Electrochemiluminescence aptamer sensor based on TbPO4:Ce for detecting lincomycin and preparation method and application thereof

Abstract

The application relates to an electrochemiluminescence aptamer sensor for detecting lincomycin based on TbPO4:Ce and a preparation method and application thereof. Specifically, in-situ doped TbPO4:Ce is used as an electrochemiluminescence sensing interface, and TbPO4:Ce has a stable electrochemiluminescence signal. The surface of TbPO4:Ce is negatively charged, and after being modified by PDDA, the surface is positively charged. The negatively charged Ag nanoparticles are combined with the modified TbPO4:Ce, the conductivity of TbPO4:Ce is improved, the catalytic activity of the electrode material is promoted, the energy transfer effect is improved, and the electrochemiluminescence signal is further amplified. The lincomycin aptamer can specifically recognize lincomycin, and the selectivity of the electrochemiluminescence sensor is improved. The linear range of the application for lincomycin detection is 1.0*10 ‑16 ~1.0*10 ‑4 mol / L, and the detection limit is 1.78*10 ‑17 mol / L.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of detection sensor preparation, and particularly relates to a preparation method of an electrochemiluminescence aptamer sensor for detecting lincomycin based on Ce-doped TbPO4 nanowires (TbPO4:Ce). BACKGROUND

[0002] Lincomycin (Lin) has become a widely used antibiotic due to its antibacterial effect on staphylococcus, streptococcus, streptococcus pneumoniae and other bacteria, and has been widely used in the breeding industry. Excessive lincomycin will enter groundwater or surface water, and the residual lincomycin in the water will not only increase bacterial resistance, but also cause damage to human liver and kidney function. Therefore, how to efficiently and sensitively detect CYFRA21-1 is a problem to be solved at present.

[0003] Traditional methods for detecting lincomycin mainly include gas chromatography, liquid chromatography-mass spectrometry, fluorescence method and photoelectrochemical method (CN202110916549.5 Preparation method of a photoelectrochemical self-powered sensor and application thereof in detection of lincomycin), etc. However, these methods have high equipment cost, complex operation, low sensitivity, high cost and great difficulty in popularization and application. Electrochemiluminescence (ECL) combines the advantages of electrochemistry and chemiluminescence, and has the characteristics of small background interference, fast response speed and high sensitivity. Electrochemiluminescent body is an important part of the preparation process of electrochemiluminescence sensor, and researchers are mainly committed to finding a simple, low-cost and environmentally friendly luminescent body. The traditional preparation process of electrochemiluminescent body is complex, often requiring a multi-step synthesis process (including hydrothermal process), and the reaction period is long and the sensor preparation process is cumbersome.

[0004] Compared with the traditional electrochemiluminescent body (CN202210640465.8 Electrochemiluminescent body, electrochemiluminescent aptamer sensor, preparation method and application thereof), lanthanide phosphate (LnPO4) has very high thermal stability, high refractive index and other fascinating properties. Most importantly, LnPO4 has low solubility in water, and can be synthesized at room temperature by solvothermal method, which has a very good prospect in electrochemiluminescence analysis and detection. However, due to the f→f transition within the lanthanide configuration and the low absorption coefficient, it is difficult for ECL to emit directly. The second element doped LnPO4 not only has the "ligand antenna effect", but also can transfer the energy absorbed by the ligand (PO4 3- ) to Ln(Ⅲ) to enhance the luminescence intensity of lanthanide (I ECL ).

[0005] Studies have shown that by combining luminophores and nanometer luminescent materials, self-enhanced nanometer luminescent structures can be manufactured, for example, using AuNPs, AgNPs, PtNPs, etc. noble metal nanoparticles to improve the sensitivity of the sensor by using their excellent electron conduction characteristics (CN202210640465.8). Silver nanoparticles have the advantages of easy availability of raw materials, high cost performance compared with other noble metal nanoparticles, and strong spectral absorption in the ultraviolet-visible light band, high chemical stability, and high surface plasmon resonance (SPR) characteristics. The luminescent material and silver nanoparticle cluster can be used as a plasmonic source to amplify the ECL signal of the luminescent material. The use of AgNPs in the ECL system can reduce the electron transfer barrier between the ECL luminophore and the electrode, accelerate electron transfer, and the plasmonic resonance effect can further improve the detection sensitivity.

[0006] Silver nanoparticles prepared by sodium citrate, sodium borohydride and hydroxylamine hydrochloride and other reducing agents are usually negatively charged, which limits the application of silver nanoparticles in ECL. Polydimethyl diallyl ammonium chloride (PDDA) is a polyelectrolyte rich in positive charges and can be used as a reducing agent and stabilizer to provide a large number of positive charges without changing the structure. By combining PDDA with luminescent materials, the firm attachment of PDDA and silver nanoparticles can be achieved by the combination of N containing lone pair electrons and Ag atoms, the surface charge structure of silver nanoparticles can be adjusted, and the stability of the combination of luminescent materials and silver nanoparticles can be improved to achieve the effect of stable catalysis.

[0007] Aptamers (apt) are gradually developed and applied to analysis and detection due to their high stability and economic characteristics. Aptamers can improve the selectivity of electrochemiluminescence sensors. When aptamers are applied to electrochemiluminescence sensors, it is necessary to consider how to stably fix the aptamers on the surface of the electrode material, otherwise stable luminescent signals cannot be obtained, which affects the detection accuracy of the sensor. Aptamers can be modified with special groups to form metal-N bonds or metal-S bonds with noble metal nanoparticles to fix the aptamers. Therefore, the combination of noble metal particles and aptamers not only can be used as a signal amplification unit to improve the sensitivity of electrochemiluminescence sensors, but also can improve the specificity of the detection performance of the modified aptamers while improving the stability of the sensor. SUMMARY

[0008] The first object of the present application is to synthesize a Ce-doped TbPO4 as a luminescent material. Due to the dual enhancement effect of ligand antenna effect and energy transfer, the doping of Ce makes the ECL signal of TbPO4:Ce significantly increase, and a higher and more stable electrochemiluminescence signal is obtained.

[0009] The second object of the application is to improve the surface charge of TbPO4: Ce, which is negatively charged itself, and after modification by PDDA, a large number of positive charges are distributed on the surface of TbPO4: Ce, so as to be stably combined with Ag NPs with negative charges to emit stable ECL signals.

[0010] The third object of the application is to synthesize Ag NPs with a particle size range of 10.5-30.0 nm, which are combined with aptamers with thiol groups through Ag-S bonds to stably combine the aptamers on the electrode surface. The Ag NPs not only amplify the ECL signal of TbPO4: Ce, but also play a role in fixing the aptamers.

[0011] In order to achieve the above object, the technical scheme adopted by the application is as follows:

[0012] 1. The preparation method of the electrochemiluminescence aptamer sensor for detecting lincomycin based on TbPO4: Ce according to the application, and the preparation steps are as follows:

[0013] (1) A glassy carbon electrode (GCE) with a diameter of 3 mm is polished and polished, and is cleaned with dilute nitric acid, anhydrous ethanol and deionized water respectively;

[0014] (2) TbPO4: Ce with a concentration of 1-10 mg / mL modified by PDDA is dropped on the surface of GCE and naturally dried to obtain GCE / TbPO4: Ce;

[0015] (3) Ag NPs with a concentration of 0.05-0.2 mg / mL are added to the electrode surface and naturally dried to obtain GCE / TbPO4: Ce / Ag NPs;

[0016] (4) Thiolated lincomycin aptamer (apt-SH) with a concentration of 1-7 μmol / L is added to the electrode surface, and is stored at 25°C for 6 h, and is cleaned with ultrapure water to obtain apt-SH / Ag NPs / TbPO4: Ce / GCE;

[0017] Further, the volume ratio of TbPO4: Ce, Ag NPs and thiolated lincomycin aptamer solution is 5:5:3.

[0018] 2. The preparation method of the electrochemiluminescence aptamer sensor for detecting lincomycin based on TbPO4: Ce according to the application, and the preparation steps of TbPO4: Ce are as follows:

[0019] Dissolve Tb(NO3)3·6H2O and Ce(NO3)3·6H2O in deionized water, wherein the mass ratio of Tb(NO3)3·6H2O to Ce(NO3)3·6H2O is 1:1, and the concentration of the mixed solution is 0.1 mol / L. 3+ : Ce 3+The molar concentration ratio was controlled to be 10:1-6:1. The transparent precursor solution was formed by stirring for 30 min. NaH2PO4·2H2O was dissolved in deionized water, and then slowly added dropwise into the precursor solution and stirred for 2 h. A white precipitate was obtained, centrifuged at 10,000 rpm for 10 min, washed with deionized water for 3 times, and freeze-dried to obtain a white TbPO4:Ce powder.

[0020] The one-step in-situ synthesis of Ce-doped TbPO4 in the application has stronger and more stable ECL signals. The Ce atoms replace the Tb atoms in TbPO4 in-situ, without changing the coordination structure of TbPO4, to ensure the stability of TbPO4 material and the excellent luminescent performance of lanthanide phosphate. The doping amount of Ce is controlled by controlling the feeding ratio during synthesis, and the TbPO4:Ce with controllable synthesis ratio is synthesized. Due to the doping of the second element Ce, the energy absorbed by Ce is transmitted to the central element Tb, which plays a role in amplifying the luminescent signal of Tb element.

[0021] TbPO4:Ce modified by PDDA: TbPO4:Ce was stirred in PDDA (polydiallyldimethylammonium chloride) (20 wt.%) for 24 h, centrifuged, washed with deionized water for 3 times to remove excess PDDA, and the product was redispersed in 2 mL of deionized water. Finally, the TbPO4:Ce with widely distributed positive charges on the surface was obtained.

[0022] The mass-volume ratio of TbPO4:Ce to PDDA (20 wt.%) is 4 mg:1-5 mL.

[0023] The TbPO4:Ce synthesized by the one-step in-situ method in the application itself has a negative charge and cannot stably coexist with negatively charged Ag NPs on the electrode surface, but after the charge is changed by PDDA, the positively charged TbPO4:Ce forms electrostatic adsorption with the negatively charged Ag NPs, and is stably combined on the electrode surface to ensure that a stable ECL signal is obtained. The PDDA used in the application has a narrow molecular weight distribution, a suitable degree of polymerization, a linear structure, small steric hindrance, and high conductivity. The preferred molecular weight range (Mw) of the application is 50,000-100,000. The TbPO4:Ce luminescent material modified by the PDDA has more positive charges on the surface, which can be more firmly combined with Ag NPs, improving the stability and luminescent efficiency of the electrode.

[0024] 3. The preparation method and application of the electrochemiluminescence aptamer sensor for detecting lincomycin based on TbPO4:Ce in the application are as follows:

[0025] 5-17 mg AgNO3 was dissolved in 100 mL deionized water, after stirring for 10 min, it was transferred into a three-necked flask, heated at 90-120 ℃ for 20 min, then 1.5-5.0 mL sodium citrate (1%, g / mL) was slowly added dropwise into the flask under stirring at a speed of 600-1140 rpm (preferably 900 rpm), and kept refluxing for 3 h, then cooled to room temperature, the solution was centrifuged at 13000 rpm for 15 min, the supernatant was removed, and the solution was re-dispersed in 100 mL deionized water at a concentration of 0.05-0.2 mg / mL, and the solution was stored at 4 ℃ in the dark.

[0026] The Ag NPs of the present application can control the particle size of the synthesized Ag NPs in the range of 10.5-30.0 nm by stirring, refluxing and dropping speed. The Ag NPs can enhance the ECL performance of TbPO4:Ce due to the small size of the nanostructure, and the reason is that: (1) most of the electrons and holes generated by small particles of 30 nm and below will not recombine before reaching the surface, and the number of electrons and holes reaching the surface is large, so the catalytic activity is high; (2) when the particle size of silver nano-ions is small, the metal micro-particles can be approximated as being in a uniform electric field in the same phase during the electrochemical excitation process, which shows a simple dipole resonance mode, and can transfer part or all of the energy of the plasmon resonance phenomenon to the TbPO4:Ce material to enhance its ECL intensity.

[0027] Further, the electrochemiluminescence aptamer sensor prepared by the AgNPs modification of the present application has a stable signal, which is due to the connection of the Ag NPs and the aptamer with thiol group (apt-SH) through Ag-S bond, and the fixation of the aptamer to prepare the ECL aptamer sensor with stable signal.

[0028] 4. The preparation method of the electrochemiluminescence aptamer sensor for detecting lincomycin based on TbPO4:Ce according to the present application, wherein the preparation method of the aptamer sensor is as follows:

[0029] The thiolated lincomycin aptamer was first centrifuged at 4000 rpm for 10 min, 440 μL Tris-HCl buffer solution was added thereto, and mixed uniformly to obtain an aptamer mother liquor with a concentration of 10 μmol / L, and when the experiment was performed, the aptamer was diluted with Tris-HCl buffer solution to obtain a low-concentration aptamer. 2.5-10 μL (preferably 5 μL) of lincomycin aptamer (apt-SH) with a concentration of 1-7 μmol / L (preferably 3 μmol / L) was added dropwise on the surface of the electrode, and the aptamer with thiol group was combined with the Ag NPs on the electrode to obtain a stable electrochemiluminescence aptamer sensor.

[0030] The 5' of the DNA strand of the thiolated aptamer (apt-SH) is connected with a thiol group (-SH), and the DNA sequence is: 5'-SH-(CH2)6-CGC GTG ATG TGG TCG ATG CGA TAC GGT GAG TCG CGC CAC GGC TAC ACACGT CTC AGCGA-3', and the lincomycin aptamer is prepared into a 10 μmol / L stock solution using a Tris-HCl solution, and then is diluted step by step for standby.

[0031] The thiol modification (-SH) of the lincomycin aptamer endows the sensor with stable and sensitive selective detection capability. Because the lincomycin aptamer can specifically recognize lincomycin, the sensor can accurately detect lincomycin in a complex water environment coexisting with other antibiotics and pollutants, and the anti-interference capability and sensitivity of the sensor are improved.

[0032] 5. The application of the electrochemiluminescence aptamer sensor for detecting lincomycin based on TbPO4: Ce, characterized in that the specific steps are as follows:

[0033] A1. Preparation of standard solutions containing lincomycin with different concentrations:

[0034] Lincomycin solid standard is prepared into a 1.0×10 -3 mol / L solution using deionized water, and is diluted into a series of lincomycin standard solutions with different concentrations, and the concentration range is 1.0×10 -3 ~ 1.0×10 -17 mol / L;

[0035] A2. Drawing of a standard curve:

[0036] A three-electrode system is adopted, the prepared apt-SH / Ag NPs / TbPO4: Ce / GCE electrochemiluminescence aptamer sensor is used as a working electrode, a platinum wire is used as a counter electrode, and Ag / AgCl is used as a reference electrode. The prepared electrochemiluminescence aptamer sensor is soaked in a series of lincomycin standard solutions with different concentrations prepared in step A1 for 30-70 min, the electrode is taken out and washed with a PBS buffer solution, and is naturally dried before electrochemiluminescence testing, a linear relationship between the lincomycin concentration and the electrochemiluminescence intensity is established, and a corresponding linear regression equation is obtained.

[0037] The parameters of the electrochemiluminescence detector were set as follows: scan rate: 100 mV / s, photomultiplier tube high voltage: 800 V; the parameters of the electrochemical workstation were set as follows: cyclic voltammetry scan voltage range: 0 to -1.8 V; scan rate: 100 mV / s; PBS buffer solution: 0.1 mol / L Na2HPO4 / NaH2PO4 buffer solution with pH 7.5 containing 0.1 mol / L K2S2O8.

[0038] A 3. Actual sample testing:

[0039] The ECL of lincomycin in the actual sample was tested using the standard addition method. The ECL intensity was calculated using the linear regression equation obtained from A2 to determine the concentration of lincomycin in the sample.

[0040] Advantages of this invention:

[0041] (1) Compared with traditional luminescent materials, the synthesis method of TbPO4:Ce is simple and easy to fix onto the electrode surface, and has good electrochemiluminescence properties. The TbPO4:Ce synthesized by in-situ one-step doping of TbPO4 with Ce atoms significantly improves the electrochemiluminescence intensity of the electrode due to energy transfer, thereby improving the sensitivity of the electrode;

[0042] (2) AgNPs of 30 nm and below improve the ECL performance of TbPO4:Ce due to their good catalytic performance and plasmon resonance effect. At the same time, AgNPs can catalyze the conversion of dissolved oxygen in solution to OH·, promoting the co-reactant S2O8. 2- To SO4 - The conversion further amplifies the electrochemiluminescence signal. PDDA-modified TbPO4:Ce further enhances its binding ability with Ag NPs, improving the stability and lifespan of the ECL electrode.

[0043] (3) The thiolized aptamer (apt-SH) is linked to AgNPs via Ag-S bonds, which improves the aptamer's adsorption strength, enhances the sensor's sensitivity and selective recognition ability, and further improves the stability of the ECL signal. The linear range for lincomycin detection in this invention is 1.0 × 10⁻⁶. -16 ~1.0×10 -4 mol / L, detection limit is 1.78 × 10⁻⁶ -17 mol / L.

[0044] The prepared TbPO4:Ce electrochemiluminescence aptamer sensor for detecting lincomycin has the characteristics of good biocompatibility, high electrode material luminescence intensity, strong anti-interference ability, good stability and long service life. The sensor can be applied to complex water environment coexisting with other antibiotics or pollutants to rapidly, sensitively and accurately detect lincomycin. BRIEF DESCRIPTION OF DRAWINGS

[0045] The accompanying drawings are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification, illustrate embodiments of the application and are included to provide a further understanding of the application, and are made a part of the specification. In the drawings:

[0046] Figure 1 is a brief flow chart of preparation of TbPO4:Ce and a brief flow chart of preparation of electrochemiluminescence sensor and detection of lincomycin; (A) is a brief flow chart of preparation of TbPO4:Ce; (B) is a brief flow chart of preparation of electrochemiluminescence sensor and detection of lincomycin.

[0047] Figure 2 is ECL intensity-time curve of TbPO4:Ce synthesized by different molar ratios of TbPO4:Ce, wherein the content represented by each curve is: a) TbPO4, b) 8:1, c) 4:1, d) CePO4. 3+ :Ce 3+

[0048] Figure 3 is ECL-time curve obtained by preparing electrochemiluminescence sensor by in-situ doping synthesis of TbPO4:Ce and stirring mixing of pure TbPO4 and pure CePO4; (a) TbPO4:Ce, b) stirring mixing of pure TbPO4 and pure CePO4.

[0049] Figure 4 is a comparison chart of ECL-time curves of GCE / TbPO4:Ce after different surface modification:

[0050] A) is GCE / TbPO4:Ce after surface modification of Ag NPs, B) is TbPO4:Ce / Ag NPs after charge improvement of PDDA.

[0051] Figure 5 ECL intensity of GCE / TbPO4:Ce / Ag NPs was tested in PBS with different pH.

[0052] Figure 6 is a linear curve of detection of lincomycin with different concentrations by using the prepared electrochemiluminescence aptamer sensor.

[0053] wherein Figure 6 ​A is the ΔECL-time curve of different concentrations of lincomycin, wherein the lincomycin concentration is 1.0 x 10 -4 ~1.0 x 10 -16 , Figure 6 B is the standard curve of ΔECL and the logarithm of lincomycin concentration, Figure 6 The inset in B is the ECL intensity-time curve of the apt-SH / AgNPs / TbPO4:Ce / GCE sensor.

[0054] Figure 7 is the selectivity histogram of the apt-SH / Ag NPs / TbPO4:Ce / GCE electrochemiluminescence aptamer sensor. DETAILED DESCRIPTION

[0055] The application will be further described below in conjunction with specific examples, which are only used to illustrate the application and not to limit the scope of the application.

[0056] Example 1

[0057] Preparation of an electrochemiluminescence sensor GCE / TbPO4:Ce for detecting lincomycin.

[0058] (1) Preparation of TbPO4:Ce

[0059] Dissolve 0.0408 g of Tb(NO3)3·6H2O and Ce(NO3)3·6H2O in 20 mL of deionized water, wherein the molar concentration ratio of Tb 3+ :Ce 3+ is 8:1. Stir thoroughly for 30 min to form a transparent precursor solution. Dissolve 0.0316 g of NaH2PO4·2H2O in 10 mL of deionized water, then slowly dropwise add to the above precursor solution and stir for 2 h. Centrifuge the white precipitate at 10000 rpm for 10 min, wash with deionized water three times, and freeze-dry to obtain a white powder of TbPO4:Ce.

[0060] (2) Preparation of an electrochemiluminescence sensor

[0061] 1) Polish a glassy carbon electrode (GCE) with a diameter of 3 mm, and ultrasonically clean the electrode with dilute nitric acid, anhydrous ethanol and deionized water for 3 min respectively to thoroughly clean the electrode;

[0062] 2) Drop 5 μL of TbPO4:Ce with a concentration of 2 mg / mL on the surface of the GCE, and naturally dry to obtain GCE / TbPO4:Ce;

[0063] 2. The electrochemiluminescence sensor described in the application is used for ECL detection

[0064] (1) The prepared GCE / TbPO4:Ce was used as the working electrode, the platinum wire as the counter electrode, and the Ag / AgCl as the reference electrode for ECL testing.

[0065] (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.

[0066] (3) The parameters of the electrochemical workstation were set as follows: the cyclic voltammetry scan voltage range was 0 to -1.8V, and the scan rate was set to 100mV / s;

[0067] (4) Record the intensity of the generated electrochemiluminescence signal using a PBS buffer solution containing K2S2O8. The PBS buffer solution is a 0.1 mol / L Na2HPO4 / NaH2PO4 buffer solution at pH 7.5 containing 0.1 mol / L K2S2O8.

[0068] Tb prepared in Example 1 3+ :Ce 3+ The results for TbPO4:Ce at a molar concentration ratio of 8:1 are as follows: Figure 2 As shown in curve b, Tb 3+ :Ce 3+ When the molar concentration ratio is 8:1, the ECL value of TbPO4:Ce is approximately 12000 a.u.

[0069] Example 2

[0070] An electrochemiluminescence sensor GCE / TbPO4:Ce / AgNPs for the detection of lincomycin was prepared.

[0071] (1) Preparation of TbPO4:Ce

[0072] Dissolve 0.0408 g of Tb(NO3)3·6H2O and Ce(NO3)3·6H2O in 20 mL of deionized water, wherein Tb 3+ :Ce 3+ The molar ratio was 8:1. The mixture was stirred thoroughly for 30 min to form a transparent precursor solution. 0.0316 g of NaH₂PO₄·2H₂O was dissolved in 10 mL of deionized water, and then slowly added dropwise to the above precursor solution while stirring for 2 h. The white precipitate was centrifuged at 10,000 rpm for 10 min, washed three times with deionized water, and freeze-dried to obtain a white TbPO₄:Ce powder.

[0073] (2) Preparation of Ag NPs

[0074] 7mg AgNO3 was dissolved in 100mL deionized water, after 10min of stirring, it was transferred into a three-necked flask, heated at 97℃ for 20min, then 2.0mL sodium citrate (1%, g / mL) was slowly added into the flask under stirring at 900rpm, kept refluxing for 3h, then cooled to room temperature, the solution was centrifuged at 13000rpm for 15min, the supernatant was removed, and the solution was re-dispersed in 100mL deionized water at a concentration of 0.1mg / mL, and the solution was stored at 4℃ in the dark.

[0075] (3) Preparation of electrochemiluminescence sensor

[0076] 1) A glassy carbon electrode (GCE) with a diameter of 3mm was polished and polished, and the electrode was thoroughly cleaned by ultrasonicating with dilute nitric acid, anhydrous ethanol and deionized water for 3min, respectively;

[0077] 2) 5μL of TbPO4:Ce with a concentration of 2mg / mL was dropped on the surface of GCE and naturally dried to obtain GCE / TbPO4:Ce;

[0078] 3) 5μL of Ag NPs with a concentration of 0.1mg / mL was dropped on the surface of the electrode and naturally dried to obtain GCE / TbPO4:Ce / Ag NPs;

[0079] 2. The electrochemiluminescence sensor of the application is used for ECL detection

[0080] (1) The prepared GCE / TbPO4:Ce / Ag NPs is used as a working electrode, a platinum wire is used as a counter electrode, and Ag / AgCl is used as a reference electrode for ECL test;

[0081] (2) The parameter setting of the electrochemiluminescence detection instrument is that the scanning rate is set to 100mV / s, and the high voltage of the photomultiplier tube is set to 800V;

[0082] (3) The parameter setting of the electrochemical workstation is that the cyclic voltammetry scanning voltage range is 0~-1.8V, and the scanning rate is set to 100mV / s;

[0083] (4) A PBS buffer solution containing K2S2O8 is used to record the generated electrochemiluminescence signal intensity; the PBS buffer solution: 0.1mol / L Na2HPO4 / NaH2PO4 buffer solution with 0.1mol / L K2S2O8 and pH 7.5.

[0084] As described in the preparation steps of the electrochemiluminescence sensor of Example 2, the content of Tb in TbPO4:Ce 3+ :Ce 3+The ECL results of GCE / TbPO4:Ce surface modified Ag NPs with a molar ratio of 8:1 are shown in Figure 2. The ECL results of GCE / TbPO4:Ce surface modified Ag NPs with a molar ratio of 8:1 are shown in Figure 2. Figure 4 As shown in Figure 2A, by comparing the ECL curves of a and b, it can be seen that the ECL intensity of TbPO4:Ce is amplified by Ag NPs, and the ECL intensity is increased to about 30000 a.u., and the RSD of stability is 2.80%. Figure 2 As shown in Figure 2A, by comparing the ECL curves of a and b, it can be seen that the ECL intensity of TbPO4:Ce is amplified by Ag NPs, and the ECL intensity is increased to about 30000 a.u., and the RSD of stability is 2.80%. Figure 4 As shown in Figure 2A, by comparing the ECL curves of a and b, it can be seen that the ECL intensity of TbPO4:Ce is amplified by Ag NPs, and the ECL intensity is increased to about 30000 a.u., and the RSD of stability is 2.80%. 2- Due to the good conductivity of noble metal Ag NPs, the electron transfer on the electrode surface is promoted; secondly, the good catalytic effect of Ag NPs catalyzes the generation of OH· from dissolved oxygen in the solution, which accelerates the generation of OH· from dissolved oxygen in the solution. ·- The generation of OH· from dissolved oxygen in the solution, which accelerates the generation of OH· from dissolved oxygen in the solution.

[0085] Example 3

[0086] Preparation of an electrochemiluminescence sensor GCE / positively charged modified TbPO4:Ce / Ag NPs for detecting lincomycin.

[0087] (1) Preparation of TbPO4:Ce

[0088] Dissolve 0.0408 g of Tb(NO3)3·6H2O and Ce(NO3)3·6H2O in 20 mL of deionized water, and the molar ratio of Tb 3+ :Ce 3+ is 8:1. Stir well for 30 min to form a transparent precursor solution. Dissolve 0.0316 g of NaH2PO4·2H2O in 10 mL of deionized water, then slowly add it to the above precursor solution and stir for 2 h. Centrifuge the white precipitate at 10000 rpm for 10 min, wash with deionized water three times, and freeze-dry to obtain a white TbPO4:Ce powder.

[0089] Weigh 4 mg of TbPO4:Ce in 2 mL of PDDA (20 wt.%), stir for 24 h, centrifuge, wash with deionized water three times to remove excess PDDA, and re-disperse the product in 2 mL of deionized water. Finally, we get TbPO4:Ce with widely distributed positive charges on the surface.

[0090] (2) Preparation of Ag NPs

[0091] 7mg AgNO3 was dissolved in 100mL deionized water, after 10min of stirring, it was transferred into a three-necked flask, heated at 97℃ for 20min, then 2.0mL sodium citrate (1%, g / mL) was slowly added into the flask under stirring at 900rpm, kept refluxing for 3h, then cooled to room temperature, the solution was centrifuged at 13000rpm for 15min, the supernatant was removed, and the solution was re-dispersed in 100mL deionized water at a concentration of 0.1mg / mL, and the solution was stored at 4℃ in the dark.

[0092] (3) Preparation of electrochemiluminescence sensor

[0093] 1) A glassy carbon electrode (GCE) with a diameter of 3mm was polished and polished, and the electrode was thoroughly cleaned by ultrasonicating with dilute nitric acid, anhydrous ethanol and deionized water for 3min respectively;

[0094] 2) 5μL of positively charged TbPO4:Ce with a concentration of 2mg / mL was dropped on the surface of GCE and naturally dried to obtain GCE / TbPO4:Ce;

[0095] 3) 5μL of Ag NPs with a concentration of 0.1mg / mL was dropped on the surface of the electrode and naturally dried to obtain GCE / TbPO4:Ce / Ag NPs;

[0096] 2. The electrochemiluminescence sensor of the application is used for ECL detection

[0097] (1) The prepared GCE / TbPO4:Ce / Ag NPs are used as a working electrode, a platinum wire is used as a counter electrode, and Ag / AgCl is used as a reference electrode, and the concentration of lincomycin is detected by electrochemiluminescence detection in a three-electrode system;

[0098] (2) The parameters of the electrochemiluminescence detection instrument are set as follows: the scanning rate is set to 100mV / s, and the high voltage of the photomultiplier tube is set to 800V;

[0099] (3) The parameters of the electrochemical workstation are set as follows: the cyclic voltammetry scanning voltage range is 0~-1.8V; and the scanning rate is set to 100mV / s;

[0100] (4) A PBS buffer solution containing K2S2O8 is used to record the electrochemiluminescence signal intensity; the PBS buffer solution is a 0.1mol / L Na2HPO4 / NaH2PO4 buffer solution containing 0.1mol / L K2S2O8 at pH 7.5.

[0101] As described in Example 3, the Tb 3+ :Ce 3+With a molar concentration ratio of 8:1, PDDA was used to alter the surface charge of TbPO4:Ce, adsorbing negatively charged Ag NPs. The final ECL results of the GCE / TbPO4:Ce / Ag NPs sensor are as follows: Figure 4 As shown in B. By comparison Figure 4 A and Figure 4 B shows that after the charge was changed by PDDA, the ECL intensity of the sensor increased to 50000 a.u. and the stability was significantly improved, with an RSD of 0.78%.

[0102] Comparative Example 1

[0103] The preparation method of Example 1 was followed, except that in step 1-(1) when preparing TbPO4:Ce, pure TbPO4 and pure CePO4 and Tb were synthesized respectively. 3+ :Ce 3+ TbPO4:Ce with a molar concentration ratio of 4:1.

[0104] The products prepared in Example 1 and Comparative Example 1 were subjected to electrochemiluminescence (ECL) testing, and the results are as follows:

[0105] The difference between Comparative Example 1 and Example 1 lies in Tb 3+ :Ce 3+ ECL intensity results for different molar concentration ratios are as follows: Figure 2 As shown. The pure TbPO4 (curve a) and pure CePO4 (curve d) prepared in Comparative Example 1, and Tb... 3+ :Ce 3+ For a TbPO4:Ce concentration ratio of 4:1 (curve c), the ECL value of pure TbPO4 is 8000 a.u., and the ECL value of pure CePO4 is approximately 4700 a.u. Tb 3+ :Ce 3+ When the molar concentration ratio is 4:1, the ECL value of TbPO4:Ce is approximately 7800 a.u., which is significantly lower than the result of Example 1.

[0106] Comparative Example 2

[0107] Pure TbPO4 and pure CePO4 were synthesized according to the synthesis method of Example 1. The two were then processed according to the Tb... 3+ :Ce 3+ The mixtures were combined in a molar ratio of 8:1 to prepare a solution with a concentration of 2 mg / mL. An electrochemiluminescence sensor was prepared according to the method described in Example 1, and ECL testing was performed.

[0108] The difference between Comparative Example 2 and Example 1 lies in Tb 3+ :Ce 3+ Different doping methods, such asFigure 3 As shown, TbPO4:Ce synthesized by Example 1 has more stable and higher electrochemiluminescence performance (curve a, RSD = 1.74%), while the ECL intensity and stability of the simple stirred mixture of pure TbPO4and pure CePO4are poor (curve b, ECL value is 4900 a.u., RSD = 3.45%), because TbPO4:Ce is Ce bonded to the structure of TbPO4by in-situ doping, which is closer to the central atom Tb and has stronger energy transfer effect, ultimately resulting in significantly improved ECL performance.

[0109] Comparative Example 3

[0110] According to the preparation method of Example 3, except that the pH of PBS in 2-(4) is 6, 6.5, 7, 8, 8.5, 9 respectively, and the other conditions are the same, ECL test is carried out.

[0111] The difference between Comparative Example 3 and Example 3 is mainly that the pH of PBS buffer is different, and the test results are as shown in Figure 5 The electrochemiluminescence test is carried out in PBS with different pH conditions, and when pH = 7.5, the ECL intensity of GCE / TbPO4:Ce is the highest. This is mainly because in acidic conditions, protons are easily reduced to hydrogen, inhibiting the reduction of S2O8 2- ; in alkaline conditions, excess anions are dispersed around the electrode, hindering the approach of SO4 ·- to the electrode surface, both of which will lead to a decrease in ECL signal intensity.

[0112] Example 4

[0113] (1) Preparation of TbPO4:Ce

[0114] Dissolve 0.0408 g of Tb(NO3)3·6H2O and Ce(NO3)3·6H2O in 20 mL of deionized water, and the molar concentration ratio of Tb 3+ :Ce 3+ is 8:1. Stir well for 30 min to form a transparent precursor solution. Dissolve 0.0316 g of NaH2PO4·2H2O in 10 mL of deionized water, then slowly add it to the above precursor solution and stir for 2 h. Centrifuge the white precipitate at 10000 rpm for 10 min, wash with deionized water three times, and freeze-dry to obtain TbPO4:Ce white powder.

[0115] Weigh 4 mg of TbPO4:Ce in 2 mL of PDDA (20 wt.%), stir for 24 h, centrifuge, wash with deionized water three times to remove excess PDDA, and redisperse the product in 2 mL of deionized water to obtain TbPO4:Ce with widely distributed positive charges on the surface.

[0116] (2) Preparation of AgNPs

[0117] 7mg AgNO3 was dissolved in 100mL deionized water, after 10min of stirring, it was transferred to a three-necked flask, heated at 97℃ for 20min, then 2.0mL sodium citrate (1%, g / mL) was slowly added dropwise to the flask under stirring at a speed of 900rpm, kept refluxing for 3h, then cooled to room temperature, the solution was centrifuged at 13000rpm for 15min, the supernatant was removed, and the solution was re-dispersed in 100mL deionized water at a concentration of 0.1mg / mL, and the solution was stored at 4℃ in the dark.

[0118] (3) Preparation of electrochemiluminescence aptamer sensor

[0119] 1) A glassy carbon electrode (GCE) with a diameter of 3mm was polished and polished, and the electrode was thoroughly cleaned by ultrasonicating with dilute nitric acid, anhydrous ethanol and deionized water for 3min respectively;

[0120] 2) 5μL of TbPO4:Ce with positive charge distribution with a concentration of 2mg / mL was dropped on the surface of GCE, and naturally dried to obtain GCE / TbPO4:Ce;

[0121] 3) 5μL of Ag NPs with a concentration of 0.1mg / mL was dropped on the surface of the electrode, and naturally dried to obtain GCE / TbPO4:Ce / Ag NPs;

[0122] 4) 3μL of thiolated lincomycin aptamer (apt-SH) with a concentration of 3μmol / L was dropped on the surface of the electrode, and stored at 25℃ for 6h, and then washed with ultrapure water to obtain apt-SH / Ag NPs / TbPO4:Ce / GCE.

[0123] 2. The electrochemiluminescence aptamer sensor according to the present application is used for the detection of lincomycin

[0124] (1) The prepared electrochemiluminescence aptamer sensor is used as a working electrode, a platinum wire is used as a counter electrode, and Ag / AgCl is used as a reference electrode, and the concentration of lincomycin is detected by electrochemiluminescence detection method in a three-electrode system;

[0125] (2) The parameters of the electrochemiluminescence detection instrument are set as follows: the scanning rate is set to 100mV / s, and the high voltage of the photomultiplier tube is set to 800V;

[0126] (3) The parameters of the electrochemical workstation are set as follows: the cyclic voltammetry scanning voltage range is 0~ -1.8V; and the scanning rate is set to 100mV / s;

[0127] (4) Using PBS buffer solution containing K2S2O8, record the electrochemiluminescence signal intensity generated by detecting different concentrations of lincomycin by electrochemiluminescence detection method; the PBS buffer solution: 0.1 mol / L K2S2O8 in 0.1 mol / L Na2HPO4 / NaH2PO4 buffer solution with pH 7.5.

[0128] (5) The prepared electrochemiluminescence aptamer sensor is immersed in 200 μL of a series of different concentrations of lincomycin standard solution for 45 minutes, and the recorded electrochemiluminescence intensity before and after immersion is recorded respectively, and the value of ΔECL (ΔECL = ECL value before immersion - ECL value after immersion) is calculated.

[0129] Figure 6 A is the ECL-time curve of the obtained electrochemiluminescence aptamer sensor for detecting a series of concentrations of lincomycin, Figure 6 B is the calculated ΔECL versus the logarithm of the concentration of lincomycin curve. Figure 6 From A, it can be seen that as the concentration of lincomycin increases, the ECL intensity of the sensor gradually decreases. Figure 6 As described in A, the concentration of Lin increases from 1×10 -16 M to 1×10 -4 M, the ECL intensity value of the sensor decreases from ≈40000 a.u. to ≈16000 a.u., and this phenomenon is due to the specific recognition of apt-SH to lincomycin, which causes apt-SH to be adsorbed on the electrode surface together with lincomycin (for details, see Figure 1 ), hindering the electron transfer on the electrode surface, resulting in a decrease in ECL value. The greater the concentration of lincomycin, the more lincomycin is adsorbed on the electrode surface, and the lower the ECL value. Before binding with lincomycin, the ECL value of the electrochemiluminescence sensor (apt-SH / Ag NPs / TbPO4:Ce / GCE electrode) is about 42000 a.u., as shown in the inset of Figure 6 B. After calculation, ΔECL has a good linear relationship with the logarithm of the concentration of lincomycin (as shown in Figure 6 B), and the linear regression equation is: ΔECL = 35095.59 + 2053.50*lgC, the correlation coefficient (R 2 ) = 0.9997, the linear range is 1.0×10 -16 ~ 1.0×10 -4 mol / L, and the detection limit is 1.78×10 -17 mol / L.

[0130] Selective detection

[0131] The electrochemiluminescence aptamer sensor described in the application is used for detecting lincomycin

[0132] (1) The prepared apt-SH / AgNPs / TbPO4:Ce / GCE was immersed in 200 μL of 1.0×10 -6 mol·L -1 Lincomycin standard solution and 200 μL of 1.0×10 -4 mol·L -1 chloramphenicol, kanamycin, tetracycline, enrofloxacin, norfloxacin and a mixture of several antibiotics. The prepared electrochemiluminescence aptamer sensor was used as a working electrode, a platinum wire was used as a counter electrode, and Ag / AgCl was used as a reference electrode. The electrochemiluminescence intensity before and after immersion was recorded in a three-electrode system in a PBS buffer solution containing K2S2O8 at pH = 7.5 by electrochemiluminescence method. The ΔECL value (ΔECL = ECL value before immersion - ECL value after immersion) was obtained, and the final results are shown in Figure 7 Figure 7 It can be seen that the concentration of the interfering substance is 100 times that of lincomycin, and the ECL quenching value of lincomycin on the aptamer sensor is significantly greater than that of other interfering substances, and the presence of other interfering substances has negligible effect on the detection of lincomycin, indicating that the sensor has good selectivity.

[0133] Actual sample detection

[0134] In order to test the practical application value of the sensor, standard addition method was used to detect Lin in river water and tap water. The apt-SH / AgNPs / TbPO4:Ce / GCE prepared in Example 4 was used as a working electrode, a platinum wire was used as a counter electrode, and Ag / AgCl was used as a reference electrode. The electrochemiluminescence method was used for detection in a three-electrode system. No Lin was found in the actual tap water and river water, and then recovery experiments were carried out, and the results are shown in Table 1. The recovery rate was between 94.4% and 103.5%, and the RSD of the test results was less than 5%. The above results show that the aptamer sensor constructed in this paper has potential applicability for analyzing the content of lincomycin in actual samples.

[0135] Table 1 Detection results of lincomycin in actual samples (n = 3)

[0136]

[0137]

[0138] ​The above shows that the application relates to a preparation method and application of an electrochemiluminescence aptamer sensor for detecting lincomycin prepared from TbPO4:Ce, on one hand, PDDA is adopted to improve the surface charge of TbPO4:Ce, and more possibilities are added to the use of TbPO4:Ce. On the other hand, the electrochemiluminescence intensity of TbPO4:Ce is greatly improved by adopting silver nanoparticles, and the sensitivity of the prepared electrochemiluminescence sensor is improved. The prepared electrochemiluminescence aptamer sensor has the advantages of high sensitivity, good selectivity and wide linear range.

Claims

1. A preparation method of an electrochemiluminescence aptamer sensor for detecting lincomycin based on TbP04:Ce, characterized in that, The preparation of the electrochemiluminescence aptamer luminescence sensor comprises the following steps: (1) drop coating the PDDA-modified TbPO4:Ce solution on the surface of the GCE, and naturally drying to obtain GCE / TbPO4:Ce; The preparation steps of the PDDA-modified TbPO4:Ce are as follows: Tb(NO3)3-6H2O and Ce(NO3)3-6H2O were dissolved in deionized water, in which the molar concentration ratio of Tb 3+ :Ce 3+ was controlled to be 10:1-6:1 to control the amount of added Ce(NO3)3-6H2O, and a transparent precursor solution was formed by sufficient stirring; NaH2PO4-2H2O was dissolved in deionized water, then slowly added dropwise into the above precursor solution and continuously stirred to obtain a white precipitate, which was centrifuged, washed, and dried to obtain a TbPO4:Ce white powder; the TbPO4:Ce was weighed and stirred in a PDDA aqueous solution, centrifuged, washed, and the washed product was redispersed in deionized water to finally obtain a PDDA-modified TbPO4:Ce; (2) drop coating the Ag NPs solution on the surface of the electrode, and naturally drying to obtain GCE / TbPO4:Ce / Ag NPs; (3) drop coating the mercapto lincomycin aptamer solution on the surface of the electrode, incubating, and washing to obtain the apt-SH / Ag NPs / TbPO4:Ce / GCE sensor.

2. The method for preparing the electrochemiluminescence aptamer sensor based on TbP04:Ce for detecting lincomycin according to claim 1, characterized in that, The concentration of the PDDA-modified TbPO4:Ce solution is 1-10 mg / mL; the concentration of the Ag NPs solution is 0.05-0.2 mg / mL; the concentration of the mercapto lincomycin aptamer solution is 1-7 μmol / L; and the volume ratio of the PDDA-modified TbPO4:Ce solution, the Ag NPs solution and the mercapto lincomycin aptamer solution is 5:5:

3.

3. The method for preparing the electrochemiluminescence aptamer sensor based on TbP04:Ce for detecting lincomycin according to claim 1, characterized in that, The mass-volume ratio of TbPO4:Ce and the PDDA aqueous solution is 4 mg:1-5 mL.

4. The method for preparing the electrochemiluminescence aptamer sensor based on TbP04:Ce for detecting lincomycin according to claim 3, characterized in that, The molecular weight of PDDA ranges from 50,000 to 100,000.

5. The method for preparing the electrochemiluminescence aptamer sensor based on TbP04:Ce for detecting lincomycin according to claim 1, characterized in that, The particle size of the Ag NPs ranges from 10.5 to 30.0 nm.

6. The method for preparing the electrochemiluminescence aptamer sensor based on TbP04:Ce for detecting lincomycin according to claim 1, wherein, The 5' end of the DNA strand of the mercapto lincomycin aptamer is connected with a mercapto group, and the DNA sequence is: 5'-SH-(CH2)6-CGC GTG ATG TGG TCG ATG CGA TAC GGT GAG TCG CGC CAC GGC TAC ACACGT CTC AGC GA-3', and the lincomycin aptamer is prepared into a 10 μmol / L stock solution by using a Tris-HCl solution, and then is diluted step by step for use.

7. The electrochemiluminescence aptamer sensor based on TbPO4:Ce for detecting lincomycin prepared by the method according to any one of claims 1-6.

8. The application of the electrochemiluminescence aptamer sensor according to claim 7 in detecting lincomycin.

9. Use according to claim 8, characterized in that: The detection steps are as follows: (1) preparation of standard solutions containing lincomycin with different concentrations: Lincomycin solid standard was prepared into 1.0 x 10 -3 mol / L solution using deionized water, and diluted into a series of lincomycin standard solutions with different concentrations, the concentration range was 1.0 x 10 -3 ~1.0 x 10 -17 mol / L. (2) drawing of a standard curve: A three-electrode system is adopted, the prepared apt-SH / Ag NPs / TbPO4:Ce / GCE electrochemiluminescence aptamer sensor is used as a working electrode, a platinum wire is used as a counter electrode, and Ag / AgCl is used as a reference electrode, the prepared electrochemiluminescence aptamer sensor is immersed in a series of lincomycin standard solutions with different concentrations prepared in step (1) for 30-70 min, the electrode is taken out and washed with a PBS buffer solution, naturally dried, and then electrochemiluminescence test is performed, a linear relationship between the lincomycin concentration and the electrochemiluminescence intensity is established, and a corresponding linear regression equation is obtained; (3) Actual sample detection: the standard addition method was used to test the lincomycin in the actual sample by ECL, and the ECL intensity obtained was calculated by the linear regression equation obtained in step (2), so as to obtain the lincomycin concentration in the sample.

10. Use according to claim 9, characterized in that: The electrochemiluminescence test parameters were set as follows: scanning rate: 100 mV / s, photomultiplier high voltage: 800 V; cyclic voltammetry scanning voltage range: 0 ~ -1.8 V; the PBS buffer solution: 0.1 mol / L Na2HPO4 / NaH2PO4 buffer solution containing 0.1 mol / L K2S2O8, pH 7.5.

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