CN112229888B - Electrochemical luminescence enzyme-linked immunosensor and preparation method and application thereof - Google Patents
Electrochemical luminescence enzyme-linked immunosensor and preparation method and application thereof Download PDFInfo
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Abstract
An electrochemical luminescence enzyme-linked immunosensor and application thereof, the electrochemical luminescence enzyme-linked immunosensor is prepared by the following method: firstly, synthesizing mesoporous silica on an ITO electrode, then attaching the mesoporous silica electrode to a paper cover plate, then reacting a biotin-labeled antibody and an alkaline phosphatase-labeled antibody with an antigen to form an antigen-antibody compound, then adding an avidin magnetic bead to separate the antigen-antibody compound, finally adding a substrate of disodium phenyl phosphate to react, and detecting the generated phenol, wherein the content of a substance to be detected is in direct proportion to the generated phenol, thereby constructing the electrochemiluminescence enzyme-linked immunosensor. The electrochemiluminescence enzyme-linked immunosensor can be applied to detection of different clinical biomarkers with different concentrations, can simply, conveniently and quickly adjust the detection range, has lower detection limit and wider detection range, and has wide application prospect in the field of realizing simultaneous detection of multiple components with larger content difference.
Description
Technical Field
The invention relates to the technical field of immunoassay, in particular to an electrochemiluminescence enzyme-linked immunosensor with an adjustable detection range and a preparation method and application thereof.
Background
The clinical biomarkers have important significance in disease diagnosis, treatment, disease detection, prognosis judgment, curative effect evaluation and the like. The combined use of multiple clinical biomarkers can provide reliable information for clinical diagnosis and treatment. However, the abundance of clinical biomarkers ranges widely from pg/mL to
the/mL level was not equal.
Currently, many detection methods are used for the detection of clinical biomarkers, including enzyme-linked immunoassay, immunochemiluminometric assay, immunocolloidal gold, and fluoroimmunoassay. The methods have the defects of high cost, complex instrument structure, high technical requirement, long detection time, narrow detection range and the like. The electrochemical luminescence sensor has the characteristics of high sensitivity, simplicity, easiness in operation, easiness in miniaturization and the like, so that a choice is provided for designing a high-sensitivity immunosensor.
Most of the constructed electrochemical luminescence immunosensors have low sensitivity and narrow detection range.
CN104849442A discloses an electrochemiluminescence immunosensor for detecting an antibody against Edwardsiella disease, and the document reports a construction method of the electrochemiluminescence sensor based on fixed and inactivated Edwardsiella on a Nafion film, which can realize the detection of the antibody against Edwardsiella disease. The method catalyzes glucose to generate hydrogen peroxide through the glucose oxidase marked on the antibody to promote the generation of electrochemiluminescence, but the detection range of the method is 7-800
mL, low sensitivity and narrow detection range.
Mesoporous silica (abbreviated as "mesoporous silicon" or "SANs") is a nano material with a diameter of about 2-3 nm and vertical to a substrate, and is widely used for constructing electrochemical luminescence sensors due to the size sieving capability and charge selectivity. However, at present, no reports related to the electrochemical luminescence enzyme-linked immunosensor based on mesoporous silica with high sensitivity and adjustable detection range exist.
Therefore, development of new methods and techniques for immunoassay of various clinical biomarkers in different concentration ranges is urgently needed.
Disclosure of Invention
The invention aims to solve the technical problem of overcoming the defects in the prior art and provides an electrochemiluminescence enzyme-linked immunosensor which can adjust the detection range, has high sensitivity and can carry out simultaneous detection on multiple indexes in different concentration ranges.
The technical scheme adopted by the invention for solving the technical problems is that the electrochemical luminescence enzyme-linked immunosensor is prepared by the following method: firstly, mesoporous silica is synthesized on an Indium Tin Oxide (ITO) electrode, then the mesoporous silica electrode is attached to a paper cover plate, then a biotin-labeled antibody and an alkaline phosphatase-labeled antibody react with an antigen to form an antigen-antibody compound, then an avidin magnetic bead is added to separate the antigen-antibody compound, finally a substrate disodium phenyl phosphate is added to react, and then generated phenol is detected, wherein the content of a substance to be detected is in direct proportion to the generated phenol, so that the electrochemiluminescence enzyme-linked immunosensor is constructed.
Further, the preparation method of the electrochemical luminescence enzyme-linked immunosensor comprises the following specific steps: (1) Cutting a customized Indium Tin Oxide (ITO) electrode into the size of 1-5 cm multiplied by 1-5 cm, soaking the electrode in an ethanol solution containing 0.1-2M (preferably 0.5-1.0) of NaOH for 1-26 h (preferably 18-24 h), sequentially ultrasonically cleaning the electrode for 1-30 min (preferably 10-20 min) by using acetone, ethanol and deionized water respectively, and then drying the electrode by using nitrogen;
(2) Synthesizing mesoporous silicon dioxide (SANs) on the ITO electrode by adopting a solution growth method: firstly weighing 0.1-0.5 g (preferably 0.16 g) Cetyl Trimethyl Ammonium Bromide (CTAB), dissolving with 10-150 mL (preferably 50-100 mL, more preferably 60-80 mL, further 70 mL) deionized water and 10-100 mL (preferably 20-50 mL, more preferably 30 mL) anhydrous ethanol mixed solution, stirring to clarify, sequentially adding 1-50 mL with micropipette
(preferably 5 to 20)
Preferably 10
) Concentrated ammonia (10-50 wt.%, preferably 15-40 wt.%, more preferably 20-30 wt.%), 10-150 wt.%
(preferably 60 to 120)
More preferably 70 to 100
Further, 80 is preferable
) Tetraethyl silicate (TEOS), stirred for 1-30 min (preferably 10-25 min; more preferably 15-20 min.); transferring the solution into a staining box; finally, the pretreated ITO glass is put into a dyeing box, sealed, and subjected to water bath for 1-48 h (preferably 10-40h, more preferably 20-30 h) at 30-150 ℃ (preferably 40-120 ℃, more preferably 50-80 ℃, and further preferably 60 ℃). In all processes, the generation of bubbles caused by oscillation is avoided;
after the water bath is finished, taking out the ITO glass growing the mesoporous silica, washing the ITO glass with deionized water, and drying the ITO glass by blowing for 5 to 48 hours (preferably 10 to 40 hours, more preferably 15 to 30 hours) at 50 to 300 ℃ (preferably 100 to 250 ℃, more preferably 150 to 200 ℃); taking out, cooling, adding into 0.1-0.5M hydrochloric acid ethanol solution, stirring for 1-30 min (preferably 10-25 min; more preferably 15-20 min), and blow-drying with nitrogen gas to obtain SANs/ITO electrode;
(3) Bonding the paper cover plate with the SANs/ITO electrodes to prepare sensors (SANs-ECL); each sensor is provided with 1-10 independent detection units; each unit comprises 1-10 working electrodes which form 1-5 micro-two-electrode systems with the counter electrode; each detection unit is respectively dripped with 1 to 15
Applying 0.5-3.0V voltage to two ends of different solutions for detection;
(4) Enzyme-linked immunosorbent assay
1) Antigen-antibody reaction: taking 10-200
0.1-1.0
mL -1 Biotinylated antibody, 10-200
0.1-1.0
mL -1 Alkaline phosphatase (ALP) -labeled antibody, 10-200
Adding human immunoglobulin (IgG) of each concentration into a centrifuge tube blocked with Bovine Serum Albumin (BSA) in advance, mixing, and incubating at 37 deg.C for 5-35 min (preferably 10-30 min);
2) Magnetic separation: taking 50-300 parts of antigen-antibody after finishing reaction
Adding the magnetic beads into a centrifugal tube, shaking uniformly, incubating at 37 ℃ for 5-30 min (10-25 min), shaking uniformly twice in the process to avoid the magnetic beads from sinking; after the reaction is completed, the supernatant is discarded by magnetic separation, and then washed 1 to 10 times (preferably 4 to 8 times) with Phosphate Buffered Saline (PBS);
3) Enzymatic reaction: adding 50-200 enzyme reaction substrates into a centrifugal tube filled with washed magnetic beads
Incubating at 37 deg.C with shaking for 5-35 min (preferably 10-30 min); and (4) carrying out magnetic separation, and taking the supernatant for electrochemical luminescence detection.
The method for adjusting the detection range of the sensor comprises the following steps: applying 0.5-3.0V voltage on the mesoporous silicon electrode by using an electrochemical workstation, and collecting a luminescence image under a chemiluminescence imaging system; firstly, adding 1-15 parts of the reagent into each detection unit
The initial luminescence intensity (I) of the substrate solution before the enzyme reaction was measured 0 ) (ii) a Adding substrate solution after enzyme reactionAnd measuring the luminescence intensity after quenching (I) 1 ) (ii) a Analysis I 1 AndI ECL = I 0 - I 1 the relationship with the enzyme content (i.e., the antigen to be detected); the concentration of the luminophor and the co-reactant is changed, the sensitivity and the linear range of detection are adjusted, and the detection of the target object with different concentration ranges is realized on the sensor.
The inventor researches and discovers that mesoporous silicon dioxide has the effect of amplifying phenol quenching electrochemiluminescence, and the phenol quenching effect on the mesoporous silicon can be dual regulated and controlled by a luminophor and a co-reactant. Based on the double regulation and control mechanism, the inventor constructs an electrochemiluminescence enzyme-linked immunosensor with adjustable detection range.
The principle of the invention is as follows: benzoquinone can quench electrochemiluminescence by two pathways of energy transfer and radical quenching of the coreactant, the quenching efficiency being determined by the luminophore (Ru (bpy) 3 2+ ) And the effect of co-reactant (DBAE) concentration. The lower the concentration of the luminophore, the higher the quenching efficiency; the higher the concentration of the co-reactant, the higher the efficiency of quenching. The mesoporous silica has the effect of amplifying phenol quenching electrochemiluminescence, and the phenol quenching effect on the mesoporous silica can be dual regulated and controlled by a luminophor and a coreactant. Based on the mechanism, a biotin-labeled antibody and an alkaline phosphatase (ALP) -labeled antibody can react with an antigen to form a sandwich structure, avidin magnetic beads are added to separate an antigen-antibody complex, finally, a substrate disodium phenyl phosphate is added to react and then the generated phenol is detected, and the content of a substance to be detected is in direct proportion to the generated phenol. Change Ru (bpy) 3 2+ And DBAE, the sensitivity and linear range of phenol detection can be changed, and the detection of target objects in different concentration ranges can be realized on the sensor. The present invention is exemplified by IgG.
The invention has the beneficial effects that: (1) The invention can simply and rapidly adjust the detection range, has lower detection limit and wider detection range, and has wide application prospect in the field of realizing simultaneous detection of multiple components with larger content difference; (2) The sensor can be used together with a commercial enzyme-linked immunosorbent assay (ELISA) kit, so that the detection range of the ELISA kit can be adjusted, and the detection range of the kit can be expanded; (3) The invention can change the simple and rapid adjusting detection range only by simply changing the concentration of the luminophor and the co-reactant, does not need special instruments and various reagents, can solve the problem of large concentration difference of different substances to be detected, and realizes the simultaneous detection of multiple indexes; (4) the sample consumption is low, and the cost is saved; (5) The method has the advantages of simplicity, visualization, high detection speed, high sensitivity, low detection limit, good stability and the like; (6) The electrochemical luminescence enzyme-linked immunosensor can be applied to various clinical biomarkers, such as tumor markers, metabolic markers and the like.
Drawings
FIG. 1 is a flow chart of the preparation process of the electrochemical luminescence enzyme-linked immunosensor of the present invention;
FIG. 2 is a flow chart of the electrochemiluminescence enzyme-linked immunosorbent assay of the invention;
FIG. 3 is a graph of the effect of luminophores of the present invention on phenol quenching of a sensor;
FIG. 4 is a graph of the effect of co-reactants of the present invention on phenol quenching of a sensor;
FIG. 5 is a graph showing the detection of enzymatic activity by a sensor according to the present invention;
FIG. 6 is a graph showing the effect of substrate concentration of an enzyme of the present invention on quenching;
FIG. 7 is a graph showing the effect of pH on quenching in the reaction of a substrate according to the present invention;
FIG. 8 shows that 50-5000 pg mL of assay is performed in accordance with the present invention -1 IgG in a concentration range;
FIG. 9 is a graph showing that 0.2-20 ng mL is measured in accordance with the present invention -1 IgG in a concentration range;
FIG. 10 shows that 2-120 ng mL of the assay of the present invention was performed -1 IgG in the concentration range.
Detailed Description
The invention is further illustrated by the following examples and figures.
Construction example of electrochemiluminescence enzyme-linked immunosensor
Referring to FIG. 1, construction of an electrochemiluminescence enzyme-linked immunosensor
(1) Cutting the customized ITO electrode into the size of 3 cm multiplied by 2.5 cm, soaking in an ethanol solution containing 1M NaOH for 24h, ultrasonically cleaning with acetone, ethanol and deionized water for 15 min respectively, and drying with nitrogen;
(2) Synthesizing SANs (mesoporous silicon dioxide) on the ITO electrode by adopting a solution growth method: firstly, weighing 0.16 g of CTAB, dissolving the CTAB in a mixed solution of 70 mL of deionized water and 30 mL of absolute ethyl alcohol, stirring the mixed solution until the mixed solution is clear, and avoiding bubbles during stirring as much as possible; sequentially adding 10 by using a micropipettor
Concentrated ammonia (concentration 30 wt.%), 80
TEOS, stirring for 10 min; transferring the solution into a staining cassette; finally, putting the pretreated ITO glass into a dyeing box, sealing, and carrying out water bath at 60 ℃ for 24 hours; in all processes, the generation of bubbles caused by oscillation is avoided;
after the water bath is finished, taking out the ITO glass growing the mesoporous silica, washing the ITO glass with deionized water, and carrying out forced air drying at 100 ℃ for 12 hours; taking out, cooling, adding into 0.1M hydrochloric acid ethanol solution, stirring for 15 min, and blow-drying with nitrogen to obtain SANs/ITO electrodes;
(3) Bonding the paper cover plate with the SANs/ITO electrodes to prepare sensors (SANs-ECL); each sensor is provided with 6 independent detection units; each unit comprises 3 working electrodes (the area is 1.5 mm multiplied by 1.5 mm) and forms three micro-two electrode systems with the counter electrode; each detection unit is respectively dripped with 15
For the different solutions, a voltage of 2.0V was applied across the ends for detection.
(4) Enzyme-linked immunosorbent assay
1) Antigen-antibody reaction: get 100
0.2 of
mL -1 Biotinylated antibody, 100
0.5 of (2)
mL -1 ALP-labeled antibody 100
Human IgG at each concentration was added to a centrifugation tube previously blocked with BSA, mixed well and incubated at 37 ℃ for 30 min.
2) Magnetic separation: taking 300 after antigen-antibody reaction
Adding the magnetic beads into a centrifuge tube, shaking uniformly, incubating for 20 min at 37 ℃, shaking uniformly twice during the incubation period, and avoiding the magnetic beads from sinking to the bottom. After the reaction was completed, the supernatant was discarded by magnetic separation and washed with a washing solution 5 times.
3) Enzymatic reaction: adding enzyme reaction substrate 100 into the centrifugal tube containing the washed magnetic beads
Incubate at 37 ℃ with shaking for 30 min. And (4) carrying out magnetic separation, and taking the supernatant for electrochemical luminescence detection.
The electrochemiluminescence enzyme-linked immunoassay comprises the following operation steps as shown in figure 2, biotin-labeled antibodies and alkaline phosphatase-labeled antibodies are used for reacting with antigens to form a sandwich structure, avidin magnetic beads are added for separating an antigen-antibody compound, finally, substrate disodium phenyl phosphate is added for reaction, the generated phenol is detected, and the content of a substance to be detected is in direct proportion to the generated phenol.
Adjustment of the detection range of the sensor: and applying a voltage of 2.0V to the electrode by using an electrochemical workstation, and acquiring a luminescence image under a chemiluminescence imaging system. Firstly, 15 is added in each detection unit
The initial luminous intensity I of the substrate solution before the enzyme reaction is measured 0 (ii) a Adding substrate solution after enzyme reaction, and measuring luminescence intensity I after quenching 1 . Analysis I 1 And Δ I ECL = I 0 - I 1 And the amount of enzyme (i.e., the antigen to be detected). The concentration of the luminophor and the co-reactant is changed, the sensitivity and the linear range of detection are adjusted, and the detection of the target objects in different concentration ranges is realized on the sensor.
The results in fig. 3 and 4 show that benzoquinone can quench electrochemiluminescence through two pathways of energy transfer and co-reactant radical quenching, the quenching efficiency being affected by the luminophore and co-reactant concentration. The lower the concentration of the luminophore, the higher the quenching efficiency; the higher the concentration of the co-reactant, the higher the efficiency of quenching. The mesoporous silicon dioxide has the effect of amplifying phenol quenching electrochemiluminescence, and the phenol quenching effect on the mesoporous silicon can be controlled by a luminophor and a co-reactant. By changing the concentration of the luminophor and the co-reactant, the sensitivity and the linear range of phenol detection can be changed, and the detection of target objects in different concentration ranges can be realized on the sensor.
Test for influence of electrochemical luminescence enzyme-linked immunosensor performance index
1. Enzyme activity detection
Detection of the specificity of the sensor for the enzyme:
get 99
Luminescent substrate (containing 0.4M disodium phenyl phosphate DPP, 10)
M Ru(bpy) 3 2+ And 10
M DBAE), magnetic beads (0.5 mg mL) were added to each -1 ，1 
) Biotin anti-human IgG (5)
mL -1 ，1 
) Human IgG (5)
mL -1 ，1 
、BSA（5 
mL -1 ，1 
）、ALP（5 U L -1 ，1 
) ALP-labeled anti-human IgG (5)
mL -1 ，1 
). The electrochemiluminescence is measured after 30min of reaction at 37 ℃.
The results in FIG. 5 show that the quenching of luminescence occurs only in solutions containing alkaline phosphatase, and thus that the substrate is catalyzed to form a substance that quenches luminescence only in the presence of alkaline phosphatase.
2. Effect of concentration of enzyme substrate on immunoassay
Detecting the effect of the concentration of the enzyme substrate on the sensor:
DPP substrates (containing 10 mM) containing substrates 1, 10, 100, 400, 600, 800 mM were prepared with 0.01M Tris buffer pH 9.0 
M Ru(bpy) 3 2+ And 10
M DBAE). Taking the above substrate solutions to respectively determine initial luminescence I 0 (ii) a 1 mL of the solution was taken and 5 was added thereto
g mL -1 ALP-anti-human IgG solution 1 of
And determining quenched luminescence I after reaction for 30min at 37 DEG C 1 。
FIG. 6 shows that the patient has a substrate concentration of less than 400 mMI ECL Gradually increasing; when the substrate concentration is higher than 400 mM, ΔI ECL And gradually decreases. The substrate concentration varied the most at 400 mM. Taken together, a substrate concentration of 400 mM was selected as the substrate concentration for the subsequent enzyme immunoreaction.
3. Effect of enzymatic reaction pH on immunoassay
The influence of the enzymatic reaction pH value on the sensor was examined:
0.4M DPP substrate luminescence solution (containing 10) is prepared by Tris-HCl buffer solution with pH 7.5, 8.0, 8.5, 9.0, 9.5 and 10.0
M Ru(bpy) 3 2+ And 10
M DBAE); taking the above substrate solutions to respectively determine initial luminescence I 0 (ii) a 1 mL of the solution was taken and 5 was added thereto
g mL -1 ALP-anti-human IgG solution 1 of (4)
Determination of quenched luminescence I after 30min of reaction at 37 deg.C 1 。
Shown in FIG. 7 result, ΔI ECL Increases with increasing pH, but no significant increase after pH 9.0, with the pH of the subsequent test substrate set to 9.0.
The concentrations of the luminophor and the coreactant are adjusted to realize the detection of IgG antibody in different concentration ranges
IgG antibodies were detected over 3 different concentration ranges:
1) Antigen-antibody reaction: get 100
0.2 of (C)
g mL -1 Biotinylated antibody, 100
0.5 of (2)
g mL -1 ALP-labeled antibody 100
Adding human IgG with various concentrations into a centrifugal tube which is sealed by BSA in advance, uniformly mixing, and then incubating for 30min at 37 ℃;
2) Magnetic separation: taking 300 out after antigen-antibody reaction
Adding the magnetic beads into a centrifuge tube, shaking uniformly, incubating at 37 ℃ for 20 min, shaking uniformly twice during the incubation period, and avoiding the magnetic beads from sinking. After the reaction is finished, magnetically separating to remove supernatant, and washing for 5 times by using a washing solution;
3) Enzymatic reaction: adding enzyme reaction substrate 100 into the centrifugal tube containing washed magnetic beads
Shaking and incubating for 30min at 37 ℃; magnetic separation, taking the supernatant for electrochemical luminescence detection;
4) Electrochemical luminescence detection: and applying a voltage of 2.0V to the electrode by using an electrochemical workstation, and acquiring a luminescence image under a chemiluminescence imaging system. Firstly, 15 is added in each detection unit
Measuring the initial luminous intensity I of the substrate solution before the enzyme reaction 0 (ii) a Adding substrate solution after enzyme reaction, and measuring luminescence intensity I after quenching 1 . Analysis I 1 AndI ECL = I 0 - I 1 and the amount of enzyme (i.e., the antigen to be detected).
The results of FIGS. 8, 9 and 10 show that the experiment is 3
M Ru(bpy) 3 2+ /30 
M DBAE detection 50-5000 pg mL -1 The IgG of (4); at 10
M Ru(bpy) 3 2+ /10 
M DBAE detection 0.2-20 ng mL -1 The IgG of (4); at 30 with
M Ru(bpy) 3 2+ /5 
M DBAE detection is 2-120 ng mL -1 For example, within 3 concentration ranges, the gray value of electrochemiluminescence imaging on the SANs electrode is in good linear correlation with the concentration of the IgG; while the gray value on the ITO varies with the concentrationSmaller (black curve), detection of three concentration ranges of IgG was not achieved.
Claims (11)
1. An electrochemiluminescence enzyme-linked immunosensor, comprising: the preparation method comprises the following steps:
(1) Synthesizing mesoporous silica on an indium tin oxide electrode: weighing 0.1-0.5 g hexadecyl trimethyl ammonium bromide, dissolving with 10-150 mL deionized water and 10-100 mL absolute ethyl alcohol mixed solution, stirring to clarify, sequentially adding 1-50% by using micropipette
Concentrated ammonia water, 10-150
Tetraethyl silicate, stirring for 1-30 min; transferring the solution into a staining cassette; finally, putting the pretreated indium tin oxide electrode glass into a dyeing box, sealing, and carrying out water bath at 30-150 ℃ for 1-48 h;
after the water bath is finished, taking out the indium tin oxide electrode glass on which the mesoporous silicon dioxide grows out, washing the indium tin oxide electrode glass clean by deionized water, and drying the indium tin oxide electrode glass by air blowing for 5 to 48 hours at the temperature of between 50 and 300 ℃; taking out, cooling, adding into 0.1-0.5M hydrochloric acid ethanol solution, stirring for 1-30 min, and blow-drying with nitrogen to obtain SANs/ITO electrodes;
(2) Adhering a paper cover plate with the SANs/ITO electrode to prepare a sensor SANs-ECL; each sensor is provided with 1-10 independent detection units; each unit comprises 1-10 working electrodes and forms 1-5 micro-two-electrode systems with the counter electrode; each detection unit is respectively dripped with 1 to 15
Applying 0.5-3.0V voltage to two ends of different solutions for detection;
(3) Reacting biotin-labeled antibody and alkaline phosphatase-labeled antibody with antigen to form an antigen-antibody complex, adding avidin magnetic beads to separate the antigen-antibody complex, and finally adding substrate disodium phenyl phosphate to react to detect the generated phenol, wherein the content of the substance to be detected is in direct proportion to the generated phenol, thereby constructing the electrochemiluminescence enzyme-linked immunosensor.
2. The electrochemiluminescence enzyme-linked immunosensor of claim 1,
in the step (1), the pretreatment method of the pretreated indium tin oxide electrode glass comprises the following steps: cutting the customized indium tin oxide electrode into the size of 1-5 cm multiplied by 1-5 cm, soaking the electrode in an ethanol solution containing 0.1-2M NaOH for 1-26 h, sequentially ultrasonically cleaning the electrode with acetone, ethanol and deionized water for 1-30 min respectively, and then drying the electrode with nitrogen.
3. The electrochemiluminescence enzyme-linked immunosensor of claim 1,
the enzyme-linked immunosorbent assay of the step (3) specifically comprises the following steps:
1) Antigen-antibody reaction: taking 10-200
0.1-1.0
mL -1 Biotinylated antibody, 10-200
0.1-1.0
mL -1 Alkaline phosphatase-labeled antibody 10-200
Adding human immunoglobulin of each concentration into a centrifuge tube sealed by bovine serum albumin in advance, uniformly mixing, and incubating at 37 ℃ for 5-35 min;
2) Magnetic separation: taking 50-300 parts of the antigen-antibody mixture after the antigen-antibody reaction is finished
Adding the magnetic beads into a centrifugal tube, shaking uniformly, incubating at 37 ℃ for 5-30 min, shaking uniformly twice during the incubation period, and avoiding the magnetic beads from sinking to the bottom; after the reaction is finished, magnetically separating to remove supernatant, and washing for 1-10 times by using phosphate buffer solution;
3) Enzymatic reaction: adding 50-200 enzyme reaction substrates into a centrifugal tube filled with washed magnetic beads
Shaking and incubating at 37 deg.C for 5-35 min; and (4) magnetically separating, and taking the supernatant for electrochemical luminescence detection.
4. The electrochemiluminescence enzyme-linked immunosensor of claim 2, wherein the ethanolic solution of NaOH comprises 0.5-1.0M NaOH.
5. The electrochemiluminescence enzyme-linked immunosensor of claim 2, wherein the soaking time is 18-24 hours.
6. The electrochemiluminescence enzyme-linked immunosensor of claim 2, wherein the time for sequentially performing ultrasonic cleaning with acetone, ethanol, and deionized water is 10-20 min during the pretreatment.
7. The electrochemiluminescence enzyme-linked immunosensor of any one of claims 1-6, wherein in step (1), 0.16 g of cetyltrimethylammonium bromide is weighed; the deionized water is 50-100 mL; the volume of the absolute ethyl alcohol is 20-50 mL; the amount of the strong ammonia water is 5 to 20
The concentration of the concentrated ammonia water is 10-50wt.%; the amount of the tetraethyl silicate is 60-120
(ii) a The stirring time is 10-25 min; the temperature of the water bath is 40-90 ℃; the water bath time is 10-40h.
8. The electrochemiluminescence enzyme-linked immunosensor of claim 7, wherein the deionized water is 60-80 mL; the concentration of the concentrated ammonia water is 15-40 wt.%; the amount of the tetraethyl silicate is 70-100
(ii) a The stirring time is 15-20 min; the temperature of the water bath is 50-80 ℃; the water bath time is 20-30 h.
9. The electrochemiluminescence enzyme-linked immunosensor of any one of claims 1-6, wherein in step (1), the temperature of the forced air drying is 150-200 ℃; the time of forced air drying is 15-30 h; stirring in hydrochloric acid ethanol solution for 15-20 min.
10. The ECL ELISA sensor of claim 3 wherein in step 1) of the ELISA reaction of step (3), the incubation time is 10-30 min; in the step 2), the incubation time is 10-25 min, and the washing times of the phosphate buffer saline solution are 4-8 times; in the step 3), the shaking incubation time is 10-25 min.
11. Use of an electrochemiluminescent enzyme-linked immunosensor according to any one of claims 1-10 for the detection of different biomarkers at different concentrations.
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|---|---|---|---|
| CN202011075117.8A CN112229888B (en) | 2020-10-09 | 2020-10-09 | Electrochemical luminescence enzyme-linked immunosensor and preparation method and application thereof |
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| CN101545913A (en) * | 2008-03-25 | 2009-09-30 | 北京科美东雅生物技术有限公司 | Chemoluminescence immunoassay measuring kit and preparation method thereof for triiodothyronine magnetic particles |
| CN102778453A (en) * | 2012-08-08 | 2012-11-14 | 济南大学 | Manufacture method and application of silver hybridization SBA-15 electrochemical luminescence immunosensor |
| CN103116023A (en) * | 2013-01-25 | 2013-05-22 | 宁波大学 | ECL (electrochemiluminescence) immunosensor for detecting tumor markers and preparation method and applications thereof |
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