Dual-signal-amplification lanthanide oxide electrochemiluminescence aptamer sensor for detecting acetamiprid, and preparation method and application of dual-signal-amplification lanthanide oxide electrochemiluminescence aptamer sensor

Abstract

The invention relates to a lanthanide oxide electrochemiluminescence aptamer sensor with dual signal amplification for detecting acetamiprid as well as a preparation method and application of the lanthanide oxide electrochemiluminescence aptamer sensor. According to the sensor, terbium-doped europium oxide (Eu2O3: Tb < 3 + >) modified by gold nanoparticles (Au NPs) is used as a luminous body, and the electrochemical luminescence performance can be remarkably improved through multiple mechanisms: on one hand, the Au NPs can effectively promote generation of more sulfate free radicals; on the other hand, the surface defect passivation effect and the efficient synergistic effect of energy transfer jointly achieve effective amplification of signals. In addition, the wool ball-shaped Ag NPs is used as a plasma resonance signal amplification source, so that the ECL intensity of the sensor is further enhanced. The prepared electrochemical luminescence sensor with the dual signal amplification effect is used for detecting acetamiprid (ACE), has a wide linear range and a low detection limit, and has a wide application prospect in detection of acetamiprid residues in a water environment.

Description

TECHNICAL FIELD

[0001] The present application relates to an electrochemiluminescence aptamer sensor for detecting acetamiprid, and belongs to the field of analytical chemistry, and particularly relates to the field of electrochemiluminescence technology and electrochemical sensors. 3+ The synergistic effect of Eu2O3:Tb @Au composite luminophore and wool ball-shaped Ag NPs-cDNA plasmonic resonance amplification source realizes high-sensitivity detection of acetamiprid. The sensor has a wide application prospect in the fields of food safety, pesticide residues and environmental safety. BACKGROUND

[0002] The monitoring and prevention of pesticide residues in water bodies have always been a hot issue of concern in environmental safety. Acetamiprid (ACE) is a systemic contact insecticide and is widely used as a substitute for traditional insecticides such as organophosphates to control piercing-sucking mouthpart pests. Its frequent and extensive use inevitably leads to the accumulation of ACE in the soil and water environment, endangering human health. Therefore, it is necessary to find an efficient, sensitive, simple, convenient and low-cost method for detecting ACE. Existing methods for detecting acetamiprid include Raman spectroscopy, fluorescence and high-performance liquid chromatography. However, these analytical methods rely on relatively expensive instruments, and the process is both time-consuming and labor-intensive. Therefore, it is necessary to develop a new ACE detection strategy.

[0003] Electrochemiluminescence combines the advantages of electrochemistry and chemiluminescence, and has the characteristics of small background interference, fast response speed and high sensitivity. Traditional electrochemiluminescence materials such as Ru(bpy)3 2+ , luminol and quantum dots. However, these traditional luminophores have complex synthesis steps, greater water solubility leading to unstable ECL efficiency, or relatively low ECL emission efficiency due to their π-π stacking effect, which seriously restricts their practical application. Therefore, it is still important to develop a simple method to prepare high-performance ECL luminophores without causing quenching due to molecular aggregation.

[0004] Lanthanide metal oxides have excellent fluorescence performance, not only showing wide application potential in energy conversion, catalysis, environmental monitoring and other fields, but also continuously becoming a hot spot in the research of electrochemiluminescence (ECL) materials as a representative of lanthanide materials. Among them, the Eu 3+ of Eu2O3 can be easily replaced by another rare-earth activating ion (such as Tb 3+ ), because they have the same valence, similar electronegativity and ion ratio. When part of the Tb 3+ ions are inserted into the crystal lattice of Eu2O3, the ECL emission of Eu 3+ is enhanced, and then the ECL intensity of Eu3+ luminescent properties. However, it is still difficult to obtain high ECL signals only by the stepwise electronic energy levels of lanthanide elements and 4 f - 4 f transitions.

[0005] In the electrochemiluminescence system, the co-reactant promoter can interact with the co-reactant to promote the generation of more reactive intermediates, thereby generating a large number of excited-state luminophores and emitting a strong ECL signal. Therefore, one of the strategies for amplifying the cathodic ECL is to promote the electroreduction decomposition of K2S2O8 on the electrode surface. Among them, Au NPs have excellent performance in catalyzing the activation of K2S2O8, and Au NPs can promote K2S2O8 to generate more SO4•−, thereby amplifying the ECL signal of the system.

[0006] In addition, the plasmonic resonance effect widely exists on the surface of noble metal nanostructures, such as gold nanoparticles (Au NPs) and silver nanoparticles (Ag NPs). When these noble metal nanoparticles are at a certain distance from the luminophore, the surface plasmon resonance (SPR) effect occurs, and the luminescence intensity is greatly improved. In particular, in the surface plasmon resonance, the collective oscillation of free electrons in the metal nanostructure excited by the electromagnetic field can induce a significant enhancement of optical signals such as electrochemiluminescence (ECL), Raman scattering or infrared absorption, thereby forming a series of plasmonic enhanced spectra.

[0007] The present application uses the noble metal Ag NPs in the form of a woolly ball as a plasmonic resonance signal amplification source to amplify the ECL signal of the lanthanide metal oxide (Eu2O3:Tb 3+ ). In addition, the Au NPs loaded on Eu2O3:Tb 3+ can enhance the ECL signal through multiple ways. On the one hand, the Au NPs can act as a co-reactant promoter to promote the generation of more free radicals such as SO4 •− and OH· to enhance the ECL intensity of Eu2O3:Tb 3+ ; on the other hand, compared with the traditional luminophore, the Au NPs can effectively inhibit the non-radiative transition of Eu2O3:Tb 3+ to enhance the ECL intensity, which is mainly because the non-radiative quenching of the traditional luminophore (such as luminol) is mainly due to the intramolecular vibration or collision with the solvent, while the non-radiative transition of Eu2O3:Tb 3+ is mainly due to the capture of the lattice defects to the excited-state energy, and the significant defect passivation of the Au NPs can improve the ECL signal of Eu2O3:Tb 3+ . In addition, the energy level matching between Eu2O3:Tb 3+ and the SPR excited state of Au NPs can directly enhance the generation of the excited state to enhance Eu2O3:Tb3+ Au NPs can also increase the biocompatibility of electrode materials, and the obtained Eu2O3:Tb 3+ @Au has stable and high-intensity ECL signals. The Ag NPs in the form of a woolly knot further amplify the ECL signals of the electrochemiluminescence sensor due to the plasmonic resonance effect. The aptamer (apt) of ACE is modified on the Eu2O3:Tb 3+ @Au through π-π conjugation for specific recognition of ACE. The Ag NPs-cDNA is used as a plasmonic resonance signal source, and is modified on the electrode surface through the hybridization of cDNA and apt to further improve the ECL signals of Eu2O3:Tb 3+ @Au. The sensor is used for ACE detection. Since the aptamer has higher specificity for ACE, the connection between the aptamer and the cDNA is disconnected, the Ag NPs-cDNA falls off from the electrode, the plasmonic resonance effect is weakened, and thus the ECL signals are reduced. The electrochemiluminescence aptamer sensor developed in the application can detect ACE with high sensitivity, and has high selectivity and stability. SUMMARY

[0008] The application aims to provide a lanthanide oxide electrochemiluminescence aptamer sensor with double signal amplification, a preparation method and application thereof, and is used for specific detection of trace acetamiprid.

[0009] The first object of the application is to synthesize a Eu2O3:Tb 3+ (Eu2O3:Tb 3+ @Au) electrochemiluminescence body modified by noble metal gold nanoparticles (Au NPs). 3+ When Tb 3+ ions are inserted into the crystal lattice of Eu2O3 as a sensitizing agent, the ECL emission of Eu 3+ is enhanced, and a layer of Au NPs is further loaded on the surface of Eu2O3:Tb 3+ . The Au NPs are used in multiple ways to enhance the ECL intensity of Eu2O3:Tb 3+ , and increase the biocompatibility of electrode materials, and the obtained Eu2O3:Tb 3+ @Au has stable and high-intensity ECL signals.

[0010] The second object of the application is to prepare a signal amplification probe (Ag NPs-cDNA) composed of silver nanoparticles and a complementary DNA chain (cDNA). The probe uses the surface plasmon resonance (SPR) effect of Ag NPs, and is combined with the luminophore Eu2O3:Tb 3+The ECL emission spectra of @Au exhibit effective overlap, and the ECL signal is further significantly enhanced through SPR energy transfer. The unique tufted structure of AgNPs provides abundant loading sites for cDNA, and the two are also stably bound by Ag-S bonds.

[0011] The third objective of this invention is to prepare an electrochemiluminescence aptamer sensor for the detection of acetamiprid. The use of the aptamer improves the specificity of the detection, enabling the identification of acetamiprid in complex environments and achieving trace detection.

[0012] To achieve the above objectives, the technical solution adopted by the present invention is as follows: 1. The present invention describes the synthesis of a method using Eu2O3:Tb 3+ @Au, as an electrochemiluminescent material, is prepared using the following steps: (1) Eu2O3:Tb 3+ Preparation Europium nitrate hexahydrate (Eu(NO3)3·6H2O) and terbium nitrate hexahydrate (Tb(NO3)3·6H2O) were weighed and dissolved in ultrapure water at a molar ratio of 100:1 to 4:1. Urea was added, and the mixture was heated to 65-100℃ and stirred for 2-8 h. The resulting white precipitate was centrifuged at 12000 rpm for 15 min and washed three times with ethanol and water, respectively. The precipitate was dried in a vacuum drying oven at 60℃ for 12 h, and finally calcined in a muffle furnace at 800℃ for 2 h. The obtained Eu2O 3: Tb 3+ Nanospheres.

[0013] The ratio of the total molar amount of urea to lanthanide nitrates is 5:1 to 20:1.

[0014] The in-situ synthesis of Tb-doped Eu₂O₃ used in this invention produces a stronger and more stable ECL signal. Tb atoms replace Eu atoms in Eu₂O₃ in situ, ensuring the stability and excellent luminescent properties of the Eu₂O₃ material without altering its morphology. The doping amount of Tb is controlled by adjusting the feed ratio during synthesis, resulting in a controllable Eu₂O₃:Tb ratio. 3+ The doping of the second element, Tb, allows the energy absorbed by Tb to be transferred to the central element, Eu, amplifying the luminescence signal of Eu. Therefore, when the Tb doping level is too low, an efficient energy transfer network cannot be formed, and Eu excitation mainly relies on its direct absorption of photons, resulting in a short excited-state lifetime, a high proportion of non-radiative relaxation, and a weak and unstable ECL signal. Conversely, when the Tb doping level is too high, Tb... 3+Excessive concentration leads to intensified energy transfer, with some energy dissipated as heat, while excessive Tb... 3+ This causes lattice distortion and increased surface defects, damaging Eu. 3+ In a complex coordination environment, the ECL signal decreases significantly with increasing doping concentration, and the material is prone to agglomeration and instability. Therefore, the molar ratio of Eu(NO3)3·6H2O to Tb(NO3)3·6H2O is controlled between 100:1 and 4:1.

[0015] (2) Preparation of Au NPs A tetrachloroauric acid (HAuCl4) aqueous solution was heated to boiling, and then sodium citrate solution was added as a reducing agent. The mixture was heated under reflux until the solution turned wine-red. The resulting Au NPs were cooled to room temperature and stored at 4°C for later use.

[0016] Furthermore, the Au NPs have a particle size in the range of 5 to 20 nm.

[0017] Furthermore, the molar ratio of sodium citrate to tetrachloroauric acid is 1.5:1 to 5:1.

[0018] The Au NPs mentioned in this invention enhance the Eu2O3:Tb composition due to their small-sized nanostructure. 3+ The reason for the superior ECL performance is that when the Au NPs particle size is controlled within the range of 5~20 nm, their surface plasmon resonance (SPR) peaks are similar to those of Eu. 3+ The emission peaks of Au and Eu are highly matched, forming a "resonance enhancement" effect—the light energy absorbed by Au NPs is efficiently converted into a local electromagnetic field through SPR, directly exciting Eu. 3+ Or enhance Tb 3+ To Eu 3+ Energy transfer, simultaneously Au and Eu2O3:Tb 3+ The electron transfer is accelerated, nonradiative relaxation is suppressed, and the ECL intensity is significantly enhanced.

[0019] Meanwhile, the electrochemiluminescence aptamer sensor prepared by Au NPs modification in this invention has a stable signal because Au NPs are connected to the aptamer with amino group (apt-NH) through Au-N bonds, and the aptamer is fixed to obtain a stable ECL aptamer sensor.

[0020] (3) Eu2O3:Tb 3+ Preparation of @Au Eu2O3:Tb 3+ Nanospheres were dispersed in deionized water, and the Au NPs solution prepared in step (2) was added. The mixture was stirred vigorously for 12 to 36 hours. After centrifugation, the precipitate was dispersed in ethanol to finally obtain Eu2O3:Tb. 3+@ Au solution.

[0021] Furthermore, Au NPs and Eu2O3:Tb 3+ The mass ratio is controlled within the range of 1:20 to 1:35; Eu2O3:Tb 3+ The concentration of Au solution is 0.5 ~ 10 mg / mL.

[0022] Furthermore, in step (3), the amount of Au NPs added is controlled to ensure that Au NPs are effective against Eu2O3:Tb. 3+ The promoting effect of Au NPs on the ECL performance of the material. When the concentration of Au NPs is too low, a small amount of Au NPs cannot promote the ECL performance of Eu2O3:Tb. 3+ It has a significant promoting effect. When the concentration of Au NPs is too high, the excess Au NPs will cause problems in the Eu2O3:Tb2O3 mixture. 3+ Surface aggregation hinders Eu2O3:Tb 3+ The excitation of Au NPs reduces the ECL efficiency of the central atom Eu, thus decreasing the ECL intensity. Therefore, the amount of Au NPs added is controlled to be 1~8 mL.

[0023] 2. The preparation steps of the yarn-like Ag NPs-cDNA used as a plasma resonance signal source in this invention are as follows: (1) Preparation of yarn-like Ag NPs Ag NO3 and PVP (Mw = 24000) were added to deionized water, and L-ascorbic acid was added under vigorous stirring. The reaction was carried out under light-protected stirring. The resulting gray precipitate was centrifuged, washed, and dispersed in a solvent for later use, yielding tufted Ag NPs with a particle size in the range of 100~200 nm.

[0024] Furthermore, the molar ratio of PVP (based on its monomer structure) to AgNO3 is controlled within the range of 0.5:1 to 5:1; the molar ratio of L-ascorbic acid to AgNO3 is controlled within the range of 1:1 to 2:1.

[0025] By controlling the ratio of PVP and ascorbic acid, uniformly morphological Ag NPs can be obtained. At an appropriate ratio, PVP can effectively inhibit aggregation and regulate the charge distribution of Ag NPs. Ascorbic acid, on the other hand, can control the charge distribution of Ag NPs. + The reduction process and particle growth rate were improved, ultimately resulting in a significant increase in the dispersion and size uniformity of Ag NPs. Therefore, the amount of PVP added was controlled at 1~5 mL, and the volume of ascorbic acid was fixed at 0.2 mL.

[0026] (2) Preparation of Ag NPs-cDNA The cDNA solution was added to the Ag NPs dispersion, and the mixture was stirred at low temperature (e.g., in an ice-water bath). The product was centrifuged at 12,000 rpm for 15 min and washed three times with deionized water. The resulting precipitate was dispersed with deionized water and adjusted to obtain an Ag NPs-cDNA dispersion with a concentration of 1–5 mg / mL.

[0027] Further, in step (1), Ag NPs-cDNA polymer is obtained by centrifugation. This step serves two purposes: firstly, to prevent excessive unbound cDNA from directly binding to apt, thus hindering the binding of Ag NPs-cDNA to apt and ultimately affecting the enhancing effect of Ag NPs-cDNA on ECL; secondly, to prevent excess ascorbic acid and other organic matter from affecting the activity of cDNA.

[0028] Furthermore, the concentration of Ag NPs-cDNA is controlled within the range of 1–5 mg / mL. Within this range, Ag NPs-cDNA is in excess, ensuring that all aptamers on the electrode surface can bind to it. If the concentration of Ag NPs-cDNA is too low, its enhancement effect will be weak, and the recognition rate and detection limit of the analyte cannot be effectively guaranteed. Therefore, it is necessary to control the concentration of Ag NPs-cDNA. However, if the concentration of Ag NPs-cDNA is too high, it will result in residues on the electrode, interfering with the ECL signal and affecting the stability and reliability of the sensor.

[0029] 3. The present invention describes the preparation of an electrochemiluminescence aptamer sensor, wherein the apt sequence of acetamiprid is: 5'-(NH2 C6)-TGT AAT TTG TCT GCA GCG GTT CTT GAT CGC TGA CAC CAT ATT ATG AAG A-3′.

[0030] The cDNA sequence is: 5′-SH-(CH2)6-TCT TCA TAA TAT GGT GTC AGC-3′ The aptamer and cDNA serve as a linker for Eu2O3:Tb. 3+ The key "bridge" between Au and Ag NPs, the chain length of which directly determines Eu2O3:Tb 3+ The spatial spacing between Au and Ag NPs (i.e., the distance between them on the electrode surface). The intensity of the SPR effect is closely related to the distribution of the local electromagnetic field around the Ag NPs—when Eu₂O₃:Tb 3+When the spacing between @Au and Ag NPs is within a specific range (approximately 10–50 nm), the SPR local electromagnetic field energy of Ag NPs is maximized within a linear range through electromagnetic induction to enhance Eu₂O₃:Tb. 3+ The luminescence efficiency of @Au can be measured. Therefore, by regulating the length of the apt and cDNA chains (regulating the number of bases), the distance between apt and Ag NPs can be precisely controlled, ensuring that the SPR effect is always within the optimal enhancement range.

[0031] 4. The Eu2O3:Tb based invention described in this invention 3+ An electrochemiluminescent aptamer sensor with dual signal amplification of @Au and its detection method are described below: (1) The glassy carbon electrode was polished with Al2O3 powder of 0.3μm and 0.05μm respectively, and then ultrasonically cleaned with 1% dilute nitric acid and deionized water for 3 min in sequence. Finally, the surface was dried with N2. (2) Take an appropriate amount (enough to completely cover the active surface of the electrode) of Eu2O3:Tb with a concentration of 0.5 ~ 10 mg / mL. 3 + @Au was added to GCE and dried under an infrared lamp to obtain Eu2O3:Tb 3+ @Au / GCE; (3) Add apt at a concentration of 1 ~ 6 μM to the surface of the modified electrode and incubate at a suitable temperature (e.g., 37℃) for a sufficient time (e.g., 2 ~ 8 hours) to allow the amino-containing aptamer to connect with Au NPs through Au-N bonds, thus obtaining apt / Eu2O3:Tb 3 + @Au / GCE; (4) Add 2 ~ 5 μL of 0.5% bovine serum albumin (BSA) to block non-specific binding sites on the electrode surface, and after natural drying, obtain / BSA / apt / Eu2O3:Tb 3+ @Au / GCE; (5) Add an appropriate amount of Ag NPs-cDNA with a concentration of 1-10 mg / mL to the electrode surface and allow it to fully bind with apt at 37℃ for 2-8 h to obtain Ag NPs-cDNA / BSA / apt / Eu2O3:Tb 3+ @Au / GCE sensor; 5. The present invention provides an electrochemiluminescence aptamer sensor for sensitive detection of acetamiprid, characterized in that the specific detection steps are as follows: A1. Preparation of standard solutions containing different concentrations of acetamiprid: The acetamiprid solid standard was prepared into a 1.0 × 10⁻⁶ ppm solution using deionized water.-3 A series of acetamiprid standard solutions of different concentrations were prepared by diluting the solutions with water, with a concentration range of 1.0 × 10⁻⁶ mol / L. -3 ~1.0×10 -17 mol / L; A2. Plotting the standard curve: A three-electrode system was used, with the prepared electrochemiluminescence aptamer sensor as the working electrode, platinum wire as the counter electrode, and Ag / AgCl as the reference electrode. The prepared electrochemiluminescence aptamer sensor was immersed in a series of standard solutions of acetamiprid at different concentrations prepared in step A1 for 10-80 min (preferably 40 min). After removal, the electrode was rinsed with PBS buffer solution with a pH of 6-9 (preferably pH 7.5) and allowed to air dry before electrochemiluminescence testing. PBS buffer solution containing K2S2O8 was used, and the intensity of the generated electrochemiluminescence signal was recorded. When acetamiprid was present, due to the stronger binding ability of the acetamiprid aptamer to ACE, the signal amplification probe Ag NPs-cDNA partially detached from the electrode surface, the plasmon resonance effect disappeared, and the electrochemiluminescence signal intensity decreased. A linear relationship between acetamiprid and the change in electrochemiluminescence intensity (ΔECL) was established, and the corresponding linear regression equation was obtained. A3. Actual sample testing: The standard addition method was used to test the ECL of acetamiprid in the actual sample. The obtained ECL intensity was calculated by the linear regression equation obtained by A2 to obtain the concentration of acetamiprid in the sample.

[0032] Furthermore, the PBS buffer solution is a 0.1 mol / L Na2HPO4 / NaH2PO4 buffer solution with pH 6 to 9.

[0033] Furthermore, the parameters of the electrochemiluminescence detector are set as follows: scan rate: 100 mV / s, photomultiplier tube high voltage: 800V; the parameters of the electrochemical workstation are set as follows: cyclic voltammetry scan voltage range: 0 ~ -1.8 V; scan rate: 100mV / s. The beneficial effects of this invention are:

[0034] (1) This invention synthesizes a rough-surfaced spherical Eu2O3:Tb by controlling the doping ratio. 3+ Material, this doping ratio of Eu2O3:Tb 3+ The material not only amplifies the luminescence signal of Eu by transferring the energy absorbed by Tb to the central element Eu due to the doping of the second element Tb, but also has a large specific surface area due to the rough surface caused by doping, which is conducive to the further assembly and synthesis of functional materials with metal ions on its surface.

[0035] (2) An electrochemiluminescent material (Eu2O3:Tb) was prepared. 3+ @Au), using precious metal gold nanoparticles (Au NPs) loaded on Eu2O3:Tb 3+ Eu2O3:Tb is formed on the surface 3+ @Au nanocomposites, Au NPs promote electron transfer, resulting in the generation of more SO4. •− The OH· radical further enhances the Eu2O3:Tb 3+ The increased ECL intensity serves as a preliminary signal amplification, broadening the application scope of lanthanide metal oxides in the field of electrochemiluminescence.

[0036] (3) A yarn-like Ag NPs-cDNA was synthesized as a plasmonic resonance signal amplification source. The UV-vis spectrum of Ag NPs-cDNA was similar to that of the luminescent material (Eu2O3:Tb). 3+ The ECL spectra of @Au show good overlap. Ag NPs and cDNA are linked by Ag-S bonds. The cDNA can specifically recognize specific DNA sequence fragments on the acetamiprid aptamer, improving the specificity and detection accuracy of the sensor.

[0037] (4) The present invention adopts a plasma resonance strategy to obtain an ECL signal with dual amplification. This strategy has the significant advantages of high sensitivity and low background noise. Attached Figure Description

[0038] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings: Figure 1 Based on Eu2O3:Tb 3+ Flowchart of an electrochemiluminescence aptamer sensor with dual signal amplification of @Au for the detection of acetamiprid (A:Eu2O3:Tb) 3+ A: Preparation diagram of Ag NPs-cDNA; B: Preparation diagram of Ag NPs-cDNA; C: Schematic diagram of materials.

[0039] Figure 2 It is Eu2O3(A), Eu2O3:Tb 3+ (20:1) (B), Eu2O3:Tb 3+ (2:1)(C)Eu2O3:Tb 3+ SEM image of @Au(D).

[0040] Figure 3 It is Eu2O3(A), Eu2O3:Tb 3+ (B) Eu2O3:Tb 3+(2:1)(C) and Eu2O3:Tb 3+ ECL-time curve of @Au(D).

[0041] Figure 4 This is a SEM image of Ag NPs.

[0042] Figure 5 Eu2O3:Tb was tested in PBS at different pH values. 3+ ECL strength of @Au.

[0043] Figure 6 ECL-time curves for different working electrodes (a: GCE, b: Eu2O3:Tb) 3+ @Au / GCE, c:apt / Eu2O3:Tb 3+ @Au / GCE,d:BSA / apt / Eu2O3:Tb 3+ @Au / GCE,e:Ag NPs-cDNA / BSA / apt / Eu2O3:Tb 3+ @Au / GCE,f:ACE / Ag NPs-cDNA / BSA / apt / Eu2O3:Tb 3+ @Au / GCE).

[0044] Figure 7 The linear curves of different concentrations of acetamiprid were detected using the prepared electrochemiluminescence aptamer sensor.

[0045] in Figure 7 In diagram A, the ECL-time curves for different concentrations of acetamiprid are shown. Figure 7 B is the standard curve of ΔECL and the logarithm of acetamiprid concentration. Detailed Implementation

[0046] The present invention will be further illustrated below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0047] Example 1:

[0048] 1. A method for preparing lanthanide oxide-based electrochemiluminescent materials, comprising the following steps: (1) Eu2O3:Tb 3+ Preparation 1.0 mmol of Eu(NO3)3·6H2O and 0.05 mmol of Tb(NO3)3·6H2O were mixed and dissolved in 70 mL of ultrapure water. 0.6 g of urea was added and the mixture was stirred for 30 min until the solution became clear. The mixture was heated to 90 °C and stirred for 4 h. The resulting white precipitate was centrifuged at 12000 rpm for 15 min and washed three times with ethanol and water. The precipitate was dried in a vacuum drying oven at 60 °C for 12 h, and finally calcined in a muffle furnace at 800 °C for 2 h.

[0049] (2) Eu2O3:Tb 3+ Preparation of Au 0.01% HAuCl4 (100 mL) was heated to boiling, and then 3.5 mL of 1% (w / v) sodium citrate was added to the solution. The mixture was heated under reflux for 30 min, and the solution turned wine red. The obtained Au NPs were cooled to room temperature and stored at 4°C for later use. The Au NPs synthesized in this invention have their particle size controlled within the range of 5–20 nm by adjusting the stirring, reflux, and dropping rate.

[0050] 10mg Eu2O3:Tb 3+ 5 mL of Au NPs at a concentration of 60 mg / L was added to 15 mL of deionized water and stirred vigorously for 24 h. After centrifugation, the precipitate was dispersed in 1 mL of ethanol to obtain Eu₂O₃:Tb at a final concentration of 2 mg / mL. 3+ @ Au.

[0051] 2. The obtained Eu2O3:Tb 3+ @ Au is used for ECL detection. (1) Fabrication of electrochemiluminescence sensor 1) The glassy carbon electrode (GCE) (5 mm in diameter) was polished with Al2O3 powder of 0.3 μm and 0.05 μm respectively, then ultrasonically cleaned with 1% dilute nitric acid and deionized water for 3 min in sequence, and finally dried with N2. 2) Add 5 μL of Eu2O3:Tb with a concentration of 2 mg / mL 3+ Au is drop-coated onto a GCE surface and allowed to dry naturally to obtain Eu2O3:Tb. 3 + @ Au / GCE; (2) The electrochemiluminescence sensor described in this invention is used for ECL detection. (1) The Eu2O3:Tb prepared 3+ @ Au / GCE was used as the working electrode, platinum wire as the counter electrode, and Ag / AgCl as the reference electrode for ECL testing; (2) The parameters of the electrochemiluminescence detector are set as follows: the scanning rate is set to 100mV / s and the photomultiplier tube voltage is set to 800V. (3) The parameters of the electrochemical workstation were set as follows: the cyclic voltammetry scan voltage range was 0 ~ -1.8V; the scan rate was set to 100mV / s. (4) Using a PBS buffer solution containing K2S2O8, record the intensity of the generated electrochemiluminescence signal; the PBS buffer solution is a 0.1 mol / L Na2HPO4 / NaH2PO4 buffer solution with pH 7.5 containing 0.1 mol / L K2S2O8.

[0052] Comparative Example 1:

[0053] The preparation method of Example 1 is followed, except that Eu2O3:Tb is prepared in step 1-(1). 3+ At that time, synthesize separately without Tb. 3 + Pure Eu2O3 and Eu 3+ :Tb 3+ Eu2O3:Tb with a molar concentration ratio of 2:1 3+ .

[0054] The products prepared in Example 1 and Comparative Example 1 were subjected to electrochemiluminescence (ECL) testing.

[0055] (1) Fabrication of electrochemiluminescence sensor (I) Polish the glassy carbon electrode (GCE) and clean it thoroughly by sonicating it for 3 minutes with dilute nitric acid, anhydrous ethanol and deionized water respectively. (II) Add 5 μL of Eu2O3 or Eu2O3:Tb solution with a concentration of 2 mg / mL to the solution respectively. 3+ Drop-coating was applied to the surface of GCE and allowed to air dry to obtain Eu2O3 / GCE and Eu2O3:Tb, respectively. 3+ / GCE; (2) The electrochemiluminescence sensor described in this invention is used for ECL detection. (I) The prepared Eu2O3 / GCE or Eu2O3:Tb 3+ Using GCE as the working electrode, platinum wire as the counter electrode, and Ag / AgCl as the reference electrode, ECL testing was performed. (II) The parameters of the electrochemiluminescence detector are set as follows: the scanning rate is set to 100mV / s, and the photomultiplier tube voltage is set to 800V. (III) The electrochemical workstation parameters were set as follows: cyclic voltammetry scan voltage range: 0 ~ -1.8V; scan rate: 100mV / s; (IV) Using a PBS buffer solution containing K2S2O8, record the intensity of the generated electrochemiluminescence signal; the PBS buffer solution is a 0.1 mol / L Na2HPO4 / NaH2PO4 buffer solution at pH 7.5 containing 0.1 mol / L K2S2O8.

[0056] Figure 2 It is the prepared Eu2O3 and Eu2O3:Tb with different doping ratios. 3+ and Eu2O3:Tb 3+ SEM image of @Au, Figure 2 Image A is a SEM image of Eu2O3, which shows a nanosphere structure with a diameter of approximately 300 nm and a smooth surface. Figure 2 B in Example 1 is Eu 3+ :Tb 3+ Eu2O3:Tb ratio of 20:1 3+ Topographic image, Tb 3+ The doping did not significantly change the morphology of Eu2O3. 3: Tb 3+ It retains the nanosphere structure with a diameter of approximately 300 nm, but its sphere surface becomes rougher compared to Eu₂O₃. When Eu 3 + :Tb 3+ When the molar ratio is 2:1, larger particles appear, causing the particles to no longer be uniform nanospheres of about 300 nm. Figure 2 C). Figure 2 D in the middle is Eu2O3:Tb 3+ SEM image of Au, showing that Eu2O is clearly visible in the image. 3: Tb 3+ Au NPs were attached to the surface (circled in red in the image), which preliminarily proves that Au NPs have been successfully loaded onto Eu2O. 3: Tb 3+ Eu2O formed on the surface. 3: Tb 3+ @Au nanocomposite materials.

[0057] Figure 3 These are the prepared Eu2O3 / GCE and Eu2O 3: Tb 3+ (20:1) / GCE, Eu2O 3: Tb 3+ (2:1) / GCE, Eu2O 3: Tb 3+(20:1) ECL-time curves obtained from ECL testing using an Au / GCE electrode. The graph shows that the ECL value of pure Eu₂O₃ is 8500 a.u. (curve a). 3+ Tb 3+ When the molar concentration ratio is 20:1, Eu2O 3: Tb 3+ The ECL value is 18200 a.u. (curve b), Eu 3+ :Tb 3+ When the molar concentration ratio is 2:1, Eu2O 3: Tb 3+ The ECL value is approximately 10000 au (curve c). This demonstrates that lanthanide elements can improve the ECL performance of lanthanide metal materials. This is because lanthanide Tb doping enables energy transfer from Tb to the central atom Eu, which is beneficial for improving ECL efficiency. It can also emit a strong signal in the K₂S₂O₈ co-reaction medium. Furthermore, when the terbium doping level is within the Eu range... 3+ Tb 3+ The effect of molar concentration ratios of Eu2O in the range of 100:1 to 4:1 3: Tb 3+ Only when the ECL strength is enhanced by doped Tb elements will the energy transfer effect be most pronounced.

[0058] In addition, Eu2O 3: Tb 3+ The ECL value of @Au / GCE is approximately 27000 a.u. (curve d). This is compared to Eu... 3+ Tb 3+ Eu2O at a molar concentration ratio of 20:1 3: Tb 3+ / GCE and Eu2O 3: Tb 3+ @Au / GCE ECL value, Eu2O 3: Tb 3+ @Au / GCE performs best. Au NPs loaded in Eu2O3:Tb 3+ In terms of surface materials, Au NPs will further enhance Eu2O 3: Tb 3+ The ECL value is due to Au NPs promoting electron transfer, resulting in the formation of more SO4. •− And OH· further enhances Eu2O3:Tb 3+ The ECL signal.

[0059] Example 2:

[0060] 1. A method for preparing Ag NPs-cDNA, comprising the following steps: (1) Preparation of Ag NPs 0.2 mL (1.0 M) Ag NO3 and 3.5 mL PVP (0.1 M, Mw = 24000) were added to 10 mL of deionized water and stirred for 5 min. Then, 0.2 mL of 1.0 M L-ascorbic acid was added under vigorous stirring, and the mixture was stirred in the dark for 30 min. The resulting gray precipitate was centrifuged at 1000 rpm for 10 min and washed three times with ethanol. The product was redispersed in 5 mL of anhydrous ethanol and stored at 4 °C for later use.

[0061] (2) Preparation of Ag NPs-cDNA 100 μL of cDNA (3 μM) was added to the Ag NPs dispersion above, and the mixture was stirred in an ice-water bath for 10 h. The product was centrifuged at 12000 rpm for 15 min and washed three times with deionized water. The resulting precipitate was dispersed with deionized water to obtain an Ag NPs-cDNA dispersion with a concentration of 1 mg / mL.

[0062] Figure 4 The image shows SEM images of Ag NPs, which appear as yarn-like nanospheres with a diameter of approximately 200 nm. This yarn-like surface provides Ag NPs with a large specific surface area, offering abundant binding sites for thiol-containing cDNA via Ag-S binding.

[0063] Comparative Example 2:

[0064] 1. A method based on Eu2O3:Tb 3+ The fabrication process of the @Au dual-signal amplified electrochemiluminescence aptamer sensor is as follows: Eu2O3:Tb was prepared according to the method in Example 1. 3+ @Au, prepare Ag NPs-cDNA according to the method in Example 2.

[0065] 5 μL of Eu2O3:Tb at a concentration of 2 mg / mL was added. 3+ @Au droplet coating on GCE surface yields Eu2O3:Tb 3+ @Au / GCE. Add 5 μL of 3 μM apt to the electrode surface and incubate at 37 °C for 6 h to obtain apt / Eu₂O₃:Tb. 3+@Au / GCE. Then, 5.0 μL of 0.5% bovine serum albumin (BSA) was added to block non-specific binding sites on the electrode surface. Next, 5 μL of Ag NPs-cDNA (1 mg / mL) was added to the electrode surface and allowed to bind fully with apt at 37°C for 2 h, yielding Ag NPs-cDNA / BSA / apt / Eu2O3:Tb. 3+ @Au / GCE; The electrochemiluminescence sensor described above was used for ECL detection. The test method of Example 1 was followed, except that the pH of PBS in steps 2-(4) was 5 and 10 respectively, and the other conditions were the same for ECL testing.

[0066] Ag NPs-cDNA / BSA / apt / Eu2O3:Tb 3+ ECL-time curves of @Au / GCE under different pH conditions are shown. Figure 5 As can be seen from the figure, the ECL value is only 20,000 a.u. at pH = 5. This is mainly because when the pH is too low, protons around the electrode are easily reduced to H2, which inhibits S2O8. 2- The reduction of SO4 leads to •− The reduced production of SO42- ultimately lowered the ECL strength. The ECL value was only 25,000 a.u. at pH = 10, because under overly alkaline conditions, excess anions were dispersed around the electrode, thus preventing SO42- from being absorbed. •− The proximity to the electrode surface caused a decrease in the ECL signal. Therefore, the pH value was selected between 6 and 9 throughout the experiment.

[0067] Example 3:

[0068] 1. A method based on Eu2O3:Tb 3+ The fabrication process of the @Au dual-signal amplified electrochemiluminescence aptamer sensor is as follows: Eu2O3:Tb was prepared according to the method in Example 1. 3+ @Au, Prepare Ag NPs-cDNA according to the method in Example 2. Add 5 μL of Eu2O3:Tb at a concentration of 2 mg / mL. 3+ @Au droplet coating on GCE surface yields Eu2O3:Tb 3+ @Au / GCE. Add 5 μL of 3 μM apt to the electrode surface and incubate at 37 °C for 6 h to obtain apt / Eu₂O₃:Tb. 3+@Au / GCE. Then, 5.0 μL of 0.5% bovine serum albumin (BSA) was added to block non-specific binding sites on the electrode surface. Next, 5 μL of 1 mg / mL Ag NPs-cDNA was added to the electrode surface and allowed to bind fully with apt at 37°C for 2 h, yielding Ag NPs-cDNA / BSA / apt / Eu2O3:Tb 3+ @Au / GCE. Finally, the obtained sensor was immersed in 10... -8 The electrode was immersed in acetamiprid (ACE) solution for 40 min, then removed and rinsed with PBS solution, and tested in PBS (0.1 M, pH = 7.5) containing 0.1 M K2S2O8.

[0069] 2. ECL Test (1) Using bare GCE and the modified electrode prepared in each step as working electrodes, platinum wire as counter electrode, and Ag / AgCl as reference electrode, ECL test was performed. (2) The parameters of the electrochemiluminescence detector are set as follows: the scanning rate is set to 100 mV / s and the photomultiplier tube voltage is set to 800V. (3) The parameters of the electrochemical workstation were set as follows: the cyclic voltammetry scan voltage range was 0 ~ -1.8V; the scan rate was set to 100mV / s. (4) Using a PBS buffer solution containing K2S2O8, record the intensity of the generated electrochemiluminescence signal; the PBS buffer solution is a 0.1 mol / L Na2HPO4 / NaH2PO4 buffer solution with pH 7.5 containing 0.1 mol / L K2S2O8.

[0070] Figure 6 The graph shows the ECL intensity curves for each modified electrode layer. As can be seen from the graph, the ECL intensity of the bare GCE is approximately 1600 a.u. (curve a). Example 1: Eu₂O₃:Tb 3+ The ECL intensity of @Au / GCE increased significantly to ≈27300 a.u. (curve b), indicating that Eu2O3:Tb 3+ @Au was successfully modified on the electrode and exhibited a strong ECL signal. After modification with apt and BSA, the ECL intensity decreased to approximately 23800 a.u. (curve c) and approximately 20400 a.u. (curve d), respectively. Subsequently, Ag NPs-cDNA was dropped onto the electrode surface, and the ECL intensity significantly increased to 51700 a.u. (curve e). This is due to the plasmon resonance effect of Ag NPs-cDNA, which enhances the ECL intensity of Eu2O3:Tb. 3+ The ECL intensity of @Au was enhanced. Finally, the prepared sensor was immersed in a solution with a concentration of 1×10⁻⁶. -8In the ACE assay of M, the electrode was removed and washed with PBS to remove incompletely bound ACE, and its ECL value was measured to be approximately 33900 a.u. (curve f). This is because apt specifically binds to the ACE analyte, replacing the weaker interaction between apt and cDNA. Ag NPs-cDNA detaches from the electrode, the plasmon resonance effect disappears, and the ECL intensity decreases. These results demonstrate that the electrochemiluminescence sensor was successfully fabricated and can be used for ACE detection.

[0071] Example 4:

[0072] 1. A method based on Eu2O3:Tb 3+ The @Au dual-signal amplification electrochemiluminescence aptamer sensor for detecting acetamiprid is prepared as follows: Eu2O3:Tb was prepared according to the method in Example 1. 3+ @Au, Prepare Ag NPs-cDNA according to the method in Example 2. Add 5 μL of Eu2O3:Tb at a concentration of 2 mg / mL. 3+ @Au droplet coating on GCE surface yields Eu2O3:Tb 3+ @Au / GCE. Add 5 μL of 3 μM apt to the electrode surface and incubate at 37 °C for 6 h to obtain apt / Eu₂O₃:Tb. 3+ @Au / GCE. Then, 5.0 μL of 0.5% bovine serum albumin (BSA) was added to block non-specific binding sites on the electrode surface. Next, 5 μL of 1 mg / mL Ag NPs-cDNA was added to the electrode surface and allowed to bind fully with apt at 37°C for 2 h, yielding Ag NPs-cDNA / BSA / apt / Eu2O3:Tb 3+ @Au / GCE.

[0073] The acetamiprid solid standard was prepared into a 1.0 × 10⁻⁶ ppm solution using deionized water. -3 A mol / L solution was prepared and diluted to create a series of acetamiprid standard solutions with different concentrations, ranging from 1.0 × 10⁻⁶. -3 ~1.0×10 -17 mol / L.

[0074] The obtained Ag NPs-cDNA / BSA / apt / Eu2O3:Tb 3+ The @Au / GCE sensor was immersed in ACE solutions of different concentrations for 40 min, then the electrode was removed and rinsed with PBS solution. The sensor was then tested in PBS (0.1 M, pH = 7.5) containing 0.1 M K2S2O8, and different ECL values ​​were obtained based on the different concentrations of ACE.

[0075] 2. ECL Test (1) Using the modified electrode as the working electrode, the platinum wire as the counter electrode, and Ag / AgCl as the reference electrode, ECL testing was performed. (2) The parameters of the electrochemiluminescence detector are set as follows: the scanning rate is set to 100 mV / s and the photomultiplier tube voltage is set to 800V. (3) The parameters of the electrochemical workstation were set as follows: the cyclic voltammetry scan voltage range was 0 ~ -1.8V; the scan rate was set to 100mV / s. (4) Using a PBS buffer solution containing K2S2O8, record the intensity of the generated electrochemiluminescence signal; the PBS buffer solution is a 0.1 mol / L Na2HPO4 / NaH2PO4 buffer solution with pH 7.5 containing 0.1 mol / L K2S2O8.

[0076] Figure 7 A is based on the luminescent material Eu2O3:Tb 3+ Electrochemiluminescent aptamer sensor prepared with @Au (Ag NPs-cDNA / BSA / apt / Eu2O3:Tb) 3+ ECL-time curves obtained using @Au / GCE for the detection of acetamiprid. The figure shows that at an acetamiprid concentration of 10... -8 ~10 -15 Within the M range, the ECL value decreases as the concentration of acetamiprid increases. Figure 7 Figure B shows the linear regression curve of the sensor's ECL change (ΔECL) and the logarithm of acetamiprid concentration. The two curves exhibit a good linear relationship, with the linear regression equation being ΔECL = 18593.7 – 1931.8 × Lg. ACE R 2 =0.9983, detection limit is 4.46×10 -16 M (3σ / slope).

[0077] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A lanthanide oxide electrochemiluminescence aptamer sensor with dual signal amplification for detecting acetamiprid, characterized in that, It comprises the following components: a conductive substrate electrode; and an electrochemiluminescent material, Eu₂O₃:Tb, composed of europium oxide doped with terbium ions and modified with gold nanoparticles. 3+ @Au was used to modify the conductive substrate electrode surface, and acetamiprid aptamers were modified on the electrochemiluminescent substrate surface. A signal amplification probe solution of Ag NPs-cDNA, composed of silver nanoparticles and complementary DNA strands, was dropped onto the electrode surface and incubated to allow hybridization with the aptamers, forming the sensor Ag NPs-cDNA / apt / Eu2O3:Tb. 3+ @Au / GCE.

2. The method for detecting acetamiprid according to claim 1, characterized in that, The electrochemiluminescent material Eu2O3:Tb 3 + @Au is used to prepare Eu2O3:Tb 3+ Eu 3+ With Tb 3+ The molar ratio is 100:1 ~ 4:

1.

3. The dual-signal amplification electrochemiluminescence aptamer sensor for detecting acetamiprid according to claim 2, characterized in that, The Eu 3+ With Tb 3+ The molar ratio is 50:1 to 10:

1.

4. The lanthanide oxide electrochemiluminescence aptamer sensor with dual signal amplification for detecting acetamiprid according to claim 2, characterized in that, The Ag NPs in the signal amplification probe Ag NPs-cDNA are tufted structures with a diameter of 100~200 nm.

5. The lanthanide oxide electrochemiluminescence aptamer sensor with dual signal amplification for detecting acetamiprid according to claim 1, characterized in that, The sequence of the acetamiprid aptamer is 5'-(NH2 C6)-TGT AAT TTG TCT GCAGCG GTT CTT GAT CGC TGA CAC CAT ATT ATG AAG A-3'; the sequence of the complementary DNA strand is 5'-SH-(CH2)6-TCT TCA TAA TAT GGT GTC AGC-3'.

6. A method for preparing a lanthanide oxide electrochemiluminescence aptamer sensor with dual signal amplification for detecting acetamiprid as described in any one of claims 1-5, characterized in that, Includes the following steps: (1) Eu2O3:Tb 3+ @Au light-emitting material is applied to the clean, conductive substrate electrode surface; (2) Add the acetamiprid aptamer solution to the modified electrode surface, fix it on the surface of the electrochemiluminescent material, and incubate for fixation; (3) Use bovine serum albumin solution to block non-specific binding sites on the electrode surface; (4) The signal amplification probe Ag NPs-cDNA solution was dropped onto the electrode surface and incubated at 37°C to hybridize with the aptamer, thus obtaining the sensor.

7. The method according to claim 6, characterized in that: In step (1), Eu2O3:Tb 3+ The preparation of the @Au luminescent material includes the following steps: (1) Dissolve Eu(NO3)3·6H2O and Tb(NO3)3·6H2O in ultrapure water at a molar ratio of 100:1 to 4:1, add urea and stir until clear, heat the mixed solution to 65 to 100°C and stir for 2 to 8 h, centrifuge, wash, dry and calcine in a muffle furnace at 800°C for 2 h to prepare Eu2O3:Tb 3+ ; (2) Heat the HAuCl4 solution to boiling, add sodium citrate solution and reflux, cool and store to prepare Au NPs; the Au NPs have a particle size range of 5~20 nm; (3) Eu2O3:Tb 3+ Au NPs were added to deionized water and stirred. After centrifugation, the mixture was dispersed in ethanol to finally obtain Eu2O3:Tb. 3+ @ Au dispersion; And / or, The preparation method of the signal amplification probe Ag NPs-cDNA in step (4) is as follows: (a) Add AgNO3 solution and PVP solution to deionized water and stir. Add L-ascorbic acid and stir in the dark. After obtaining the precipitate, centrifuge, wash and disperse in anhydrous ethanol for storage to obtain a yarn ball-shaped Ag NPs nanosphere dispersion. (b) Add cDNA to Ag NPs nanosphere dispersion, stir at low temperature, centrifuge, wash, and disperse in deionized water to obtain Ag NPs-cDNA dispersion.

8. The method according to claim 7, characterized in that: Eu2O3:Tb 3+ The concentration of Au dispersion is 0.5 ~ 10 mg / mL; In the preparation of Ag NPs-cDNA, the molar ratio of PVP to AgNO3, based on its monomer structure, was controlled within the range of 0.5:1 to 5:1; the molar ratio of L-ascorbic acid to AgNO3 was controlled within the range of 1:1 to 2:1; and the concentration of Ag NPs-cDNA dispersion was controlled within the range of 1 to 5 mg / mL.

9. The application of the lanthanide oxide electrochemiluminescence aptamer sensor with dual signal amplification according to any one of claims 1-5 in the detection of acetamiprid.

10. The application according to claim 9, characterized in that: The steps for detecting acetamiprid are as follows: (1) Prepare a 1.0×10⁻⁶ solid standard of acetamiprid using deionized water. -3 A mol / L solution was prepared and serially diluted to a concentration range of 1.0 × 10⁻⁶. -3 ~ 1.0×10 -17 mol / L acetamiprid standard solution; (2) The prepared electrochemiluminescence aptamer sensor was used as the working electrode, the platinum wire as the counter electrode, and Ag / AgCl as the reference electrode. A PBS buffer solution containing K2S2O8 was used to record the electrochemiluminescence signal intensity generated by detecting standard solutions containing different concentrations of acetamiprid using the electrochemiluminescence detection method. A standard curve was plotted based on the linear relationship between the obtained electrochemiluminescence signal and the logarithm of the concentration of acetamiprid. The PBS buffer solution was a 0.1 mol / L Na2HPO4 / NaH2PO4 buffer solution with pH 6 ~ 9. (3) The standard addition method was used to test the ECL of acetamiprid in the actual sample. The ECL intensity was calculated by the obtained linear regression equation to obtain the concentration of acetamiprid in the sample.

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