sample

[0098]

[0099] Remark: aThe average of three measurements

[0100] As shown in Table 1, the samples were measured in parallel three times. The spiked recovery rate detected in Example 1 was between 97% and 101%, with a relative standard deviation of less than 5%, indicating good recovery performance. The above experimental results show that 4-CEC cannot be detected when the FTO electrode is modified with any two of the apt / PCN-224 / SIOPCs composite materials alone and then assembled into a sensing element. Therefore, the sensor of the present invention can be used to detect 4-CEC in electronic cigarettes.

[0101] Based on the above verification, this invention utilizes the electrochemiluminescence quenching effect of 4-CEC on the apt / PCN-224 / SIOPCs / FTO system to construct a novel method for rapid and sensitive detection of 4-CEC. Various materials are combined through electrostatic interactions during electrode assembly, achieving efficient and stable electrochemiluminescence performance. SIOPCs, on the one hand, effectively immobilize PCN-224 using their unique inverse opal cavity structure, achieving ECL signal stability; on the other hand, they enhance the ECL signal by reflecting light of the same wavelength as the luminescent material PCN-224 using the photonic bandgap of the photonic crystal. After adding the analyte to synthesize cathinone 4-CEC, the ECL signal value of the electrochemiluminescence aptamer sensor exhibits significant quenching, and ΔECL shows a good linear relationship with the logarithm of the 4-CEC concentration, enabling the detection of 4-CEC. The introduction of the aptamer can significantly improve the specificity of the aptamer sensor. In addition, by utilizing the different binding forces of apt to SRB and 4-CEC, colorimetric detection of 4-CEC was successfully achieved through the change in UV-Vis absorbance intensity caused by the substitution of SRB and 4-CEC. The electrochemiluminescence method used in this invention not only has the advantages of high sensitivity, fast detection speed, good selectivity and wide linear range, but also has great application potential for the quantitative analysis of 4-CEC in electronic cigarettes.

[0102] The above embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various corresponding changes without departing from the scope of the invention. Therefore, all technical solutions formed by equivalent substitutions or equivalent modifications are within the protection scope of the invention.

Claims

1. An electrochemiluminescence aptamer sensor for detecting synthetic cathinone 4-CEC, characterized in that: The electrochemiluminescence aptamer sensor is formed by loading the aptamer aptamer onto the surface of conductive glass FTO modified with composite material PCN-224 / SIOPCs; PCN-224 is a metal-organic framework synthesized with Zr as the central metal ion and medium-tetra(4-carboxyphenyl)porphyrin (TCPP) and benzoic acid as organic ligands; SIOPCs is a SiO2 inverse opal photonic crystal synthesized with polystyrene microspheres as templates and tetraethyl orthosilicate as raw material; the aptamer aptamer is an aptamer containing the 5'-GGCAC TTACG ACCTT AAGTG GGGTT CGGGTGGAGT TTATG GGGTC GTAAG -3' base sequence.

2. The method for preparing the electrochemiluminescence aptamer sensor for detecting synthetic cathinone 4-CEC as described in claim 1, characterized in that: Includes the following steps: (1) Preparation of PS template: Pretreated hydrophilic FTO was placed vertically in an ethanol suspension containing PS photonic crystals, kept at 60 °C until the suspension was completely dry, and heated at 80 °C for 1 h to obtain a tightly packed PS template. (2) Preparation of SIOPCS: Silica sol was dropped onto a PS template. After it was fully wetted, excess silica sol was removed. After air drying, it was heated at 90 °C for 1 h and calcined at 500 °C for 2 h to etch the PS template. After washing and drying, a SIOPCs / FTO modified electrode with a three-dimensional ordered macroporous structure was obtained. The main components of the silica sol included 0.1 mol / L HCl, tetraethyl orthosilicate and anhydrous ethanol in a mass ratio of 1:1.5:

1. (3) Preparation of PCN-224: TCPP, ZrCl4 and benzoic acid were dissolved in DMF and reacted at 115~125 °C for 23~25 h. After natural cooling, the mixture was centrifuged, washed and freeze-dried to obtain dark red powder PCN-224. (4) Disperse PCN-224 in DMF and sonicate it to make it uniformly to obtain PCN-224 dispersion; drop the PCN-224 dispersion onto the prepared SIOPCs / FTO modified electrode surface and air dry it to obtain PCN-224 / SIOPCs / FTO electrode; modify the PCN-224 / SIOPCs / FTO surface with apt and incubate it at room temperature to obtain electrochemiluminescence aptamer sensor apt / PCN-224 / SIOPCs / FTO.

3. The method for preparing the electrochemiluminescence aptamer sensor for detecting synthetic cathinone 4-CEC according to claim 2, characterized in that: The mass concentration of the PS photonic crystal in the ethanol suspension of the PS photonic crystal in step (1) is 0.1~0.5 wt%.

4. The method for preparing the electrochemiluminescence aptamer sensor for detecting synthetic cathinone 4-CEC according to claim 2, characterized in that: The pretreatment steps for the hydrophilic FTO electrode in step (1) include: ultrasonically cleaning the FTO glass with water, ethanol, and acetone in sequence to remove surface grease and impurities, and drying it at room temperature; immersing the cleaned FTO in a dilute basic H2O2 solution for hydrophilic treatment, and washing and drying to obtain the hydrophilic FTO electrode; wherein, the mass fraction of the dilute basic H2O2 solution is 3wt%, pH=12; the temperature condition for hydrophilic treatment is heating at 80 ℃ for 1 h.

5. The method for preparing an electrochemiluminescence aptamer sensor for detecting synthetic cathinone 4-CEC according to claim 2, characterized in that: In step (4), the concentration of the PCN-224 dispersion is 0.5~2 mg / mL, and the modification amount is 40 μL / cm. 2 .

6. The method for preparing an electrochemiluminescence aptamer sensor for detecting synthetic cathinone 4-CEC according to claim 2, characterized in that: Modification method of aptamer: 30 μL of Tris-HCl buffer solution with apt concentration of 3 μmol / L was drop-coated onto the surface of PCN-224 / SIOPCs / FTO and incubated for 6-8 h.

7. A method for detecting synthetic cathinone 4-CEC, characterized in that: An electrochemiluminescence detection method was adopted, using the electrochemiluminescence aptamer sensor for detecting synthetic cathinone 4-CEC as described in claim 1 as the working electrode, Ag / AgCl as the reference electrode, and a platinum wire electrode as the counter electrode to form a three-electrode system. The detection of 4-CEC was achieved by detecting the change in the luminescence signal before and after the binding of the sample to the test sample.

8. The method for detecting synthetic cathinone 4-CEC according to claim 7, characterized in that: In the electrochemiluminescence detection: using a PBS buffer solution containing K2S2O8 as the electrolyte, cyclic voltammetry was performed within the electrochemical window range of -1.8 to 0 V, with a photomultiplier tube voltage of 800 V and a scan rate of 0.1 V / s. The luminescence intensity-time curve was recorded, and the difference in luminescence intensity before and after binding to 4-CEC was established. The concentration of synthesized cathinone 4-CEC in the sample was then calculated based on the linear regression equation. The PBS buffer solution containing K2S2O8 was prepared by using a 0.1 mol / L PBS buffer solution with a pH of 7.4 to form a PBS buffer solution containing 0.05 mol / L K2S2O8.

9. A method for detecting synthetic cathinone 4-CEC, characterized in that: The colorimetric detection method is adopted. The electrochemiluminescence aptamer sensor for detecting synthetic cathinone 4-CEC as described in claim 1 is used as the capture unit. It first binds to sulfonylrhodamine B, and then soaks in the sample solution to allow 4-CEC to undergo a displacement reaction with sulfonylrhodamine B. The detection of 4-CEC is achieved by detecting the change in UV-Vis absorbance value before and after the reaction.

10. The method for detecting synthetic cathinone 4-CEC according to claim 9, characterized in that: The ultraviolet detection wavelength is 200-800 nm.

Images