CN110702758B

CN110702758B - Method for enhancing luminous intensity of squamous cell carcinoma antigen in electrochemical luminescence detection

Info

Publication number: CN110702758B
Application number: CN201910923971.6A
Authority: CN
Inventors: 邓必阳; 莫桂春; 郑向菲; 冯金素
Original assignee: Guangxi Normal University
Current assignee: Guangxi Normal University
Priority date: 2019-09-27
Filing date: 2019-09-27
Publication date: 2021-04-30
Anticipated expiration: 2039-09-27
Also published as: CN110702758A

Abstract

The invention discloses a method for enhancing the luminous intensity of squamous cell carcinoma antigen in electrochemical luminescence detection, which mainly comprises the following stepsA construction step comprising an electrochemiluminescence sensor, the construction step comprising: preparing SCCA solution with a certain concentration, dropwise adding the SCCA solution to a polymer molecular imprinting modification electrode for incubation, washing with water after incubation is finished, and then dropwise adding Fe-MIL-88-NH on the electrode₂The @ ZnSe/Ab/BSA compound is incubated, and after incubation, the electrochemical luminescence sensor is obtained by washing and drying; the prepared electrochemical luminescence sensor is placed in PBS buffer solution containing co-reactant for electrochemical luminescence detection, so that the luminous intensity of SCCA in electrochemical luminescence detection can be effectively enhanced. The electrochemical luminescence sensor constructed by the invention has good selectivity and high sensitivity, and when the electrochemical luminescence sensor is used for detecting SCCA, the linear range of the method is effectively enlarged and the detection sensitivity is improved.

Description

Method for enhancing luminous intensity of squamous cell carcinoma antigen in electrochemical luminescence detection

Technical Field

The invention relates to a method for enhancing the luminous intensity of squamous cell carcinoma antigen in electrochemical luminescence detection, belonging to the technical field of biological detection.

Background

Cervical cancer is a common female cancer worldwide. Squamous Cell Carcinoma Antigen (SCCA) is an isoform glycoprotein having a molecular weight of about 45 to 55kDa and first isolated from squamous cell carcinoma tissue (Wu L, Hu Y F, Sha Y H, Li W R, Yan T, Wang S, Li X, Guo Z Y, Zhou J, Su X R.an "in-electrode" -type immunizing expression for the detection of squamos cell carcinoma antigen derived from AuNPs/g-C₃N₄nanocomposites[J]Talanta,2016,160: 247-. Originally, SCCA was found to be a good specific antigen for the diagnosis and monitoring of cervical squamous cell carcinoma. Later, SCCA was found to be associated with other types of cancer, including lung cancer, head and neck cancer, melanoma, hepatocellular carcinoma, and the like. To date, various immunoassays have been developed for the detection of SCCA in serum, such as enzyme-linked immunosorbent assay (ELISA), Radioimmunoassay (RIA), chemiluminescence immunoassay (CLIA), photoelectrochemical immunoassay, and electrochemical immunoassay. In the existing analysisSome methods require expensive experimental equipment and high technical requirements, and other methods are complicated and are not favorable for rapid analysis. The electrochemiluminescence analysis method has the advantages of high analysis speed, simple operation, low cost, small reagent dosage, high detection sensitivity and the like, and is widely applied to clinical diagnosis (Ma H M, Zhao Y H, Liu Y, Zhang Y, Wu D, Li H, Wei Q.A compatible sensitivity enhancement sequences for electrochemiluminescence analysis on the biological membrane-like location [ J H M].Analytical Chemistry,2017,89:13049-13053.Wang H,Pu G Q,Devaramani S,Wang Y F,Yang Z F,Li L F,Ma X F,Lu X Q.Bimodal electrochemiluminescence of g-CNQDs in the presence of double coreactants for ascorbic acid detection[J].Analytical Chemistry,2018,90:4871-4877.)。

Molecularly Imprinted Polymers (MIPs) have high specificity for recognizing target molecules. Binding sites of blots in MIPs are complementary in shape and size to the template analyte. The molecularly imprinted polymer is widely applied to detecting small organic molecules, proteins and biomarkers. Inspired by the marine mussel bioadhesive principle, Messermith and colleagues discovered that Dopamine (Dopamine, DA) can spontaneously polymerize under alkaline conditions and form a potentially reactive Polydopamine (PDA) coating on a variety of substrates (Wang J L, Li B C, Li Z J, Ren K F, Jin L J, Zhang S M, Chang H, Sun Y X, Ji J. electropolymerization of Dopamine for surface modification of complex-shaped cardiac scaffolds [ J ] Biomaterials,2014,35: 7679-. Due to the advantages of simple structure, good biocompatibility and the like, PDA attracts extensive attention and research in the aspect of biomaterial surface modification in recent years.

Metal-organic frameworks (MOFs) are porous materials composed of metal ions and organic ligands, and have wide applications in the fields of gas storage, catalysis, sensing, separation and the like. Fe-MIL-88MOFs is an MOF (Xie S B, Ye J W, Yuan Y L, Chai Y Q, Yuan R. Amultifunctional hemin @ metal-organic frame and its application to structural an electrochemical cell) which is easy to synthesize, controllable in size and good in water solubilitymical aptasensor for thrombin detection[J]Nanoscale,2015,7: 18232-. Meanwhile, Fe-MIL-88MOFs is a class of crystal microporous materials and can be used as carriers for loading quantum dots. However, Fe-MIL-88-NH is not found at present₂The compound obtained by loading ZnSe quantum dots on the metal organic framework is combined with an electro-deposition molecular imprinting technology to construct an electrochemical luminescence sensor so as to enhance the relevant report of the luminous intensity of the squamous cell carcinoma antigen during electrochemical luminescence detection.

Disclosure of Invention

The invention aims to provide a method capable of effectively enhancing the luminous intensity of a squamous cell carcinoma antigen in electrochemical luminescence detection.

In order to solve the technical problems, the invention adopts the following technical scheme:

a method for enhancing the luminous intensity of squamous cell carcinoma antigen in electrochemical luminescence detection mainly comprises the construction step of an electrochemical luminescence sensor, and the construction step of the electrochemical luminescence sensor comprises the following steps: preparing a squamous cell carcinoma antigen solution with a certain concentration, dripping the squamous cell carcinoma antigen solution on a polymer molecular imprinting modification electrode for incubation, washing with water after the incubation is finished, and dripping Fe-MIL-88-NH on the electrode₂The @ ZnSe/Ab/BSA compound is incubated, and after incubation, the compound is washed with water and dried, and the dried electrode is the electrochemical luminescence sensor;

the prepared electrochemical luminescence sensor is placed in PBS buffer solution containing a coreactant for electrochemical luminescence detection, so that the luminous intensity of the squamous cell carcinoma antigen in the electrochemical luminescence detection can be effectively enhanced; wherein the content of the first and second substances,

the polymer molecularly imprinted modified electrode is prepared by the following method: placing a working electrode in dopamine solution containing squamous cell carcinoma antigens, scanning by cyclic voltammetry to form a polymer molecular imprinting film on the working electrode, taking out, washing with water, eluting the squamous cell carcinoma antigens on the electrode, washing with water again, and airing to obtain a polymer molecular imprinting modified electrode;

the Fe-MIL-88-NH₂@ ZnSe/Ab/BSA ComplexThe preparation method comprises the following steps: firstly obtaining Fe-MIL-88-NH₂Fe-MIL-88-NH of ZnSe quantum dot loaded on metal organic framework₂@ ZnSe complex, incubating in EDC solution, adding antibody (Ab) corresponding to squamous cell carcinoma antigen, incubating, centrifuging, and collecting precipitate as Fe-MIL-88-NH₂@ ZnSe/Ab compound, and then Bovine Serum Albumin (BSA) is used for blocking the non-specific binding site of the compound, thus obtaining Fe-MIL-88-NH₂@ ZnSe/Ab/BSA complex, and storing the obtained complex in PBS buffer solution for later use;

the PBS buffer solution involved in the method is the PBS buffer solution with the concentration of 0.1-0.12 mol/L, pH-7.3-7.5.

In the above method, the EDC is 1-ethyl-3- (3-dimethylaminopropyl carbodiimide), the BSA is bovine serum albumin, the PBS buffer is preferably a PBS buffer with a concentration of 0.1mol/L, pH ═ 7.4, and the working electrode may be a common working electrode such as a Glassy Carbon Electrode (GCE) or a gold electrode.

In the construction steps of the electrochemical luminescence sensor, preferably, a squamous cell carcinoma antigen solution with the concentration of 1ng/mL is prepared, the time for incubating the squamous cell carcinoma antigen solution by dripping the squamous cell carcinoma antigen solution on the polymer molecular imprinting modification electrode is preferably 40-60 min, and then Fe-MIL-88-NH is dripped on the electrode₂The incubation time after @ ZnSe/Ab/BSA complex is preferably 50-90 min.

The test result of the applicant shows that the concentration of the co-reactant in the PBS buffer solution containing the co-reactant in the method is 0.09-0.11 mol/L, and preferably 0.1 mol/L. The co-reactant may be any conventional co-reactant suitable for ZnSe quantum dots, such as hydrogen peroxide or potassium persulfate, preferably potassium persulfate. The conditions under which the electrochemiluminescence detection is carried out are preferably: the high voltage of the photomultiplier is 800V, the scanning potential is 0-2.0V, and the scanning speed is 0.1V/s.

In the above method for preparing the polymer molecularly imprinted modified electrode, the ZnSe quantum dots can be synthesized by referring to the existing literature (Mo G C, He X, Zhou C Q, Ya D M, Feng J S, Yu ch,Deng B Y.Sensitive detection of hydroquinone based on electrochemiluminescence energy transfer between the exited ZnSequantum dots and benzoquinone[J]sensors and activators B,2018,266: 784-792), Fe-MIL-88-NH₂The metal organic framework can be synthesized according to the existing literature or by design, and is preferably synthesized according to the following method:

dispersing polyvinylpyrrolidone (PVP) in a mixed solvent (the volume ratio of DMF to ethanol is 2-4: 1-3) composed of N, N-Dimethylformamide (DMF) and ethanol (the dosage ratio of PVP to the mixed solvent is calculated according to 0.2 g: 20-40 mL) to obtain a mixed solution A; simultaneously, FeCl is added₃·6H₂O and 2-Aminoterephthalic acid (2-NH)₂-BDC)(FeCl₃·6H₂O and 2-NH₂-BDC mass ratio of 1: 1) dispersed in DMF (the amount of DMF is 16.25mg FeCl per DMF)₃·6H₂O and 16.5mg 2-NH₂-5-10 mL of BDC is shared) to obtain a mixed solution B; uniformly mixing the mixed solution A and the mixed solution B to obtain a mixed solution C (the mixing ratio of the mixed solution A to the mixed solution B is controlled to control PVP and FeCl in the obtained mixed solution C₃·6H₂O and 2-NH₂-mass ratio of BDC 0.2 g: 16.25 mg: 16.25mg), the mixed solution C is transferred to a reaction kettle to react for 18-22 h (preferably 20h) under the condition of 80-120 ℃ (preferably 100 ℃) after ultrasonic (ultrasonic is preferably 30-40 min), after the reaction is finished, the reactant is cooled to room temperature, then the mixed solution C is centrifuged (the rotating speed during centrifugation is 8000-10000 rpm, the time is 5-10 min), washed (usually washed by DMF), dried (usually dried at 60-80 ℃), and Fe-MIL-88-NH is obtained₂(the morphology is in an octahedral structure). Wherein: FeCl₃·6H₂Fe in O³⁺As a central ion, 2-NH₂BDC as ligand, DMF as solvent, PVP as activator (or plasticizer).

In the method for preparing the polymer molecularly imprinted modified electrode, the concentration of the dopamine solution is 7.5-12.5 mmol/L, and preferably 10 mmol/L. The squamous cell carcinoma antigen is dispersed in a PBS buffer solution and then placed in a dopamine solution, and the concentration of the squamous cell carcinoma antigen in the dopamine solution is 990-1010 ng/mL, preferably 1000 ng/mL. When the cyclic voltammetry is used for scanning, the scanning potential range is-0.5V, the number of scanning circles is 30-50 circles, and the preferred number of scanning circles is 40 circles. The squamous cell carcinoma antigen on the electrode can be eluted by using the conventional solution, such as acetic acid-sodium dodecyl sulfate solution (wherein acetic acid is used as a solvent, sodium dodecyl sulfate is used as a solute, the concentration of acetic acid is 0.1mol/L, and the concentration of sodium dodecyl sulfate is 5 wt%), hydrochloric acid-sodium dodecyl sulfate solution (wherein hydrochloric acid is used as a solvent, sodium dodecyl sulfate is used as a solute, the concentration of hydrochloric acid is 0.1mol/L, and the concentration of sodium dodecyl sulfate is 0.5 wt%), and the like.

In the above preparation of Fe-MIL-88-NH₂In the method of @ ZnSe/Ab/BSA complex, the concentration of the EDC solution is 100 to 110mmol/L, preferably 100 mol/L. The Fe-MIL-88-NH₂The concentration of the @ ZnSe complex in the EDC solution is 1.15-1.25 mg/mL, preferably 1.2 mg/mL.

Compared with the prior art, the invention adopts Fe-MIL-88-NH for the first time₂The ZnSe quantum dots loaded on the metal organic framework are combined with the electro-deposition molecular imprinting technology to accelerate the electron transfer rate of ZnSe and a co-reactant, and the electrochemical luminescence intensity of the ZnSe quantum dots is effectively improved, so that the electrochemical luminescence sensor with good selectivity and high sensitivity is constructed, and when the electrochemical luminescence sensor is used for detecting SCCA (liquid chromatography-tandem junction array) by electrochemical luminescence, the linear range of the method is enlarged and the detection sensitivity of the method is improved by effectively enhancing the electrochemical luminescence intensity.

Drawings

FIG. 1 shows ZnSe quantum dots, metal organic framework Fe-MIL-88-NH₂And an electron microscope image of the polymer molecular imprinting modified electrode and a particle size distribution diagram of ZnSe quantum dots, wherein FIG. 1(a) is a transmission electron microscope image of the ZnSe quantum dots, and FIG. 1(b) is a metal organic framework Fe-MIL-88-NH₂Fig. 1(c) is a scanning electron microscope image of the polymer molecular imprinting modified electrode, and fig. 1(d) is a particle size distribution diagram of ZnSe quantum dots.

FIG. 2 shows Fe-MIL-88-NH₂Scanning electron micrograph and energy spectrum analysis of @ ZnSe Complex, in which FIG. 2(a) is Fe-MIL-88-NH₂Scanning electron micrograph of @ ZnSe Complex, in which FIG. 2(b) is Fe-MIL-88-NH₂@ ZnSe ComplexEnergy spectrum analysis chart of (1).

FIG. 3 is a cyclic voltammogram of an electropolymerization of dopamine and SCCA, wherein a is the cyclic voltammogram of cycle 1, b is the cyclic voltammogram of cycle 5, c is the cyclic voltammogram of cycle 10, d is the cyclic voltammogram of cycle 20, e is the cyclic voltammogram of cycle 30, and f is the cyclic voltammogram of cycle 40.

FIG. 4 is a cyclic voltammogram of different modified electrodes, wherein a is the cyclic voltammogram of a bare GCE, b is the cyclic voltammogram of a polymer molecularly imprinted modified electrode, c is the cyclic voltammogram of a polymer molecularly imprinted modified electrode eluting template molecule SCCA, and d is the cyclic voltammogram of a polymer molecularly imprinted modified electrode reconnecting the template molecule SCCA.

FIG. 5 is the electrochemical impedance spectrum of different modified electrodes, wherein a is the electrochemical impedance spectrum of bare GCE, b is the electrochemical impedance spectrum of polymer molecular engram modified electrode, c is the electrochemical impedance spectrum of polymer molecular engram modified electrode elution template molecule SCCA, and d is the electrochemical impedance spectrum of polymer molecular engram modified electrode reconnected with template molecule SCCA.

FIG. 6 is an ECL intensity-scan time curve of different modified electrodes, wherein a is an ECL intensity-scan time curve of a bare GCE, i.e., a background baseline, b is an ECL intensity-scan time curve of MIP-SCCA @ ZnSe/Ab/GCE (i.e., a glassy carbon electrode loaded with only quantum dots and SCCA and simultaneously sealed with BSA), and c is MIP-SCCA-Fe-MIL-88-NH₂@ ZnSe/Ab/ASB/GCE (i.e., loaded with Fe-MIL-88-NH)₂Electrode of @ ZnSe/Ab/BSA complex and SCCA) electrochemical luminescence intensity-scan time curve of glassy carbon electrode.

FIG. 7 is a graph of dopamine concentration versus electrochemiluminescence intensity of an electrochemiluminescence sensor.

FIG. 8 is a graph showing the relationship between the incubation time and the electrochemiluminescence intensity of the electrochemiluminescence sensor after the dropwise addition of the squamous cell carcinoma antigen solution.

FIG. 9 is a plot of the concentration of squamous cell carcinoma antigen as a function of electrochemiluminescence intensity of an electrochemiluminescence sensor, where a is 0.0001ng/mL, b is 0.001ng/mL, c is 0.01ng/mL, d is 0.1ng/mL, e is 1ng/mL, f is 10ng/mL, and g is 100 ng/mL.

FIG. 10 is a plot of electrochemiluminescence intensity versus log of squamous cell carcinoma antigen concentration.

FIG. 11 shows an electrochemical hair sensor constructed according to the present invention for detecting 1ng/mL SCCA (containing 0.1 mol. L^-1K₂S₂O₈In PBS buffer (0.1mol/L, pH 7.4), the electrochemiluminescence intensity was scanned 10 times within 400 s.

FIG. 12 shows that the electrochemical hair sensor constructed according to the present invention was stored at 4 ℃ for 1 week, 2 weeks, 3 weeks, and 4 weeks, respectively, and then continuously used for detecting 1ng/mL SCCA (containing 0.1 mol. L^-1K₂S₂O₈In PBS buffer (0.1mol/L, pH 7.4) solution) of (a).

FIG. 13 is a bar graph of the electrochemiluminescence intensity of the electrochemical luminescence sensor constructed by the present invention for detecting blank, Bovine Serum Albumin (BSA), carcinoembryonic antigen (CEA), CA15-3 and Alpha Fetoprotein (AFP).

Detailed Description

The present invention will be better understood from the following detailed description of specific examples, which should not be construed as limiting the scope of the present invention.

Example 1

1. Experimental methods

1.1 reagents

Selenium powder (Aladdin reagent); sodium borohydride (Shanghai Tianlian Fine chemical Co., Ltd.); 3-mercaptopropionic acid (MPA), 1-ethyl-3- (3-dimethylaminopropyl carbodiimide) (EDC), polyvinylpyrrolidone (PVP), FeCl₃·6H₂O, 2-amino terephthalic acid (2-NH)₂BDC), Dopamine (DA) (Aladdin reagent), zinc chloride, sodium hydroxide, disodium hydrogen phosphate, sodium dihydrogen phosphate, N-Dimethylformamide (DMF), ethanol (chemical industry, ltd, west longshou, guangdong); sodium lauryl sulfate, Bovine Serum Albumin (BSA) (beijing solibao technologies ltd); acetic acid (Guangdong Guanghua science and technology Co., Ltd.); squamous Cell Carcinoma Antigen (SCCA), mouse anti-SCCA antigen, alpha-fetoprotein (A)FP), CA15-3, carcinoembryonic antigen (CEA) (Beijing Boaosen Biotechnology Co., Ltd.).

1.2 instruments

MPI-B type multiparameter chemical analysis and detection system (Simanimey analytical instruments, Inc.); CHI660 electrochemical workstation (shanghai chenhua); the electrochemical detection adopts a three-electrode system: the working electrode is a 3mm glassy carbon electrode; the reference electrode is an Ag/AgCl electrode, and the auxiliary electrode is a platinum wire electrode; model SK3310HP ultrasonic cleaner (shanghai kodao ultrasonic instruments ltd); a Vario Micro Cube field emission environment scanning electron microscope (Elementar, germany); SeikoSPA400 scanning probe microscope/atomic force microscope (japan SeikoSPA); tecnai G2F 20S-TWIN field emission transmission electron microscope (FEI corporation, USA).

1.3 preparation of ZnSe Quantum dots

33.7mg of Se and 32.4mg of NaBH are taken₄Disperse to 10mL H₂And reacting in O at 0 ℃ for 1h under the protection of nitrogen. 122.2mg ZnCl₂And 174. mu.L of MPA (3-mercaptopropionic acid) were dispersed in 50mL of H₂In O, 1 mol. L^-1The pH was adjusted to 11 with NaOH and deoxygenated with nitrogen for 30 min. Adding the newly prepared oxygen-free NaHSe solution into the Zn-MPA solution without peroxide, and refluxing for 8h at 105 ℃ to obtain ZnSe nano particles. The product was centrifuged at 10000rpm, washed with absolute ethanol 3 times, and purified 50mg ZnSe was dispersed in 10mL of water and stored in a refrigerator at 4 ℃ in the dark.

1.4 Fe-MIL-88-NH₂Synthesis of @ ZnSe

0.2g of polyvinylpyrrolidone (PVP) was added to 18mL of N, N-Dimethylformamide (DMF) and 11mL of ethanol, and then 1mL of a 5mg/mL ethanol solution of ZnSe quantum dots was added dropwise to the solution while stirring constantly to obtain a mixed solution A. At the same time, 16.25mg FeCl₃·6H₂O and 16.5mg of 2-aminoterephthalic acid (2-NH)₂-BDC) into 6mL DMF to obtain mixed solution B, and mixing mixed solution B and mixed solution a uniformly to obtain mixed solution C. The mixed solution C is subjected to ultrasonic treatment for 30min, and then transferred to a 50mL reaction kettle to react for 20h at 100 ℃. Cooling to room temperature, centrifuging the obtained suspension at 10000rpm for 5min, washing with DMF for 3 times, and oven drying at 70 deg.C to obtain Fe-MIL-88-NH₂@ ZnSe complex. To investigate Fe-MIL-88-NH₂The shape is that in the preparation process, ZnSe quantum dots are not added, and the other are the same, and Fe-MIL-88-NH is synthesized₂Metal organic framework (shape is octahedral structure).

1.5 preparation of Polymer molecularly imprinted modified electrode

First, a glassy carbon electrode was coated with 0.3, 0.05 μm Al in this order on a polishing cloth₂O₃Polishing the powder into a mirror surface, washing with ethanol, washing with sub-boiling water, and drying with nitrogen for later use. Then, 5mL of a 10mmol/L Dopamine (DA) solution containing 1 μ g/mL SCCA PBS buffer (0.1mol/L, pH 8) was deoxygenated with nitrogen for 15min, and then the polished glassy carbon electrode was immersed in the above solution, scanned for 40 cycles with cyclic voltammetry within a potential range of-0.5 to 0.5V to form a polymer molecularly imprinted membrane, and then the electrode was washed with water, and finally immersed in an acetic acid-sodium dodecyl sulfate solution (in which acetic acid is a solvent, sodium dodecyl sulfate is a solute, acetic acid is 0.1mol/L acetic acid, and the concentration of sodium dodecyl sulfate is 5 wt%) for 12h, and SCCA on the template was eluted, washed with water, and dried at room temperature to obtain a polymer molecularly imprinted modified electrode.

1.6 preparation of Fe-MIL-88-NH₂@ ZnSe/Ab/ASB complex

Taking a proper amount of Fe-MIL-88-NH₂@ ZnSe Complex was placed in 300. mu.L of EDC at a concentration of 100mmol/L (control of Fe-MIL-88-NH)₂@ 1.2mg/mL concentration of ZnSe complex in EDC) was incubated at 25 deg.C for 20min, then 100. mu.L of antibody (Ab) against squamous cell carcinoma antigen was added to the solution and incubated for 2h, centrifuged, and the lower precipitate, i.e., Fe-MIL-88-NH, was collected₂@ ZnSe/Ab complex, and added to 1mL of PBS buffer (0.1mol/L, pH 7.4). Following incubation for 1h with 20 μ L of 5 wt% BSA, non-specific binding sites were blocked and centrifuged, and the precipitate was collected and dispersed in 1mL buffer (0.1mol/L, pH 7.4) and stored at 4 ℃ until use.

1.7 construction and detection of an electrochemiluminescence sensor (also referred to herein as an electrodeposited molecularly imprinted immunosensor)

A series of SCCA solutions (0.0001ng/mL,0.001ng/mL,0.01ng/mL,0.1ng/mL,1ng/mL,10ng/mL,100ng/mL) with different concentrations were prepared.

Dropping a series of SCCA solutions with different concentrations 6 μ L onto the modified electrode of the polymer molecularly imprinted membrane, incubating for 40min, washing with water to remove unbound SCCA, and dropping 6 μ L of Fe-MIL-88-NH₂Incubation for 2h with @ ZnSe/Ab solution, washing with water, and air drying; the electrode was then placed at a temperature of 0.1mol/L K₂S₂O₈In 0.1mol/L PBS buffer solution for electrochemical luminescence detection. The high voltage of the photomultiplier is 800V, the scanning potential is 0 to-2.0V (vs. Ag/AgCl), and the scanning speed is 0.1V/s.

2. Results and discussion

2.1 characterization of the synthetic Material

FIG. 1 shows a transmission electron microscope and a metal organic framework Fe-MIL-88-NH of ZnSe quantum dots₂Scanning electron microscope images of the polymer molecular imprinting modified electrode and particle size distribution images of the ZnSe quantum dots. As shown in fig. 1(a), which is a transmission electron microscope image of ZnSe quantum dots, the ZnSe quantum dots present well-dispersed spherical particles; FIG. 1(b) shows a metal organic framework Fe-MIL-88-NH₂FIG. 1(b) shows that the metal-organic framework is Fe-MIL-88-NH₂Presenting a very regular rectangular pyramid morphology; FIG. 1(c) is a scanning electron microscope image of the polymer molecularly imprinted modified electrode, and it can be seen from FIG. 1(c) that the template molecules are uniformly distributed on the molecularly imprinted membrane; FIG. 1(d) is a particle size distribution diagram of ZnSe quantum dots, the particle size distribution range is 1.7-2.6 nm, and the average particle size is 2.0 nm.

FIG. 2 shows Fe-MIL-88-NH₂Scanning electron micrograph and energy spectrum analysis of @ ZnSe Complex, in which FIG. 2(a) is Fe-MIL-88-NH₂Scanning electron micrograph of @ ZnSe Complex, in which FIG. 2(b) is Fe-MIL-88-NH₂Energy spectrum analysis diagram of @ ZnSe complex. As can be seen from FIG. 2(a), many small particles are attached to the surface of the rectangular pyramid, which shows that ZnSe quantum dots are successfully loaded to Fe-MIL-88-NH₂A surface of (a); as shown in FIG. 2(b), Fe-MIL-88-NH₂The @ ZnSe complex contains elements such as C, N, O, Fe, Zn, Se and the like, and explains Fe-MIL-88-NH₂The @ ZnSe complex was successfully synthesized.

Cyclic voltammetry is a common method for electrochemically polymerizing electrochemically active materials on the surface of an electrode. The experiment utilizes cyclic voltammetry to prepare the polydopamine molecularly imprinted membrane. FIG. 3 is a cyclic voltammogram of electropolymerization of 10mmol/L DA and 1. mu.g/mL SCCA on a bare glassy carbon electrode. In the first cycle (oxidation peak potential of 0.38V (Pa1)), which indicates that dopamine oxidizes to dopaquinone, reduction peak potentials of 0.08V (Pc1) and-0.29V (Pc2) are the potentials of the dopanechrome couple and dopanechrome. However, as the number of cycles increased, all peak currents decreased, indicating that a polydopamine imprinted membrane was gradually formed and that the membrane was electrochemically inert, preventing electrochemical oxidation of dopamine at the electrode-solution interface. During electropolymerization, SCCA is combined with amino groups of dopamine through hydrogen bonds, and after an SCCA template is eluted, imprinting sites are formed on a polydopamine imprinting membrane, so that the SCCA can be identified.

FIG. 4 shows cyclic voltammograms of different modified electrodes (detection conditions: 5mmol/L K)₃[Fe(CN)₆]/K₄[Fe(CN)₆]0.1mol/L KCl, potential scanning range: -0.2-0.6V, scan rate 100mV/s), where a is the cyclic voltammetry curve of bare glassy carbon electrode, in this case Fe (CN)₆ ^3-/4-A pair of reversible redox peaks appear, b is a cyclic voltammetry curve of a polymer molecular imprinting modified electrode of the electro-deposition polydopamine molecule, and due to poor conductivity of a molecular imprinting film and a template molecule SCCA, Fe (CN)₆ ^3-/4-The redox peak current of (a) is drastically reduced. When the template molecule SCCA is eluted, Fe (CN) is enhanced₆ ^3-/4-Diffusion in the cavity of a molecularly imprinted membrane, Fe (CN)₆ ^3-/4-The redox peak current of (c) was increased (as shown in curve c), however, when the target molecule SCCA was reconnected to the molecularly imprinted membrane, the kinetic rate of electron transfer was slowed, Fe (CN)₆ ^3-/4-The redox peak current of (a) decreases again (as shown in curve d).

Electrochemical impedance is an effective detection method for detecting changes in surface characteristics of modified electrodes during polymerization. Electrochemical Impedance Spectroscopy (EIS) consists of two parts, one of which is linear and the other of which is semicircular. The straight-line part being governed by diffusion theory. The semicircular part is kinetically controlled and its diameter is equal to the resistance (R) of the electron transfer_ct)，R_ctThe value is inversely proportional to the electrochemical activity of the sample. The smaller the area of the semicircle in the impedance diagram is, the smaller the obstruction effect of the modified electrode on electron transfer is, and the better the conductivity of the electrode is. The different modified electrodes contained 5.0mmol/L K₃[Fe(CN)₆]/K₄[Fe(CN)₆]And the electrochemical impedance spectrum of the 0.1mol/L KCl solution is shown in FIG. 5 (detection condition: 5mmol/L K)₃[Fe(CN)₆]/K₄[Fe(CN)₆]0.1mol/L KCl, impedance frequency of 0.01 Hz-10⁵Hz, amplitude 5mV), curve a is the impedance spectrum of the bare glassy carbon electrode, which has a small semicircle in the high frequency region, indicating the resistance to electron transfer (R) of the bare electrode_ct) Small, curve b is the impedance spectrum of the electrode modified by the polymer molecular imprinting of the electro-deposited polydopamine molecule, the resistance of the electron transfer (R)_ct) The increase is rapid because the polydopamine molecularly imprinted membrane is successfully modified on the surface of the electrode, the electrochemical activity of the molecularly imprinted membrane and the template molecule SCCA is poor, and the transfer resistance of electrons on the surface of the electrode is increased. Curve c is the impedance spectrum of the eluted SCCA as the resistance to electron transfer (R)_ct) This is significantly reduced because the barrier to electron transfer is reduced when the template molecule is eluted. When the target molecule SCCA is reconnected to the molecularly imprinted membrane, the resistance (R) of electron transfer is increased_ct) And increases again (curve d), indicating successful attachment of the target molecule to the electrodeposited polydopamine molecularly imprinted membrane.

2.2 electrochemiluminescence behavior of differently modified electrodes

The polymer molecularly imprinted electrode is incubated in 1ng/mL SCCA for 40min to form MIP-SCCA-Fe-MIL-88-NH₂@ ZnSe/Ab/GCE and MIP-SCCA-ZnSe/Ab/GCE, in a concentration of 0.1mol/L K₂S₂O₈The electrochemiluminescence of the sample was detected in an electrolyte solution of PBS buffer (0.1mol/L, pH 7.4), and the result is shown in FIG. 6 (detection conditions: 0.1mol/L K)₂S₂O₈PBS buffer (0.1mol/L, pH 7.4), potential sweep range: 0 to-2.0V, a scan rate of 100mV/s, and an SCCA concentration of 1 ng/mL). MIP-SCCA-Fe-MIL-88-NH₂The electrochemiluminescence intensity of @ ZnSe/Ab/GCE (curve c) is 8 times that of MIP-SCCA-ZnSe/Ab/GCE (curve b) due to the metal organic framework Fe-MIL-88-NH₂The surface area is large, more ZnSe quantum dots can be loaded, and the surface area is Fe-MIL-88-NH₂Contains organic ligand 2-amino terephthalic acid (2-NH)₂BDC) as a novel co-reaction catalyst for promoting S₂O₈ ^2-Conversion to SO₄ ^·-Thereby enhancing the quantum dots and S₂O₈ ^2-Electrochemiluminescence of the system.

2.3 Condition optimization

The experiment was optimized mainly for dopamine concentration, as shown in FIG. 7 (detection conditions: 0.1mol/L K)₂S₂O₈PBS buffer (0.1mol/L, pH 7.4), potential sweep range: 0 to-2.0V, a scan rate of 100mV/s, and an SCCA concentration of 1 ng/mL). As the concentration of dopamine is increased from 2.5mmol/L to 10mmol/L, SCCA connected on the polydopamine molecularly imprinted membrane is increased, and the electrochemical luminescence intensity is increased. When the concentration of the dopamine is more than 10mmol/L, the electrochemical luminescence intensity is gradually reduced, because when the concentration of the dopamine is higher, the formed electro-deposition molecular imprinting film is thicker, and the electron transfer is hindered, so that the electrochemical luminescence intensity is reduced. Therefore, a dopamine concentration of 10mmol/L was chosen for this experiment.

The electrochemiluminescence intensity of the molecularly imprinted immunosensor is also influenced by the incubation time for reconnecting the electrodeposited molecularly imprinted membrane to SCCA, and the result is shown in FIG. 8 (detection condition: 0.1mol/L K)₂S₂O₈PBS buffer (0.1mol/L, pH 7.4), potential sweep range: 0 to-2.0V, a scan rate of 100mV/s, and an SCCA concentration of 1 ng/mL). The electrochemiluminescence intensity is increased along with the increase of the incubation time, when the incubation time is 40min, the electrochemiluminescence intensity reaches the maximum, the incubation time is prolonged, the electrochemiluminescence intensity does not increase any more, which indicates that the SCCA fixed on the surface of the electrodeposited molecular imprinting membrane is saturated, and therefore, the optimal incubation time is selected to be 40 min.

2.4 analytical Properties

Under the optimal experimental conditions, the prepared electrochemistryThe light-emitting sensor was used to detect Squamous Cell Carcinoma Antigen (SCCA), and the results are shown in FIG. 9 (detection conditions: 0.1mol/L K)₂S₂O₈PBS buffer (0.1mol/L, pH 7.4), potential sweep range: 0 to-2.0V, and a scanning rate of 100 mV/s). The electrochemiluminescence intensity of the sensor gradually increases along with the increase of the concentration of the squamous cell carcinoma antigen, and the electrochemiluminescence intensity is in linear relation with the logarithm of the concentration of the squamous cell carcinoma antigen in the concentration range of 0.0001ng/mL to 100 ng/mL. As can be seen from FIG. 10 (detection conditions: 0.1mol/L K)₂S₂O₈PBS buffer (0.1mol/L, pH 7.4), potential sweep range: 0 to-2.0V, the scanning rate is 100mV/s), and the linear equation is I_ECL1866.6logC +8545.2, the correlation coefficient is 0.9963. Calculation was performed according to 3 σ (where σ is the standard deviation of blanks, and n ═ 11) to give a detection limit of squamous cell carcinoma antigen of 31 fg/mL. The electrochemiluminescence sensor prepared by the method has a wider linear range and a lower detection limit, and is specifically shown in the following table 1.

Table 1: comparison of different methods for detecting SCCA Performance

2.5 reproducibility of the electrochemiluminescence sensor

The electrochemiluminescence sensor prepared in the above way is used for detecting 1ng/mL SCCA (containing 0.1 mol. L)^-1K₂S₂O₈In PBS buffer (0.1mol/L, pH 7.4), 10 cycles of scanning were performed within 400s (detection conditions: 0.1mol/L K₂S₂O₈PBS buffer (0.1mol/L, pH 7.4), potential sweep range: 0 to-2.0V, the scanning rate is 100mV/s, the SCCA concentration is 1ng/mL), the electrochemical luminescence signal is not obviously reduced (as shown in figure 11), the relative standard deviation is 2.4 percent, and the result shows that the electrochemical luminescence sensor prepared by the invention has the advantages of high sensitivity, highThe device has good reproducibility. The prepared electrochemiluminescence sensor was stored at 4 ℃ for 1 week, 2 weeks, 3 weeks, and 4 weeks, and cyclic scanning was performed again (detection condition: 0.1mol/L K)₂S₂O₈PBS buffer (0.1mol/L, pH 7.4), potential sweep range: 0 to-2.0V, the scanning rate of 100mV/s, the SCCA concentration of 1ng/mL) and the electrochemical luminescence signal of 93 percent (as shown in figure 12) of the original value indicate that the electrochemical luminescence sensor constructed by the invention has good stability.

2.6 Selectivity of the electrochemiluminescence sensor

In order to examine the selectivity of the electrochemiluminescence sensor, the applicant detected 10ng/mL Bovine Serum Albumin (BSA), carcinoembryonic antigen (CEA), CA15-3 and Alpha Fetoprotein (AFP) with the prepared electrochemiluminescence sensor, and the results are shown in FIG. 13 (detection conditions: 0.1mol/L K)₂S₂O₈PBS buffer (0.1mol/L, pH 7.4), potential sweep range: 0 to-2.0V, a scan rate of 100mV/s, and an SCCA concentration of 1 ng/mL). As can be seen from fig. 13, the electrochemiluminescence response values of other proteins other than SCCA were close to the blank value, indicating that the electrochemiluminescence sensor has good selectivity.

Experimental example 1: the method is used for analyzing actual sample samples, and in order to examine whether the electrochemical luminescence sensor constructed by the invention can be used for analyzing complex samples, the applicant uses the sensor to analyze human serum samples. The human serum sample comes from the fifth people hospital in Guilin city, and is directly analyzed according to the immunoassay method without any pretreatment after being retrieved, the result is shown in Table 2, the standard recovery rate of the human serum sample is 96.08-105.1% and the RSD is less than 7% through analysis, and the electrochemical luminescence sensor constructed by the invention is suitable for analyzing complex biological samples.

Table 2: measurement result and recovery rate of SCCA in human serum sample (n ═ 3)

Claims

1. A method for enhancing the luminous intensity of squamous cell carcinoma antigen in electrochemical luminescence detection mainly comprises the construction steps of an electrochemical luminescence sensor, and is characterized in that:

the construction steps of the electrochemical luminescence sensor comprise: preparing a squamous cell carcinoma antigen solution with a certain concentration, dripping the squamous cell carcinoma antigen solution on a polymer molecular imprinting modification electrode for incubation, washing with water after the incubation is finished, and dripping Fe-MIL-88-NH on the electrode₂The @ ZnSe/Ab/BSA compound is incubated, and after incubation, the compound is washed with water and dried, and the dried electrode is the electrochemical luminescence sensor;

the Fe-MIL-88-NH₂The @ ZnSe/Ab/BSA complex was prepared as follows: firstly obtaining Fe-MIL-88-NH₂Fe-MIL-88-NH of ZnSe quantum dot loaded on metal organic framework₂The @ ZnSe complex is placed in EDC solution for incubation, then the antibody corresponding to the squamous cell carcinoma antigen is added into the solution for incubation, centrifugation is carried out after the incubation is finished, and the precipitate is collected, namely Fe-MIL-88-NH₂@ ZnSe/Ab Complex, blocking Fe-MIL-88-NH with bovine serum Albumin₂The non-specific binding site of the @ ZnSe/Ab complex is obtained, namely Fe-MIL-88-NH₂@ ZnSe/Ab/BSA Complex to obtain Fe-MIL-88-NH₂The @ ZnSe/Ab/BSA complex was stored in PBS buffer for use;

2. The method of claim 1, wherein: in the construction step of the electrochemical luminescence sensor, a squamous cell carcinoma antigen solution with the concentration of 1ng/mL is prepared.

3. The method of claim 1, wherein: in the construction step of the electrochemical luminescence sensor, the squamous cell carcinoma antigen solution is dripped onto the polymer molecular imprinting modification electrode for incubation for 40-60 min.

4. The method of claim 1, wherein: in the construction step of the electrochemical luminescence sensor, Fe-MIL-88-NH is dripped on an electrode₂The incubation time after @ ZnSe/Ab/BSA complex is 50-90 min.

5. The method of claim 1, wherein: in the PBS buffer solution containing the co-reactant, the concentration of the co-reactant in the PBS buffer solution is 0.09-0.11 mol/L.

6. The method of claim 1, wherein: the conditions for performing the electrochemiluminescence detection are as follows: the high voltage of the photomultiplier is 800V, the scanning potential is 0-2.0V, and the scanning speed is 0.1V/s.

7. The method according to any one of claims 1 to 6, wherein: in the method for preparing the polymer molecular imprinting modified electrode, the concentration of the dopamine solution is 7.5-12.5 mol/L, and the concentration of squamous cell carcinoma antigen in the dopamine solution is 990-1010 ng/mL.

8. The method according to any one of claims 1 to 6, wherein: in the method for preparing the polymer molecular imprinting modified electrode, when cyclic voltammetry is used for scanning, the scanning potential range is-0.5V, and the number of scanning circles is 30-50 circles.

9. The method according to any one of claims 1 to 6, wherein: in the preparation of Fe-MIL-88-NH₂In the method of @ ZnSe/Ab/BSA compound, the concentration of the EDC solution is 100-110 mmol/L, and the Fe-MIL-88-NH₂The concentration of the @ ZnSe complex in the EDC solution is 1.15-1.25 mg/mL.