CN110687175A

CN110687175A - Construction method of electrochemical luminescence sensor based on cerium dioxide and nano-silver dual-enhanced perylene tetracarboxylic acid luminescence

Info

Publication number: CN110687175A
Application number: CN201911092291.0A
Authority: CN
Inventors: 宋先震; 魏琴; 刘蕾; 刘雪静; 王雪莹; 罗川南; 任祥
Original assignee: University of Jinan
Current assignee: University of Jinan
Priority date: 2019-11-11
Filing date: 2019-11-11
Publication date: 2020-01-14
Anticipated expiration: 2039-11-11
Also published as: CN110687175B

Abstract

The invention discloses a construction method of an electrochemical luminescence sensor based on cerium dioxide and nano-silver dual-enhanced perylene tetracarboxylic acid luminescence. In the invention, the PTCA serving as a luminous body is directly loaded on the surface of the MWCNTs of the carbon nano tube to form the PTCA @ MWCNTs nano composite material. Cerium oxide CeO₂And nano silver AgNPs as perylene tetracarboxylic acid-potassium persulfate PTCA-K₂S₂O₈Novel co-reaction promoter in system for catalyzing co-reaction agent K₂S₂O₈Produce more sulfate radical SO₄ ^•‑The luminous intensity of PTCA is greatly enhanced. Procalcitonin PCT with different concentrations can be combined with different amounts of secondary antibody marker gold-hybridized perylene tetracarboxylic acid-carbon nanotube Ab₂Au-PTCA @ MWCNTs, thereby causing a change in the sensor's luminescence intensity, enabling detection of PCT. Line for PCT detection of the inventionThe sex range is 50 fg/mL-100 ng/mL, and the detection limit is 16 fg/mL.

Description

Construction method of electrochemical luminescence sensor based on cerium dioxide and nano-silver dual-enhanced perylene tetracarboxylic acid luminescence

Technical Field

The invention discloses a construction method of an electrochemical luminescence sensor based on cerium dioxide and nano-silver dual-enhanced perylene tetracarboxylic acid luminescence, and particularly relates to an AgNPs @ CeO electrochemical luminescence sensor taking PTCA as a luminescent material₂A double-enhanced electrochemical luminescence sensor for detecting PCT is prepared as a co-reaction promoter, and belongs to the technical field of electrochemical luminescence detection.

Background

Sepsis is a systemic inflammatory response caused by infection with bacteria, fungi, etc., and is considered as a global life-threatening disease. Studies have shown that PCT reflects the activity of the systemic inflammatory response and has been explored as a reliable prognostic and therapeutic index for sepsis. Therefore, it would be of great interest to develop a novel and sensitive immunoassay for the rapid detection of PCT.

In recent years, electrochemiluminescence ECL has attracted much research interest as a highly sensitive and selective analytical method. Electrochemiluminescence refers to a luminescence phenomenon generated by electrochemically generating electric biomass and then reacting among the electric biomass or between the electric biomass and other substances, and is a product of combining a chemiluminescence method and an electrochemical method. The electrochemical luminescence analysis has high sensitivity and wide linear range; the analysis speed is high, and the application range is wide; is beneficial to research on the advantages of rapid luminescence reaction, luminescence reaction mechanism and the like, and has been developed into a branch subject of analytical chemistry.

PTCA is receiving increasing attention as an emerging electrochemiluminescent reagent due to its stable luminescent signal. However, PTCA by itself cannot generate ECL signals strong enough to meet the requirements of trace analysis, and it is therefore of particular importance to develop novel co-reaction promoter-catalyzed co-reactants to enhance ECL signals of PTCA. CeO (CeO)₂As an oxide of a rare earth element, there has been a wide interest in view of its unique property in redox reactions, and Ce therein³⁺/Ce⁴⁺The redox couple can be rapidly and reversibly converted, and has high catalytic activity and electron conversionThe rate of shift. AgNPs have good conductivity and can catalyze a co-reactant K₂S₂O₈More SO is generated₄ ^•-. The invention adopts PTCA as a luminous body to obtain a stable luminous signal and CeO₂And AgNPs are used as a co-reaction promoter to catalyze the co-reactant, so that a luminescence signal is enhanced to meet the requirement of trace analysis, and the construction of the electrochemical luminescence sensor for enhancing the luminescence of the perylene tetracarboxylic acid by using cerium dioxide and nano silver is realized.

Disclosure of Invention

One of the purposes of the invention is to synthesize a luminescent material with stable signals and a co-reaction promoter with good catalytic performance.

Another object of the present invention is to construct a CeO-based alloy₂And AgNPs double-enhanced perylene tetracarboxylic acid luminescence electrochemical luminescence sensor.

The third purpose of the invention is to realize high-sensitivity detection of PCT by the constructed electrochemical luminescence sensor.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

1. pretreating a glassy carbon electrode with the diameter of 4 mm by using polishing powder to obtain a mirror-like surface, and washing the mirror-like surface by using ultrapure water; immersing the treated electrode into HAuCl with the mass fraction of 1%₄In the solution, a layer of AuNPs is electrodeposited at a constant voltage of-0.2V for 30 s; 8 mu L of AgNPs @ CeO with the concentration of 1-5 mg/mL₂Dripping the solution on the surface of an electrode, and storing at room temperature until the solution is dried; mu.L of 1 mg/mL Ab₁The solution is applied by dripping on the surface of the electrode, 4^oC, storing in a refrigerator until the refrigerator is dry, and cleaning with ultrapure water; mu.L of 1% BSA by mass fraction was applied dropwise to the electrode surface to block Ab₁Upper non-specific active site, 4^oC, storing in a refrigerator until the refrigerator is dry, and cleaning with ultrapure water; 6 μ L of PCT at different concentrations were applied drop-wise to the electrode surface, 4^oC, storing in a refrigerator until the refrigerator is dry, and cleaning with ultrapure water; ab of 8 mu L3-7 mg/mL₂Dropping Au-PTCA @ MWCNTs solution on the surface of the electrode, 4^oC, storing the refrigerator until the refrigerator is dried, and cleaning the refrigerator with ultrapure water to realize the construction of the electrochemical luminescence sensor for enhancing the luminescence of the perylene tetracarboxylic acid by using cerium dioxide and nano silver; the invention utilizes a luminophor PTCA stable luminescent signal and coreaction promoter CeO₂And AgNPs have excellent catalytic performance to prepare an electrochemical luminescence sensor based on cerium dioxide and nano-silver double enhanced perylene tetracarboxylic acid luminescence; in the preparation process of the sensor, a layer of AuNPs is electrodeposited at first, so that the luminous efficiency can be enhanced, and AgNPs @ CeO can be better adsorbed₂And Ab₁(ii) a Ab Synthesis Using luminophore PTCA as Secondary antibody marker₂The Au-PTCA @ MWCNTs can load more luminophors to enhance a luminescence signal, and can enable the sensor to react more sensitively to PCT concentration change, so that high-sensitivity detection of PCT is realized.

2. 20 mL of a 10 mmol/L sodium citrate solution was mixed with 85 mL of a 0.1 mol/L urea solution, followed by the addition of 0.5 g CeCl₃And 0.5 mL of 30% volume fraction H₂O₂Stirring for 1 h to fully dissolve the mixture, adding the dissolved solution into a polytetrafluoroethylene reaction kettle, and stirring for 180 hours^oC, reacting for 20 hours; after the reaction, the resulting solution was washed by centrifugation, 60%^oC is dried to realize CeO₂Preparing a nano material; the invention prepares CeO₂The nanometer material is used as a co-reaction promoter, and the specific Ce of the nanometer material³⁺/Ce⁴⁺The redox couple can be rapidly reversibly converted, has high catalytic activity, and can catalyze S₂O₈ ^2-More SO is generated₄ ^•-Thereby enhancing the luminous intensity of the constructed sensor;

mixing Na₂S、PVP、AgNO₃Respectively dissolving in ethylene glycol to obtain 3 mmol/L Na₂S solution, 20 mg/mL PVP solution, 282 mmol/L AgNO₃A solution; 30 mL of ethylene glycol was poured into a round bottom flask, 150^oHeating for 1 h under C, and sequentially adding prepared Na₂S, PVP and AgNO₃A solution; dropwise adding a sodium citrate solution until the solution becomes brown, and then carrying out cooling reaction by using an ice water bath; the solution is added in 4^oCentrifuging under C, and alternately washing with ethanol and water to realize the preparation of AgNPs; finally dispersing the prepared AgNPs into ultrapure water to obtain a 4 mg/mL AgNPs dispersion; the sodium citrate solution takes glycol as a solvent, and the sodium citrate solution is the sodium citrate solutionCan be used as a stabilizer to prevent coagulation of AgNPs, and the oxide of the AgNPs can be chelated with the AgNPs to enable the AgNPs to be negatively charged; AgNPs prepared by the method are used as a co-reaction promoter, have good conductivity and can catalyze a co-reactant K₂S₂O₈More SO is generated₄ ^•-Thereby enhancing the luminous intensity of the constructed sensor;

dissolving 10 mg of PTCDA in 10 mL of 1 mol/L NaOH solution, heating until the PTCDA is completely dissolved and the color of the solution is changed into yellow green, then dropwise adding 1 mol/L HCl until the color of the solution is changed from yellow green into red, and centrifugally washing the obtained solution until the PH is 7.4, thereby realizing the preparation of the PTCA nano material; the PTCA prepared by the invention is used as a luminophor, the luminescence signal of the PTCA is stable, and the special structure can reduce biological toxicants and increase hydrophobicity, so that the PTCA is used as the luminophor to enhance the stability of constructing the sensor.

3. 1mL of a PDDA solution having a mass fraction of 1% was added to 2 mL of the prepared CeO₂In the solution, ultrasonic treatment is carried out for 30 min to fully mix the mixture, and centrifugal washing is carried out to obtain PDDA modified CeO₂Precipitating; dissolving the precipitate obtained by centrifugation in ultrapure water, adding 0.3 mL of the prepared AgNPs dispersoid, and stirring for 3 h to obtain AgNPs @ CeO₂A solution; AgNPs @ CeO₂The solution is centrifugally washed, and then the precipitate obtained by centrifugation is redispersed in ultrapure water, so that AgNPs @ CeO is realized₂Preparation of the solution and in 4^oC, storing for later use; the PDDA is a cationic polymer electrolyte material, which can be mixed with CeO₂Binding makes it positively charged, so that the CeO is positively charged₂Can generate electrostatic adsorption with AgNPs with negative charges to obtain AgNPs @ CeO₂A nanocomposite; the invention combines two coreaction accelerators, AgNPs @ CeO₂The luminous signal enhancement effect on the luminous body is better, so that the luminous intensity of the constructed sensor is greatly enhanced.

4. Dispersing 0.05 g of MWCNTs in 50 mL of ultrapure water, performing ultrasonic treatment for 1 h to fully dissolve the MWCNTs, adding the prepared PTCA nano material, and performing ultrasonic treatment for 2 h to fully mix the PTCA and the MWCNTs to obtain a PTCA @ MWCNTs nano composite material; 50 mg of the prepared PTCA @ MWCNTs nanocomposite is dissolved in ultrapure waterUltrasonic treating for 1 h to fully dissolve, and then adding 1mL of HAuCl with the mass fraction of 2%₄And 10 mg PVP is added to the solution, after stirring for 6 h, 4 mL of 50 mmol/L sodium citrate solution and a small amount of NaBH are added₄Continuing stirring for 6 h, and finally magnetically separating to remove unbound AuNPs to obtain the PTCA-Au @ MWCNTs nano composite material; PTCA-Au @ MWCNTs were dispersed in 5 mL PBS pH 7.4, and 500. mu.L of 500. mu.g/mL Ab was added₂And is in 4^oC incubation for 12 h in a constant temperature shaking chamber, then 100 mul BSA with mass fraction of 1% is added to block the nonspecific active site, thus realizing Ab₂Preparation of Au-PTCA @ MWCNTs solution, and in 4^oC, storing for later use; the PBS was washed with 1/15 mol/L Na₂HPO₄And 1/15 mol/L KH₂PO₄Preparing; ab is prepared by the invention₂the-Au-PTCA @ MWCNTs is used as a secondary antibody marker, the MWCNTs have large specific surface area and good conductivity, more luminophors can be loaded, and the stability and the luminous efficiency of the constructed sensor are obviously improved.

5. A silver/silver chloride Ag/AgCl electrode is used as a reference electrode, a platinum wire electrode is used as a counter electrode, a construction sensor is used as a working electrode to construct a three-electrode system, the three electrodes are connected in a cassette of a chemiluminescence detector, an electrochemical workstation is connected with the chemiluminescence detector, the high voltage of a photomultiplier is set to be 600V, the scanning voltage is set to be-1.4-0V, and the scanning speed is set to be 0.1V/s; using a K of 10-100 mmol/L₂S₂O₈The solution is used as a base solution, and a three-electrode system is utilized to detect the intensity of electrochemical luminescence signals generated under PCT with different concentrations; drawing a working curve according to the linear relation between the obtained electrochemical luminescence signal intensity value and the logarithm of the PCT concentration; said K₂S₂O₈The pH of the solution is 6.0-8.5, and the solution is treated with 10-100 mmol/L K₂S₂O₈And KCl of 100 mmol/L in PBS.

6. Different concentrations of PCT were added to the diluted serum samples and the relative standard deviation and average recovery of PCT in the serum samples were determined using standard addition methods. As can be seen from Table 1, the relative standard deviation of PCT in the serum sample is 1.05-1.14%, and the recovery rate is 99.6-101%, which indicates that the invention can be applied to the detection of actual biological samples, and the result is accurate and reliable.

Advantageous results of the invention

1. The PTCA-Au @ MWCNTs nano composite material is synthesized, so that the sensitivity and the luminous efficiency of the sensor are improved; CeO with excellent catalytic performance is synthesized₂And AgNPs as novel co-reaction promoters with K₂S₂O₈The reaction produces more SO₄ ^•-Thereby obviously enhancing the luminous intensity of PTCA and meeting the requirement of trace analysis.

2. The invention successfully constructs the electrochemical luminescence sensor based on the double enhanced luminescence of the perylene tetracarboxylic acid by the cerium dioxide and the nano silver.

3. According to the invention, the constructed electrochemical luminescence sensor realizes high-sensitivity detection on PCT, the detection result has excellent reproducibility and stability, the linear range of detection is 50 fg/mL-100 ng/mL, and the detection limit is 16 fg/mL.

Detailed Description

The present invention is further described with reference to the following examples, which are not intended to limit the scope of the present invention, and modifications of the technical solutions of the present invention by those skilled in the art are within the scope of the present invention.

Example 1

Pretreating a glassy carbon electrode with the diameter of 4 mm by using polishing powder to obtain a mirror-like surface, and washing the mirror-like surface by using ultrapure water; immersing the treated electrode into HAuCl with the mass fraction of 1%₄In the solution, a layer of AuNPs is electrodeposited at a constant voltage of-0.2V for 30 s; mu.L of 1 mg/mL AgNPs @ CeO₂Dripping the solution on the surface of an electrode, and storing at room temperature until the solution is dried; mu.L of 1 mg/mL Ab₁The solution is applied by dripping on the surface of the electrode, 4^oC, storing in a refrigerator until the refrigerator is dry, and cleaning with ultrapure water; mu.L of 1% BSA by mass fraction was applied dropwise to the electrode surface to block Ab₁Upper non-specific active site, 4^oC, storing in a refrigerator until the refrigerator is dry, and cleaning with ultrapure water; 6 μ L of PCT at different concentrations were applied drop-wise to the electrode surface, 4^oStoring in refrigerator until dry and ultrapure waterCleaning; ab of 8 mu L3 mg/mL₂Dropping Au-PTCA @ MWCNTs solution on the surface of the electrode, 4^oAnd C, storing the mixture in a refrigerator until the mixture is dried, and cleaning the mixture with ultrapure water to realize the construction of the electrochemical luminescence sensor for enhancing the luminescence of the perylene tetracarboxylic acid by using the cerium dioxide and the nano silver.

Example 2

Pretreating a glassy carbon electrode with the diameter of 4 mm by using polishing powder to obtain a mirror-like surface, and washing the mirror-like surface by using ultrapure water; immersing the treated electrode into HAuCl with the mass fraction of 1%₄In the solution, a layer of AuNPs is electrodeposited at a constant voltage of-0.2V for 30 s; mu.L of 3 mg/mL AgNPs @ CeO₂Dripping the solution on the surface of an electrode, and storing at room temperature until the solution is dried; mu.L of 1 mg/mL Ab₁The solution is applied by dripping on the surface of the electrode, 4^oC, storing in a refrigerator until the refrigerator is dry, and cleaning with ultrapure water; mu.L of 1% BSA by mass fraction was applied dropwise to the electrode surface to block Ab₁Upper non-specific active site, 4^oC, storing in a refrigerator until the refrigerator is dry, and cleaning with ultrapure water; 6 μ L of PCT at different concentrations were applied drop-wise to the electrode surface, 4^oC, storing in a refrigerator until the refrigerator is dry, and cleaning with ultrapure water; ab of 8 mu L5 mg/mL₂Dropping Au-PTCA @ MWCNTs solution on the surface of the electrode, 4^oAnd C, storing the mixture in a refrigerator until the mixture is dried, and cleaning the mixture with ultrapure water to realize the construction of the electrochemical luminescence sensor for enhancing the luminescence of the perylene tetracarboxylic acid by using the cerium dioxide and the nano silver.

Example 3

Pretreating a glassy carbon electrode with the diameter of 4 mm by using polishing powder to obtain a mirror-like surface, and washing the mirror-like surface by using ultrapure water; immersing the treated electrode into HAuCl with the mass fraction of 1%₄In the solution, a layer of AuNPs is electrodeposited at a constant voltage of-0.2V for 30 s; 8 uL of 5 mg/mL AgNPs @ CeO₂Dripping the solution on the surface of an electrode, and storing at room temperature until the solution is dried; mu.L of 1 mg/mL Ab₁The solution is applied by dripping on the surface of the electrode, 4^oC, storing in a refrigerator until the refrigerator is dry, and cleaning with ultrapure water; mu.L of 1% BSA by mass fraction was applied dropwise to the electrode surface to block Ab₁Upper non-specific active site, 4^oC, storing in a refrigerator until the refrigerator is dry, and cleaning with ultrapure water; 6 microlitres of PCT with different concentrations are dripped on the surface of the electrode,4^oc, storing in a refrigerator until the refrigerator is dry, and cleaning with ultrapure water; ab of 8 mu L7 mg/mL₂Dropping Au-PTCA @ MWCNTs solution on the surface of the electrode, 4^oAnd C, storing the mixture in a refrigerator until the mixture is dried, and cleaning the mixture with ultrapure water to realize the construction of the electrochemical luminescence sensor for enhancing the luminescence of the perylene tetracarboxylic acid by using the cerium dioxide and the nano silver.

Example 4

20 mL of a 10 mmol/L sodium citrate solution was mixed with 85 mL of a 0.1 mol/L urea solution, followed by the addition of 0.5 g CeCl₃And 0.5 mL of 30% volume fraction H₂O₂Stirring for 1 h to fully dissolve the mixture, adding the dissolved solution into a polytetrafluoroethylene reaction kettle, and stirring for 180 hours^oC, reacting for 20 hours; after the reaction, the resulting solution was washed by centrifugation, 60%^oC is dried to realize CeO₂Preparing a nano material;

mixing Na₂S、PVP、AgNO₃Respectively dissolving in ethylene glycol to obtain 3 mmol/L Na₂S solution, 20 mg/mL PVP solution, 282 mmol/L AgNO₃A solution; 30 mL of ethylene glycol was poured into a round bottom flask, 150^oHeating for 1 h under C, and sequentially adding prepared Na₂S, PVP and AgNO₃A solution; dropwise adding a sodium citrate solution until the solution becomes brown, and then carrying out cooling reaction by using an ice water bath; the solution is added in 4^oCentrifuging under C, and alternately washing with ethanol and water to realize the preparation of AgNPs; finally dispersing the prepared AgNPs into ultrapure water to obtain a 4 mg/mL AgNPs dispersion; the sodium citrate solution takes ethylene glycol as a solvent, can be used as a stabilizer to prevent AgNPs from coagulation, and can be chelated with the AgNPs to enable the AgNPs to be negatively charged;

dissolving 10 mg of PTCDA in 10 mL of 1 mol/L NaOH solution, heating until the PTCDA is completely dissolved and the color of the solution is changed into yellow green, then dropwise adding 1 mol/L HCl until the color of the solution is changed from yellow green to red, and centrifugally washing the obtained solution until the pH value is 7.4, thereby realizing the preparation of the PTCA nano material.

Example 5

1mL of a PDDA solution having a mass fraction of 1% was added to 2 mL of the prepared CeO₂In solution, sonicationFully mixing for 30 min, and centrifugally washing to obtain PDDA modified CeO₂Precipitating; dissolving the precipitate obtained by centrifugation in ultrapure water, adding 0.3 mL of the prepared AgNPs dispersoid, and stirring for 3 h to obtain AgNPs @ CeO₂A solution; AgNPs @ CeO₂The solution is centrifugally washed, and then the precipitate obtained by centrifugation is redispersed in ultrapure water, so that AgNPs @ CeO is realized₂Preparation of the solution and in 4^oC, storing for later use; the PDDA is a cationic polymer electrolyte material, which can be mixed with CeO₂Binding makes it positively charged, so that the CeO is positively charged₂Can generate electrostatic adsorption with AgNPs with negative charges to obtain AgNPs @ CeO₂A nanocomposite material.

Example 6

Dispersing 0.05 g of MWCNTs in 50 mL of ultrapure water, performing ultrasonic treatment for 1 h to fully dissolve the MWCNTs, adding the prepared PTCA nano material, and performing ultrasonic treatment for 2 h to fully mix the PTCA and the MWCNTs to obtain a PTCA @ MWCNTs nano composite material; 50 mg of the prepared PTCA @ MWCNTs nanocomposite is dissolved in ultrapure water, the mixture is subjected to ultrasonic treatment for 1 hour to be fully dissolved, and then 1mL of HAuCl with the mass fraction of 2 percent is added₄And 10 mg PVP is added to the solution, after stirring for 6 h, 4 mL of 50 mmol/L sodium citrate solution and a small amount of NaBH are added₄Continuing stirring for 6 h, and finally magnetically separating to remove unbound AuNPs to obtain the PTCA-Au @ MWCNTs nano composite material; PTCA-Au @ MWCNTs were dispersed in 5 mL PBS pH 7.4, and 500. mu.L of 500. mu.g/mL Ab was added₂And is in 4^oC incubation for 12 h in a constant temperature shaking chamber, then 100 mul BSA with mass fraction of 1% is added to block the nonspecific active site, thus realizing Ab₂Preparation of Au-PTCA @ MWCNTs solution, and in 4^oC, storing for later use; the PBS was washed with 1/15 mol/L Na₂HPO₄And 1/15 mol/L KH₂PO₄And (4) preparing.

Example 7

The method comprises the following steps of constructing a three-electrode system by taking a silver/silver chloride Ag/AgCl electrode as a reference electrode, a platinum wire electrode as a counter electrode and a constructed sensor as a working electrode, connecting the three electrodes into a cassette of a chemiluminescence detector, connecting an electrochemical workstation and the chemiluminescence detector together, and carrying out photomultiplierThe high voltage of the tube is set to 600V, the scanning voltage is set to-1.4-0V, and the scanning speed is set to 0.1V/s; using 10 mmol/L of K₂S₂O₈The solution is used as a base solution, and a three-electrode system is utilized to detect the intensity of electrochemical luminescence signals generated under PCT with different concentrations; drawing a working curve according to the linear relation between the obtained electrochemical luminescence signal intensity value and the logarithm of the PCT concentration; said K₂S₂O₈Solution, pH 6.0, with 10 mmol/L K₂S₂O₈And KCl of 100 mmol/L in PBS.

Example 8

A silver/silver chloride Ag/AgCl electrode is used as a reference electrode, a platinum wire electrode is used as a counter electrode, a construction sensor is used as a working electrode to construct a three-electrode system, the three electrodes are connected in a cassette of a chemiluminescence detector, an electrochemical workstation is connected with the chemiluminescence detector, the high voltage of a photomultiplier is set to be 600V, the scanning voltage is set to be-1.4-0V, and the scanning speed is set to be 0.1V/s; using 50 mmol/L of K₂S₂O₈The solution is used as a base solution, and a three-electrode system is utilized to detect the intensity of electrochemical luminescence signals generated under PCT with different concentrations; drawing a working curve according to the linear relation between the obtained electrochemical luminescence signal intensity value and the logarithm of the PCT concentration; said K₂S₂O₈Solution, pH 7.4, with 50 mmol/L K₂S₂O₈And KCl of 100 mmol/L in PBS.

Example 9

A silver/silver chloride Ag/AgCl electrode is used as a reference electrode, a platinum wire electrode is used as a counter electrode, a construction sensor is used as a working electrode to construct a three-electrode system, the three electrodes are connected in a cassette of a chemiluminescence detector, an electrochemical workstation is connected with the chemiluminescence detector, the high voltage of a photomultiplier is set to be 600V, the scanning voltage is set to be-1.4-0V, and the scanning speed is set to be 0.1V/s; using 100 mmol/L of K₂S₂O₈The solution is used as a base solution, and a three-electrode system is utilized to detect the intensity of electrochemical luminescence signals generated under PCT with different concentrations; according to the linearity of the obtained electrochemical luminescence signal intensity value and the logarithm of the PCT concentrationDrawing a working curve according to the relation; said K₂S₂O₈Solution, pH 8.5, with 100 mmol/L K₂S₂O₈And KCl of 100 mmol/L in PBS.

Example 10

Different concentrations of PCT were added to the diluted serum samples and the relative standard deviation and average recovery of PCT in the serum samples were determined using standard addition methods. As can be seen from Table 1, the relative standard deviation of PCT in the serum sample is 1.05-1.14%, and the recovery rate is 99.6-101%, which indicates that the invention can be applied to the detection of actual biological samples, and the result is accurate and reliable.

Table 1: and (3) detecting the PCT in a serum sample by using the cerium dioxide and nano-silver dual-enhanced perylene tetracarboxylic acid luminescent electrochemical luminescence sensor.

Claims

1. A construction method of an electrochemical luminescence sensor based on cerium dioxide and nano-silver dual-enhanced perylene tetracarboxylic acid luminescence is characterized by comprising the following steps:

pretreating a glassy carbon electrode with the diameter of 4 mm by using polishing powder to obtain a mirror-like surface, and washing the mirror-like surface by using ultrapure water; immersing the treated electrode into HAuCl with the mass fraction of 1%₄In the solution, a layer of gold nano-particle AuNPs is deposited at a constant voltage of-0.2V for 30 s; 8 mu L of AgNPs @ CeO with the concentration of 1-5 mg/mL₂Dripping the solution on the surface of an electrode, and storing at room temperature until the solution is dried; mu.L of 1 mg/mL primary anti-Ab₁The solution is applied by dripping on the surface of the electrode, 4^oC, storing in a refrigerator until the refrigerator is dry, and cleaning with ultrapure water; mu.L of bovine serum albumin BSA with mass fraction of 1% was applied dropwise to the electrode surface to block Ab₁Upper non-specific active site, 4^oC, storing in a refrigerator until the refrigerator is dry, and cleaning with ultrapure water; 6 μ L of PCT at different concentrations were applied drop-wise to the electrode surface, 4^oC, storing in a refrigerator until the refrigerator is dry, and cleaning with ultrapure water; ab of 8 mu L3-7 mg/mL₂Dropping Au-PTCA @ MWCNTs solution on the surface of the electrode, 4^oAnd C, storing the mixture in a refrigerator until the mixture is dried, and cleaning the mixture with ultrapure water to realize the construction of the electrochemical luminescence sensor for enhancing the luminescence of the perylene tetracarboxylic acid by using the cerium dioxide and the nano silver.

2. A construction method of an electrochemical luminescence sensor based on cerium dioxide and nano-silver dual-enhanced perylene tetracarboxylic acid luminescence is characterized by comprising the following steps:

20 mL of 10 mmol/L sodium citrate solution was mixed with 85 mL of 0.1 mol/L urea solution, followed by the addition of 0.5 g of cerium chloride CeCl₃And 0.5 mL of 30% by volume hydrogen peroxide H₂O₂Stirring for 1 h to fully dissolve the mixture, adding the dissolved solution into a polytetrafluoroethylene reaction kettle, and stirring for 180 hours^oC, reacting for 20 hours; after the reaction, the resulting solution was washed by centrifugation, 60%^oC is dried to realize CeO₂Preparing a nano material;

sodium sulfide Na₂S, polyvinylpyrrolidone PVP and silver nitrate AgNO₃Respectively dissolving in ethylene glycol to obtain 3 mmol/L Na₂S solution, 20 mg/mL PVP solution, 282 mmol/L AgNO₃A solution; 30 mL of ethylene glycol was poured into a round bottom flask, 150^oHeating for 1 h under C, and sequentially adding prepared Na₂S, PVP and AgNO₃A solution; dropwise adding a sodium citrate solution until the solution becomes brown, and then carrying out cooling reaction by using an ice water bath; the solution is added in 4^oCentrifuging under C, and alternately washing with ethanol and water to realize the preparation of AgNPs; finally dispersing the prepared AgNPs into ultrapure water to obtain a 4 mg/mL AgNPs dispersion; the sodium citrate solution takes ethylene glycol as a solvent, can be used as a stabilizer to prevent AgNPs from coagulation, and can be chelated with the AgNPs to enable the AgNPs to be negatively charged;

dissolving 10 mg of perylenetetracarboxylic dianhydride PTCDA in 10 mL of 1 mol/L sodium hydroxide NaOH solution, heating until the PTCDA is completely dissolved and the color of the solution is changed into yellow green, then dropwise adding 1 mol/L hydrochloric acid HCl until the color of the solution is changed from yellow green into red, and centrifugally washing the obtained solution until the pH value is 7.4, thereby realizing the preparation of the PTCA nano material.

3. The method of claim 1, wherein the AgNPs @ CeO is used as a material for the electrochemical luminescence sensor based on the double enhancement of cerium oxide and nano-silver by perylene tetracarboxylic acid luminescence₂A solution characterized by:

1mL of poly (diallyldimethylammonium chloride) (PDDA) solution with the mass fraction of 1% is added into 2 mL of prepared CeO₂In the solution, ultrasonic treatment is carried out for 30 min to fully mix the mixture, and centrifugal washing is carried out to obtain PDDA modified CeO₂Precipitating; dissolving the precipitate obtained by centrifugation in ultrapure water, adding 0.3 mL of the prepared AgNPs dispersoid, and stirring for 3 h to obtain AgNPs @ CeO₂A solution; AgNPs @ CeO₂The solution is centrifugally washed, and then the precipitate obtained by centrifugation is redispersed in ultrapure water, so that AgNPs @ CeO is realized₂Preparation of the solution and in 4^oC, storing for later use; the PDDA is a cationic polymer electrolyte material, which can be mixed with CeO₂Binding makes it positively charged, so that the CeO is positively charged₂Can generate electrostatic adsorption with AgNPs with negative charges to obtain AgNPs @ CeO₂A nanocomposite material.

4. The method for constructing an electrochemiluminescence sensor based on double enhanced luminescence of perylene tetracarboxylic acid by cerium oxide and nano-silver as claimed in claim 1, wherein Ab₂-Au-PTCA @ MWCNTs solution characterized by:

dispersing 0.05 g of MWCNTs in 50 mL of ultrapure water, performing ultrasonic treatment for 1 h to fully dissolve the MWCNTs, adding the prepared PTCA nano material, and performing ultrasonic treatment for 2 h to fully mix the PTCA and the MWCNTs to obtain a PTCA @ MWCNTs nano composite material; 50 mg of the prepared PTCA @ MWCNTs nanocomposite is dissolved in ultrapure water, the mixture is subjected to ultrasonic treatment for 1 hour to be fully dissolved, and then 1mL of HAuCl with the mass fraction of 2 percent is added₄And 10 mg of PVP are added into the solution, after stirring for 6 hours, 4 mL of 50 mmol/L sodium citrate solution and a small amount of sodium borohydride NaBH are added₄Continuing stirring for 6 h, and finally magnetically separating to remove unbound AuNPs to obtain the PTCA-Au @ MWCNTs nano composite material; PTCA-Au @ MWCNTs were dispersed in 5 mL phosphate buffer PBS at pH 7.4, 500. mu.L Ab 500. mu.g/mL was added₂And is in 4^oC, culturing for 12 h in a constant-temperature shaking box, and then100 μ L of 1% by mass BSA was added to block the non-specific active sites, realizing Ab₂Preparation of Au-PTCA @ MWCNTs solution, and in 4^oC, storing for later use; the PBS was treated with 1/15 mol/L disodium hydrogen phosphate Na₂HPO₄And 1/15 mol/L potassium dihydrogen phosphate KH₂PO₄And (4) preparing.

5. The method for constructing the electrochemiluminescence sensor based on the double enhanced perylene tetracarboxylic acid luminescence of cerium dioxide and nano silver according to claim 1, wherein the detection of the PCT is characterized in that:

a silver/silver chloride Ag/AgCl electrode is used as a reference electrode, a platinum wire electrode is used as a counter electrode, a construction sensor is used as a working electrode to construct a three-electrode system, the three electrodes are connected in a cassette of a chemiluminescence detector, an electrochemical workstation is connected with the chemiluminescence detector, the high voltage of a photomultiplier is set to be 600V, the scanning voltage is set to be-1.4-0V, and the scanning speed is set to be 0.1V/s; using a K of 10-100 mmol/L₂S₂O₈The solution is used as a base solution, and a three-electrode system is utilized to detect the intensity of electrochemical luminescence signals generated under PCT with different concentrations; drawing a working curve according to the linear relation between the obtained electrochemical luminescence signal intensity value and the logarithm of the PCT concentration; said K₂S₂O₈The pH of the solution is 6.0-8.5, and the solution is treated with 10-100 mmol/L K₂S₂O₈And KCl of 100 mmol/L in PBS.

6. The method for constructing the electrochemiluminescence sensor based on the double enhanced perylene tetracarboxylic acid luminescence of cerium dioxide and nano-silver according to claim 1, which is used for detecting PCT in a serum sample, and is characterized in that:

the relative standard deviation and mean recovery of PCT in serum samples were determined by standard addition methods with different concentrations of PCT added to diluted serum samples: as can be seen from Table 1, the relative standard deviation of PCT in the serum sample is 1.05-1.14%, and the recovery rate is 99.6-101%, which indicates that the invention can be applied to the detection of actual biological samples, and the result is accurate and reliable.