CN114965644A - Electrochemical luminophor, electrochemical luminescence aptamer sensor, and preparation method and application thereof - Google Patents
Electrochemical luminophor, electrochemical luminescence aptamer sensor, and preparation method and application thereof Download PDFInfo
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- CN114965644A CN114965644A CN202210640465.8A CN202210640465A CN114965644A CN 114965644 A CN114965644 A CN 114965644A CN 202210640465 A CN202210640465 A CN 202210640465A CN 114965644 A CN114965644 A CN 114965644A
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- 108091023037 Aptamer Proteins 0.000 title claims abstract description 42
- 238000004020 luminiscence type Methods 0.000 title claims abstract description 21
- 238000002360 preparation method Methods 0.000 title claims abstract description 21
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- OJMMVQQUTAEWLP-UHFFFAOYSA-N Lincomycin Natural products CN1CC(CCC)CC1C(=O)NC(C(C)O)C1C(O)C(O)C(O)C(SC)O1 OJMMVQQUTAEWLP-UHFFFAOYSA-N 0.000 claims abstract description 84
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- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y20/00—Nanooptics, e.g. quantum optics or photonic crystals
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Abstract
The invention belongs to the field of electrochemical luminescence detection, and relates to an electrochemical luminous body, an electrochemical luminescence aptamer sensor, a preparation method and application thereof, wherein the electrochemical luminous body is formed by loading a lincomycin aptamer on the surface of a glassy carbon electrode modified by UCNPs @ Au/PTCA-PATP nano composite material, and the UCNPs @ Au/PTCA-PATP nano composite material is a product formed by combining gold-sulfur bonds between conversion nanoparticles modified by gold nanoparticles and sulfhydrylation perylene tetracarboxylic acid; lincomycin aptamer nucleosidesThe sequence is apt: 5'-CGCG TGAT GTGG TCGA TGCG ATAC GGTG AGTC GCGC CACG GCTA CACA CGTC TCAG CGA-3'. The sensor can realize sensitive detection on lincomycin, and the minimum detection limit is 2.4 multiplied by 10 ‑16 mol/L。
Description
Technical Field
The invention belongs to the field of electrochemical luminescence detection, and relates to an electrochemical luminous body, an electrochemical luminescence aptamer sensor, and a preparation method and application thereof.
Background
Lincomycin (Lincomycin, Lin for short) as a lincosamide antibiotic produced by fermentation of streptomyces has strong bacteriostatic activity and wide antibacterial spectrum, and is widely applied to livestock breeding. The problem of lincomycin residues in water environments has become a public health problem. The highest residual limit of lincomycin in animal food is 100ng/mL specified by the ministry of agriculture in China.
The common methods for detecting lincomycin at present comprise the following steps: the method comprises the following steps of photoelectric method, enzyme-linked immunosorbent assay, molecular imprinting, high performance liquid chromatography, fluorescence method and the like, and the methods have the defects of expensive instrument and equipment, complex sample pretreatment, low sensitivity, poor stability and the like. Therefore, the development of a convenient and efficient analysis method for detecting the lincomycin content in the water body is of great significance.
Electrochemiluminescence (ECL) is also called electrochemiluminescence, and combines wide electrochemical detection range, simple instrument and high sensitivity of chemiluminescence, good reproducibility, stable reagent and easy control. Moreover, the anti-interference capability of the sensor can be enhanced by introducing the nucleic acid aptamer chain with low price and strong specificity. ECL is a method for realizing trace analysis by acquiring a luminous intensity spectrum with the aid of optical instruments such as a photomultiplier and establishing the relationship between the luminous intensity spectrum and an object to be detected.
Disclosure of Invention
The invention aims to provide an electrochemical luminophor, an electrochemical luminescence aptamer sensor, and preparation methods and applications thereof, aiming at the defects of lincomycin detection in the prior art. The electrochemical luminophor is a UCNPs @ Au/PTCA-PATP nano composite material, and is a substance which is formed by modifying up-conversion nano particles UCNPs by gold nanoparticles and combining the up-conversion nano particles UCNPs with sulfhydrylation perylene tetracarboxylic acid PTCA-PATP through gold-sulfur bonds. The UCNPs @ Au and the PTCA-PATP are tightly combined through a gold-sulfur bond, the perylene substance has good electrochemical activity and good film forming property, the gold nanoparticles have excellent conductivity, the relay barrier between a luminescent material and an electrode can be reduced, the up-conversion nanoparticles (UCNPs) have stable cathode signals and excellent electro-optical characteristics, the combination of the UCNPs @ Au and the PTCA-PATP has a good synergistic effect, and the sensitivity and the strong anti-interference capability can be effectively improved.
When the sensor is used as an electrochemical luminophor of an electrochemical luminescence aptamer sensor, the sensitivity and stability of electrochemical luminescence are obviously improved, and the electrochemical luminescence aptamer sensor (apt/UCNPs @ Au/PTCA-PATP/GCE sensor for short) is obtained by loading the aptamer matched with a target molecule to be detected through electrostatic action, so that the sensor can specifically identify the target molecule and improve the selectivity of the target molecule (such as the detection of antibiotics such as lincomycin).
Further, gold nanoparticle modified up-conversion nanoparticles UCNPs (UCNPs @ Au) are obtained by the following method: uniformly dispersed with upconverting nanoparticles UCNPs and HAuCl 4 The ethylene glycol solution is stirred and reacted for at least 30min at the temperature of 160-180 ℃ under the protection of argon, and then is gradually cooled to the room temperature during stirring.
More specifically, the reaction product of Y 2 O 3 、Yb 2 O 3 、Tm 2 O 3 Respectively dissolving in trifluoroacetic acid, stirring under sealed condition, filtering, and drying to obtain white solid (Y (CF) 3 COO) 3 、Yb(CF 3 COO) 3 、Tm(CF 3 COO) 3 ). Dispersing the solid in oleic acid and 1-octadecene according to a certain mass ratio, stirring in vacuum, and then adding NH 4 And F and a methanol solution of NaOH, continuously stirring, centrifuging, washing and drying to obtain a white solid, namely UCNPs. Finally, UCNPs and HAuCl are mixed 4 Mixing, ultrasonic processing, stirring, centrifuging, washing and drying to obtain dark purple solid, namely UCNPs @ Au.
Further, gold nanoparticle modified up-conversion nanoparticles UCNPs and PTCA-PATP are respectively dispersed in DMF, and then the mass ratio of UCNPs to PTCA-PATP is 1: 2, mixing, performing ultrasonic thorough mixing in an ice-water bath, stirring at room temperature until the mixture is fully combined, collecting the precipitate, and performing vacuum drying to obtain the UCNPs @ Au/PTCA-PATP.
More specifically, UCNPs @ Au and PTCA-PATP are respectively dispersed in DMF to form 1mg/mL of UCNPs @ Au-DMF solution and PTCA-PATP-DMF solution, the two solutions are ultrasonically mixed (m (UCNPs @ Au)/m (PTCA-PATP) ═ 1: 2), stirred (the time is generally 16h), and after the two solutions are fully reacted, the product, namely UCNPs @ Au/PTCA-PATP, is obtained through centrifugation, washing and drying.
Further, the preparation method of the PTCA-PATP comprises the following steps: mixing perylene-3, 4,9, 10-tetracarboxylic dianhydride, imidazole and 4-aminothiophenol, heating and stirring under the protection of argon (preferably 100 ℃, 3h), cooling, dispersing in ethanol, adding a certain amount of hydrochloric acid, continuously stirring (preferably 16h), filtering, washing and drying to obtain the PTCA-PATP.
The invention also provides an electrochemiluminescence aptamer sensor for specifically detecting lincomycin, which is formed by loading an aptamer containing 5'-CGCG TGAT GTGG TCGA TGCG ATAC GGTG AGTC GCGC CACG GCTA CACA CGTC TCAG CGA-3' base sequence on the surface of a glassy carbon electrode modified by UCNPs @ Au/PTCA-PATP nano composite material.
The invention also provides a preparation method of the electrochemical luminescence aptamer sensor for specifically detecting lincomycin, which comprises the following steps:
preparation of apt/UCNPs @ Au/PTCA-PATP/GCE sensor:
polishing the glassy carbon electrode, sequentially performing ultrasonic treatment on the glassy carbon electrode by using nitric acid, absolute ethyl alcohol and deionized water respectively, naturally drying the glassy carbon electrode for later use, transferring ultrapure water dispersion (1mg/mL) of UCNPs @ Au/PTCA-PATP to the surface of the clean glassy carbon electrode, and drying the ultrapure water dispersion at room temperature to obtain the UCNPs @ Au/PTCA-PATP modified glassy carbon electrode; and then, dropwise adding the aptamer to the surface of UCNPs @ Au/PTCA-PATP/GCE, electrostatically combining the aptamer and the UCNPs @ Au/PTCA-PATP, and then placing the apt/UCNPs @ Au/PTCA-PATP/GCE modified electrode in a refrigerator for 6-8 hours to obtain the ECL aptamer sensor.
Finally, the present invention provides a method for the use of an electrochemiluminescent aptamer sensor for the specific detection of lincomycin, comprising the steps of:
firstly, preparing a series of lincomycin standard solutions with different concentrations, preparing lincomycin into a solution by using water, and then adding K 2 S 2 O 8 In PBS buffer solution to obtain a series of lincomycin standard solutions with different concentrations, wherein the concentration range is 1.0 multiplied by 10 -15 mol/L～1.0×10 -7 mol/L。
Then establishing a linear regression equation, taking the apt/UCNPs @ Au/PTCA-PATP/GCE sensor as a working electrode, a platinum electrode as an auxiliary electrode and Ag/AgCl as a reference electrode to form a three-electrode system, and placing the three-electrode system in the series of lincomycin standard solutions with different concentrations and the K-containing lincomycin standard solution 2 S 2 O 8 Soaking the solution in PBS buffer solution, performing cyclic voltammetry scanning on the solution at a scanning speed of 0.1V/s and a photomultiplier tube high voltage of 700V within an electrochemical window range of-1.8-0V, recording a potential-luminous intensity curve (E-ECL), and establishing a linear relation between a luminous intensity difference before and after adding lincomycin and a lincomycin concentration logarithm value to obtain a corresponding linear regression equation.
And finally, carrying out sample detection: the sample to be tested is pretreated (the pretreatment is mainly to filter impurities, such as taking 200mL of pond water sample, naturally standing for a period of time, centrifuging at 6000rpm for 10min, sucking supernatant, filtering with 0.22 μm filter membrane), and testing according to the same electrochemiluminescence test conditions (the filtrate is prepared to contain 0.05mol/L K) 2 S 2 O 8 Taking 25mL of the obtained solution in a detection pool, recording the luminous intensity, and calculating the concentration of lincomycin in the sample to be detected by using a linear regression equation corresponding to the obtained standard curve.
Further, containing K 2 S 2 O 8 The preparation method of the PBS buffer solution comprises the following steps: by using0.05mol/L K in 0.1mol/L PBS buffer solution at pH7.4 2 S 2 O 8 PBS buffer solution of (1).
Further, in an electrochemiluminescence test, the photomultiplier has a high voltage of 700V, a sweeping speed of 0.1V/s and a soaking time of 35min (a detection value is stable at 35 min).
Compared with the prior art, the invention has the following beneficial effects: the invention designs an electrochemiluminescence aptamer sensor of perylene derivatives based on up-conversion nanoparticles, UCNPs @ Au and PTCA-PATP are combined by gold-sulfur bonds, an electrochemiluminescence body with high strength and stable performance can be obtained, the invention fully utilizes the advantages of the aptamer and the electrochemiluminescence sensor, and realizes the sensitive detection of lincomycin by the mechanism of the enhancement of the ECL signal strength of the system by the lincomycin, and the detection range of the invention is 1.0 multiplied by 10 -15 ～1.0×10 -7 mol/L, minimum detection limit of 2.4X 10 -16 mol/L. The method for detecting lincomycin has the advantages of simple operation, good selectivity, low detection cost, high sensitivity and good stability.
Drawings
FIG. 1 shows the scanning electron micrographs of PTCA-PATP (A), UCNPs @ Au (B), UCNPs @ Au/PTCA-PATP (C), and the EDS chart of UCNPs @ Au/PTCA-PATP (D).
FIG. 2 is a graph of ECL-potential curves for different concentrations of lincomycin.
The concentration of the lincomycin is as follows from front to back according to the peak value of the curve: 1.0X 10 -15 mol/L、1.0×10 -14 mol/L、1.0×10 -13 mol/L、1.0×10 -12 mol/L、1.0×10 -11 mol/L、1.0×10 -10 mol/L、1.0×10 -9 mol/L、1.0×10 -8 mol/L、1.0×10 -7 mol/L。
FIG. 3 is a standard curve of the difference in luminescence intensity before and after addition of lincomycin and the logarithm of the lincomycin concentration.
FIG. 4 is the specific detection of lincomycin by the sensor of example 1.
FIG. 5 is ECL-voltage diagram of several modified electrodes of GCE, UCNPs @ Au/GCE, PTCA-PATP/GCE, Au/PTCA-PATP/GCE, UCNPs/PTCA-PATP/GCE and UCNPs @ Au/PTCA-PATP/GCE in the invention.
Detailed Description
The present invention is not limited to the following embodiments, and those skilled in the art can implement the present invention in other embodiments according to the disclosure of the present invention, or make simple changes or modifications on the design structure and idea of the present invention, and fall into the protection scope of the present invention. It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict.
The invention is described in more detail below with reference to the following examples:
the embodiment provides an electrochemiluminescence aptamer sensor for specifically detecting lincomycin, which is formed by loading an aptamer containing 5'-CGCG TGAT GTGG TCGA TGCG ATAC GGTG AGTC GCGC CACG GCTA CACA CGTC TCAG CGA-3' base sequence on the surface of a glassy carbon electrode modified by UCNPs @ Au/PTCA-PATP nano composite material, wherein the UCNPs @ Au/PTCA-PATP nano composite material is a product combined by gold-sulfur bonds between up-conversion nanoparticles (UCNPs @ Au) and thiolated perylene tetracarboxylic acid (PTCA-PATP), and the electrochemiluminescence aptamer sensor can be obtained by the method described in the embodiment 1, and specifically comprises the following steps:
example 1:
(1) preparation of UCNPs @ Au:
firstly weighing 0.3388g Y 2 O 3 ，0.2758g Yb 2 O 3 ，0.1158g Tm 2 O 3 Respectively dissolving in 14mL/8mL/4mL 50% trifluoroacetic acid water solution, stirring at 85 deg.C under sealed condition until the solution becomes clear, filtering to remove insoluble substances, slowly evaporating the obtained clear solution at 100-120 deg.C to obtain white solid, namely Y (CF) 3 COO) 3 、Yb(CF 3 COO) 3 、Tm(CF 3 COO) 3 . Subsequently, 0.5950g Y (CF) was added to a flask containing 12mL oleic acid and 15mL 1-octadecene 3 COO) 3 、0.3072g Yb(CF 3 COO) 3 、0.0052g Tm(CF 3 COO) 3 Stirring vigorously under vacuum, heating the mixture at 120 deg.C to remove water and oxygen until no bubbles are formed, and removing the solvent from the mixtureThis formed a clear solution containing the lanthanide-oleate complex. The solution was then allowed to cool naturally to room temperature under an argon atmosphere. Thereafter, 12mL of a solution containing NH was added 4 A solution of F (0.2963g) and NaOH (0.2100g) in methanol was stirred at 50 ℃ for 20min, the methanol was evaporated, then the solution was heated to 300 ℃ under argon atmosphere for 1h, and then the solution was rapidly cooled to room temperature. The upconverting nanoparticles obtained by precipitation by adding ethanol to the reaction solution were finally centrifuged for 10min by 9000rmp, washed three times with ethanol/cyclohexane (v: v ═ 1: 1), dried for 12h at 60 ℃ in vacuo to give white solids, i.e. UCPNs. 50mg of UCNPs were dispersed in 10mL of ethylene glycol and 20. mu.M of HAuCl was added 4 Mixing 10mL of ethylene glycol (mixed with 300mg of PVP) with the solution, heating to 180 ℃ under the protection of argon, keeping the temperature and stirring for 30min, continuously stirring for 16h, gradually cooling to room temperature, washing with acetone, centrifuging, and drying in vacuum to obtain a dark purple solid, namely UCNPs @ Au.
(2) Preparation of PTCA-PATP:
0.3209g of perylene-3, 4,9, 10-tetracarboxylic dianhydride (PTCDA), 6.1566g of imidazole and 0.7850g of 4-aminothiophenol are heated to 100 ℃ under argon protection and stirred for 3 h. After the reaction was cooled to room temperature, 100mL of ethanol and 300mL of hydrochloric acid (2M) were added, stirring was continued for 16h, followed by vacuum filtration through a 0.45 μ M membrane filter, washing with ultrapure water until the pH was neutral, and the solid was placed in a vacuum oven and dried at 60 ℃ for 12h to obtain a wine-red solid, i.e., PTCA-PATP.
(3) Preparation of UCNPs @ Au/PTCA-PATP:
respectively dispersing the UCNPs @ Au and the PTCA-PATP prepared in the steps (1) and (2) in DMF to form a 1mg/mL UCNPs @ Au-DMF solution and a PTCA-PATP-DMF solution, ultrasonically mixing the two solutions (m (UCNPs @ Au)/m (PTCA-PATP) ═ 1: 2) uniformly, stirring for 16h, and after the two solutions fully react, centrifuging, washing and drying to obtain a product, namely the UCNPs @ Au/PTCA-PATP.
(4) Preparation of modified electrode apt/UCNPs @ Au/PTCA-PATP/GCE:
polishing the glassy carbon electrode, sequentially performing ultrasonic treatment on the glassy carbon electrode by using nitric acid, absolute ethyl alcohol and deionized water respectively, naturally drying the glassy carbon electrode for later use, transferring 5 mu L of solution of UCNPs @ Au/PTCA-PATP to the surface of the clean glassy carbon electrode by using a liquid transfer gun, and drying the solution at room temperature to obtain the UCNPs @ Au/PTCA-PATP modified glassy carbon electrode. Then, 5. mu.L of aptamer of 3. mu. mol/L is dripped on the surface of UCNPs @ Au/PTCA-PATP/GCE, and the aptamer and the UCNPs @ Au/PTCA-PATP are combined through static electricity to obtain apt/UCNPs @ Au/PTCA-PATP/GCE. And finally, placing the apt/UCNPs @ Au/PTCA-PATP/GCE modified electrode in a refrigerator at 4 ℃ for 6-8h to obtain the ECL aptamer sensor.
The recognition molecule apt sequence is: apt: 5'-CGCG TGAT GTGG TCGA TGCG ATAC GGTG AGTC GCGC CACG GCTA CACA CGTC TCAG CGA-3' (manufacturer is Industrial bioengineering (Shanghai) GmbH)
(5) Drawing of standard curve
Forming a three-electrode system by taking a modified electrode apt/UCNPs @ Au/PTCA-PATP/GCE as a working electrode, a platinum electrode as an auxiliary electrode and Ag/AgCl as a reference electrode, and placing the three-electrode system in a series of lincomycin concentrations (1.0 multiplied by 10) -15 mol/L、1.0×10 -14 mol/L、1.0×10 -13 mol/L、1.0×10 -12 mol/L、1.0×10 -11 mol/L、1.0×10 -10 mol/L、1.0×10 -9 mol/L、1.0×10 -8 mol/L and 1.0X 10 -7 mol/L) contains 0.05mol/L of K 2 S 2 O 8 Soaking the solution in 0.1mol/L PBS buffer solution with the pH of 7.4 for 35min, performing cyclic voltammetry scanning on the solution at the high voltage of 700V and the scanning speed of 0.1V/s of a photomultiplier tube within the electrochemical window range of-1.8-0V, recording a potential-luminous intensity curve (E-ECL), and establishing a linear relation between the luminous intensity difference before and after adding lincomycin and the log value of the lincomycin concentration to obtain a corresponding linear regression equation: delta I ECL 6240.1245+1243.3891lgC (mol/L), the correlation coefficient (R) is 0.9993. The detection range of the linear regression equation is 1.0 multiplied by 10 -15 ～1.0×10 -7 mol/L, minimum detection limit of 2.4X 10 -16 mol/L。
(6) Detection of samples
Collecting water sample of a certain pond, standing naturally for a period of time, centrifuging to absorb supernatant, filtering with 0.22 μm filter membrane to collect filtrate, and adding water containing 0.05mol/LK 2 S 2 O 8 And (3) adjusting the pH value of the 0.1mol/L PBS buffer solution to 7.4, taking 25mL of the obtained solution for electrochemical luminescence analysis, testing according to the electrochemical luminescence test conditions in the step (4), recording the luminescence intensity, calculating the concentration of lincomycin in the sample to be detected according to the linear regression equation obtained in the step (5), and obtaining the result shown in Table 1.
Compared with the common electrochemical luminescence sensor, the electrochemical luminescence sensor for detecting lincomycin and the preparation method thereof have the following two remarkable advantages: firstly, UCNPs @ Au and PTCA-PATP are tightly combined through a gold-sulfur bond, and the synergistic effect of the UCNPs @ Au and the PTCA-PATP is embodied in ECL; secondly, the perylene substance has good electrochemical activity and good film-forming property, the gold nanoparticles have excellent conductivity, the relay barrier between the luminescent material and the electrode can be reduced, and the up-conversion nanoparticles (UCNPs) have stable cathode signals and excellent electro-optical characteristics. The invention fully utilizes the advantages of the aptamer and the electrochemical luminescence sensor, successfully realizes the sensitive detection of the lincomycin by the mechanism of the enhancement of the ECL signal intensity of the lincomycin to the system, and the sensing platform can specifically identify the lincomycin serving as the detection object and has high selectivity. The invention has important significance for popularizing the practical application of the aptamer sensor in the aspects of water environment monitoring and the like.
And the electrochemical luminescence sensor for detecting lincomycin prepared in example 1 is further subjected to anti-interference detection, wherein working electrodes after incubation of the aptamer are respectively arranged at 10 -6 M Ciprofloxacin (CIP), Chloramphenicol (CAP), Kanamycin (KAN), aureomycin (CTC), Streptomycin (STP) interferents, and at 10 -8 M lincomycin (ENR) standard solution, and detecting the working electrode in the mixed solution of the substances, wherein the detection result is shown in figure 4.
As can be seen from FIG. 4, the modified electrode with excellent electrochemical properties has a selective recognition effect on lincomycin after incubation of the aptamer, and the interference substances with 100 times concentration after mixing have a slight effect on lincomycin detection. Therefore, the working electrode can realize anti-interference selective detection of lincomycin.
Comparative example 1:
(1) preparation of UCNPs @ Au/PTCA-PATP/GCE modified electrode
Polishing the glassy carbon electrode, respectively performing ultrasonic treatment on the polished glassy carbon electrode by using nitric acid, absolute ethyl alcohol and deionized water in sequence, and naturally drying the polished glassy carbon electrode for later use. And (3) transferring 5.0 mu L of 1.0mg/mL aqueous solution of the UCNPs @ Au/PTCA-PATP material onto the surface of a clean glassy carbon electrode by using a liquid transfer gun, and drying at room temperature to obtain the UCNPs @ Au/PTCA-PATP/GCE modified electrode as a working electrode for an electrochemiluminescence test.
(2) Drawing of standard curve
A UCNPs @ Au/PTCA-PATP/GCE modified electrode is used as a working electrode, a platinum electrode is used as an auxiliary electrode, Ag/AgCl is used as a reference electrode to form a three-electrode system, and K containing 0.05mol/L 2 S 2 O 8 The pH of the solution (2) is 7.4, 0.1mol/L PBS buffer solution is used as a blank solution to detect the luminous intensity, and the three-electrode system is placed in a series of lincomycin concentrations (1.0 multiplied by 10) - 15 mol/L、1.0×10 -14 mol/L、1.0×10 -13 mol/L、1.0×10 -12 mol/L、1.0×10 -11 mol/L、1.0×10 - 10 mol/L、1.0×10 -9 mol/L、1.0×10 -8 mol/L and 1.0X 10 -7 mol/L) contains 0.05mol/L of K 2 S 2 O 8 In the 0.1mol/L PBS buffer solution with the pH of 7.4, within an electrochemical window range of-1.8-0V, the photomultiplier tube has high voltage of 700V and the sweep rate of 0.1V/s, cyclic voltammetry scanning is carried out, an E-ECL curve is recorded, a linear relation between the luminous intensity difference before and after adding lincomycin and the log value of the lincomycin concentration is established, and a corresponding linear regression equation is obtained.
(3) Detection of samples
25mL of the treated pond water was added to a solution containing 0.05mol/L of K 2 S 2 O 8 The pH of the sample solution is 7.4, and the concentration of lincomycin in the sample to be detected is calculated according to the linear regression equation corresponding to the step (2) when the sample solution is used for electrochemiluminescence detection, and the result is shown in Table 1.
Comparative example 2:
(1) preparation of apt/UCNPs @ Au/GCE modified electrode
Polishing the glassy carbon electrode, respectively performing ultrasonic treatment on the polished glassy carbon electrode by using nitric acid, absolute ethyl alcohol and deionized water in sequence, and naturally drying the polished glassy carbon electrode for later use. 5.0 mu L of 1.0mg/mL aqueous solution of UCNPs @ Au material is dripped on the surface of a clean glassy carbon electrode by using a pipetting gun, and the UCNPs @ Au/GCE modified electrode is obtained after drying at room temperature; 5.0 muL of 3.0 muM aptamer (same as example 1) is dripped on the surface of the UCNPs @ Au/GCE modified electrode, and the electrode is placed in a refrigerator at 4 ℃ for 6h to obtain the apt/UCNPs @ Au/GCE sensor which is used as a working electrode for electrochemiluminescence test.
(2) Drawing of standard curve
An apt/UCNPs @ Au/GCE modified electrode is used as a working electrode, a platinum electrode is used as an auxiliary electrode, Ag/AgCl is used as a reference electrode to form a three-electrode system, and K containing 0.05mol/L 2 S 2 O 8 0.1mol/L PBS buffer solution with pH of 7.4 as blank solution, and the three-electrode system is placed in a series of lincomycin concentrations (1.0 × 10) -15 mol/L、1.0×10 -14 mol/L、1.0×10 -13 mol/L、1.0×10 -12 mol/L、1.0×10 -11 mol/L、1.0×10 -10 mol/L、1.0×10 -9 mol/L、1.0×10 -8 mol/L and 1.0X 10 -7 mol/L) contains 0.05mol/L of K 2 S 2 O 8 In the 0.1mol/L PBS buffer solution with the pH of 7.4, within an electrochemical window range of-1.8-0V, the photomultiplier tube has high voltage of 700V and the sweep rate of 0.1V/s, cyclic voltammetry scanning is carried out, an E-ECL curve is recorded, a linear relation between the luminous intensity difference before and after adding lincomycin and the log value of the lincomycin concentration is established, and a corresponding linear regression equation is obtained.
(3) Detection of samples
25mL of the treated pond water was added to a solution containing 0.05mol/L of K 2 S 2 O 8 The pH of the sample solution (3) is 7.4, and the sample solution is used for electrochemical luminescence detection, the concentration of lincomycin in the sample to be detected is calculated according to the linear regression equation corresponding to the step (2), and the result is listed in Table 1.
Comparative example 3:
(1) preparation of apt/PTCA-PATP/GCE modified electrode
Polishing the glassy carbon electrode, respectively performing ultrasonic treatment on the polished glassy carbon electrode by using nitric acid, absolute ethyl alcohol and deionized water in sequence, and naturally drying the polished glassy carbon electrode for later use. Transferring 5.0 mu L of PTCA-PATP aqueous solution to the surface of a clean glassy carbon electrode by using a liquid transfer gun, and drying at room temperature to obtain a PTCA-PATP/GCE modified electrode; 5.0 mu L of 3.0 mu M aptamer (same as example 1) is dripped on the surface of the PTCA-PATP/GCE modified electrode, and the electrode is placed in a refrigerator at 4 ℃ for 6h to obtain the apt/PTCA-PATP/GCE sensor which is used as a working electrode for electrochemiluminescence test.
(2) Drawing of standard curve
An apt/PTCA-PATP/GCE modified electrode is used as a working electrode, a platinum electrode is used as an auxiliary electrode, Ag/AgCl is used as a reference electrode to form a three-electrode system, and K containing 0.05mol/L 2 S 2 O 8 0.1mol/L PBS buffer solution with pH of 7.4 as blank solution, and the three-electrode system is placed in a series of lincomycin concentrations (1.0 × 10) -15 mol/L、1.0×10 -14 mol/L、1.0×10 -13 mol/L、1.0×10 -12 mol/L、1.0×10 -11 mol/L、1.0×10 -10 mol/L、1.0×10 -9 mol/L、1.0×10 -8 mol/L and 1.0X 10 -7 mol/L) of the sample solution for 35min, taking out and leaching the sample solution to be used as a working electrode, performing cyclic voltammetry scanning on the sample solution within an electrochemical window range of-1.8-0V at a photomultiplier tube high voltage of 700V and a scanning speed of 0.1V/s, recording an E-ECL curve, and establishing a linear relation between a luminous intensity difference value before and after adding lincomycin and a lincomycin concentration logarithm value to obtain a corresponding linear regression equation.
(3) Detection of samples
25mL of the treated pond water was added to a solution containing 0.05mol/L of K 2 S 2 O 8 The pH of the sample solution is 7.4, and the concentration of lincomycin in the sample to be detected is calculated according to the linear regression equation corresponding to the step (2) when the sample solution is used for electrochemiluminescence detection, and the result is shown in Table 1.
Comparative example 4:
(1) preparation of Au/PTCA-PATP/GCE modified electrode
Polishing the glassy carbon electrode, respectively performing ultrasonic treatment on the polished glassy carbon electrode by using nitric acid, absolute ethyl alcohol and deionized water in sequence, and naturally drying the polished glassy carbon electrode for later use. And (3) transferring 5.0 mu L of 1.0mg/mL aqueous solution of the Au/PTCA-PATP material onto the surface of a clean glassy carbon electrode by using a liquid transfer gun, and drying at room temperature to obtain the Au/PTCA-PATP/GCE modified electrode serving as a working electrode for the electrochemiluminescence test.
(2) Drawing of standard curve
An Au/PTCA-PATP/GCE modified electrode is used as a working electrode, a platinum electrode is used as an auxiliary electrode, Ag/AgCl is used as a reference electrode to form a three-electrode system, and K containing 0.05mol/L 2 S 2 O 8 The pH of the solution (2) is 7.4, 0.1mol/L PBS buffer solution is used as a blank solution to detect the luminous intensity, and the three-electrode system is placed in a series of lincomycin concentrations (1.0 multiplied by 10) -15 mol/L、1.0×10 -14 mol/L、1.0×10 -13 mol/L、1.0×10 -12 mol/L、1.0×10 -11 mol/L、1.0×10 -10 mol/L、1.0×10 -9 mol/L、1.0×10 -8 mol/L and 1.0X 10 -7 mol/L) contains 0.05mol/L of K 2 S 2 O 8 In the 0.1mol/L PBS buffer solution with the pH of 7.4, within an electrochemical window range of-1.8-0V, the photomultiplier tube has high voltage of 700V and the sweep rate of 0.1V/s, cyclic voltammetry scanning is carried out, an E-ECL curve is recorded, a linear relation between the luminous intensity difference before and after adding lincomycin and the log value of the lincomycin concentration is established, and a corresponding linear regression equation is obtained.
(3) Detection of samples
25mL of the treated pond water was added to a solution containing 0.05mol/L of K 2 S 2 O 8 The pH of the sample solution is 7.4, and the concentration of lincomycin in the sample to be detected is calculated according to the linear regression equation corresponding to the step (2) when the sample solution is used for electrochemiluminescence detection, and the result is shown in Table 1.
Comparative example 5:
(1) preparation of UCNPs/PTCA-PATP/GCE modified electrode
Polishing the glassy carbon electrode, respectively performing ultrasonic treatment on the polished glassy carbon electrode by using nitric acid, absolute ethyl alcohol and deionized water in sequence, and naturally drying the polished glassy carbon electrode for later use. And (3) transferring 5.0 mu L of 1.0mg/mL aqueous solution of the UCNPs/PTCA-PATP material onto the surface of a clean glassy carbon electrode by using a liquid transfer gun, and drying at room temperature to obtain the UCNPs/PTCA-PATP/GCE modified electrode as a working electrode for an electrochemiluminescence test.
(2) Drawing of standard curve
A UCNPs/PTCA-PATP/GCE modified electrode is used as a working electrode, a platinum electrode is used as an auxiliary electrode, Ag/AgCl is used as a reference electrode to form a three-electrode system, and K containing 0.05mol/L 2 S 2 O 8 The pH of the solution (2) is 7.4, 0.1mol/L PBS buffer solution is used as a blank solution to detect the luminous intensity, and the three-electrode system is placed in a series of lincomycin concentrations (1.0 multiplied by 10) -15 mol/L、1.0×10 -14 mol/L、1.0×10 -13 mol/L、1.0×10 -12 mol/L、1.0×10 -11 mol/L、1.0×10 -10 mol/L、1.0×10 -9 mol/L、1.0×10 -8 mol/L and 1.0X 10 -7 mol/L) contains 0.05mol/L of K 2 S 2 O 8 In the 0.1mol/L PBS buffer solution with the pH of 7.4, within an electrochemical window range of-1.8-0V, the photomultiplier tube has high voltage of 700V and the sweep rate of 0.1V/s, cyclic voltammetry scanning is carried out, an E-ECL curve is recorded, a linear relation between the luminous intensity difference before and after adding lincomycin and the log value of the lincomycin concentration is established, and a corresponding linear regression equation is obtained.
(3) Detection of samples
25mL of the treated pond water was added to a solution containing 0.05mol/L of K 2 S 2 O 8 The pH of the sample solution is 7.4, and the concentration of lincomycin in the sample to be detected is calculated according to the linear regression equation corresponding to the step (2) when the sample solution is used for electrochemiluminescence detection, and the result is shown in Table 1.
TABLE 1 determination of lincomycin in Pond waters
Remarking: a is determined three timesMean value
As shown in Table 1, the samples were tested in parallel for 3 times with a relative standard deviation of less than 5% and a recovery rate of 95% -101%. The results show that the composite electrode material is feasible for detecting lincomycin in pond water, and the sensor element is further assembled after the glassy carbon electrode modified by UCNPs @ Au/PTCA-PATP instead of the UCNPs @ Au/PTCA-PATP composite material is singly used for detecting the lincomycin.
Although the present invention has been described with reference to a preferred embodiment, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (10)
1. An electrochemical light emitter, comprising: the gold nanoparticle modified UCNPs are modified and converted nanoparticles, and are combined with the thiol-perylene tetracarboxylic acid PTCA-PATP through gold-sulfur bonds.
2. The electrochemical light emitter of claim 1, wherein: the gold nanoparticle modified up-conversion nano particle UCNPs are obtained by the following method: uniformly dispersed with upconverting nanoparticles UCNPs and HAuCl 4 The ethylene glycol solution is stirred and reacted for at least 30min at the temperature of 160-180 ℃ under the protection of argon, and then is gradually cooled to the room temperature during stirring.
3. The electrochemical light emitter of claim 1, wherein: UCNPs @ Au/PTCA-PATP is obtained by the following method: respectively dispersing gold nanoparticle modified up-conversion nanoparticles UCNPs and PTCA-PATP in DMF (dimethyl formamide), and then mixing the gold nanoparticle modified up-conversion nanoparticles UCNPs and PTCA-PATP in a mass ratio of 1: 2, mixing, performing ultrasonic thorough mixing in an ice-water bath, stirring at room temperature until the mixture is fully combined, collecting the precipitate, and performing vacuum drying to obtain the UCNPs @ Au/PTCA-PATP.
4. The electrochemical light emitter of claim 1, wherein: the preparation method of the PTCA-PATP comprises the following steps: mixing perylene-3, 4,9, 10-tetracarboxylic dianhydride, imidazole and 4-aminothiophenol, heating and stirring under the protection of argon, cooling, dispersing in ethanol, adding a certain amount of hydrochloric acid, continuously stirring, filtering, washing and drying to obtain the PTCA-PATP.
5. Use of an electrochemical light emitter according to any one of claims 1 to 4, wherein: an electrochemiluminescent aptamer sensor.
6. An electrochemiluminescence aptamer sensor for specifically detecting lincomycin, characterized in that: is formed by loading lincomycin aptamer on the surface of a glassy carbon electrode modified by an electrochemical luminophor of any one of claims 1 to 4;
the nucleotide sequence of the lincomycin aptamer is shown as follows:
apt:5′-CGCG TGAT GTGG TCGA TGCG ATAC GGTG AGTC GCGC CACG GCTA CACA CGTC TCAG CGA-3′。
7. the method for preparing an electrochemiluminescence aptamer sensor for specific detection of lincomycin according to claim 6, wherein the electrochemiluminescence aptamer sensor comprises: the method comprises the following steps:
polishing and cleaning a glassy carbon electrode, then dropwise adding a DMF (dimethyl formamide) dispersion liquid of UCNPs @ Au/PTCA-PATP onto the surface of GCE, naturally drying to obtain a UCNPs @ Au/PTCA-PATP/GCE modified electrode, then, dropwise adding an aptamer to obtain an apt/UCNPs @ Au/PTCA-PATP/GCE modified electrode, and storing at a low temperature for 6-8h for later use to obtain the ECL aptamer sensor.
8. The use of an electrochemiluminescent aptamer sensor for specific detection of lincomycin in water according to claim 6, comprising the steps of:
(1) preparation of a composition containing K 2 S 2 O 8 Phosphate PBS buffer solution;
(2) contains lincomycin with different concentrations and 0.05mol/L K 2 S 2 O 8 Preparing a PBS buffer solution;
preparing lincomycin aqueous solution, and adding a certain amount of lincomycin aqueous solution into the lincomycin aqueous solution containing 0.05mol/L K 2 S 2 O 8 0.1mol/L PBS buffer solution with pH value of 7.4 to obtain a series of lincomycin standard solutions with different concentrations, wherein the concentration range is 1.0 multiplied by 10 -15 ～1.0×10 -7 mol/L；
(3) Drawing of standard curve
Forming a three-electrode system by taking a modified electrode apt/UCNPs @ Au/PTCA-PATP/GCE as a working electrode, a platinum electrode as an auxiliary electrode and Ag/AgCl as a reference electrode, and placing the three-electrode system in a system containing lincomycin with a series of different concentrations and 0.05mol/L K 2 S 2 O 8 The PBS buffer solution is soaked for a certain time and K with the concentration of 0.05mol/L 2 S 2 O 8 The luminescence intensity was measured using 0.1mol/L PBS buffer solution with pH of 7.4 as a blank solution; in the electrochemical window range of-1.8-0V, carrying out cyclic voltammetry scanning on a photomultiplier at a high voltage of 700V and a scanning speed of 0.1V/s, recording a potential-luminous intensity curve (E-ECL), and establishing a linear relation between a luminous intensity difference value before and after adding lincomycin and a lincomycin concentration logarithm value to obtain a corresponding linear regression equation;
(4) actual sample detection
And (3) carrying out pretreatment and pH adjustment on the actual sample detection, testing according to the same electrochemical luminescence test conditions in the step (3), recording the luminescence intensity, and calculating the concentration of lincomycin in the sample to be detected by using a linear regression equation corresponding to the obtained standard curve of the obtained luminescence intensity.
9. The use of an electrochemiluminescent aptamer sensor for the specific detection of lincomycin according to claim 8 in lincomycin detection in water, wherein: and (3) soaking the modified electrode apt/UCNPs @ Au/PTCA-PATP/GCE for 35 min.
10. The use of an electrochemiluminescent aptamer sensor for the specific detection of lincomycin according to claim 8 in lincomycin detection in water, wherein: the pretreatment method in the step (4) comprises the following steps: after the sample to be tested is naturally stood for a period of time, the supernatant fluid is centrifugally absorbed and then filtered by a 0.22 mu m filter membrane.
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