CN112858416B - Preparation method of coreactant-free electrochemical luminescence immunosensor - Google Patents

Preparation method of coreactant-free electrochemical luminescence immunosensor Download PDF

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CN112858416B
CN112858416B CN202110152918.8A CN202110152918A CN112858416B CN 112858416 B CN112858416 B CN 112858416B CN 202110152918 A CN202110152918 A CN 202110152918A CN 112858416 B CN112858416 B CN 112858416B
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zinc sulfide
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CN112858416A (en
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邹桂征
傅莉
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Shandong University
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Abstract

The invention belongs to the field of analytical technique and method, and relates to a preparation method of a co-reactant-free electrochemical luminescence immunosensor, which mainly comprises (1) preparing an oil-soluble indium phosphide nanocrystalline dispersion solution by using indium bromide, tri (dimethylamino) phosphine, zinc bromide and oleylamine as raw materials; (2) preparing oil-soluble indium phosphide/zinc sulfide nanocrystals by using the oil-soluble indium phosphide nanocrystals, zinc acetate and n-dodecyl mercaptan as raw materials; (3) dissolving oil-soluble indium phosphide/zinc sulfide nanocrystals in dichloromethane, and adding N, N-dimethylformamide, mercaptopropionic acid and thiomalic acid to prepare a water-soluble indium phosphide/zinc sulfide nanocrystal solution; (4) preparing a secondary antibody marked by water-soluble indium phosphide/zinc sulfide nanocrystals; (5) and preparing the electrochemical luminescence immunosensor without the co-reactant. The preparation method does not need to add a coreactant in the preparation process, and avoids the damage to biological substances and cells caused by the coreactant.

Description

Preparation method of co-reactant-free electrochemical luminescence immunosensor
Technical Field
The invention belongs to the field of analysis technical methods, and relates to a preparation method of a co-reactant-free electrochemical luminescence immunosensor taking an indium phosphide/zinc sulfide nano material as a marker.
Background
Electrochemiluminescence (ECL) is a technology combining chemiluminescence and electrochemiluminescence, and has attracted much attention in bioanalysis, environmental monitoring, and clinical diagnosis due to its fast response, high sensitivity, low cost, and easy control. In ECL detection, high sensitivity detection of a target depends not only on high luminous efficiency of the luminophores, but also on the effective signal switching pattern.
The co-reactant type electrochemiluminescence can realize ECL by means of one-way anode or one-way cathode electrochemical driving, has high radiation efficiency and high intensity, is the most common electrochemiluminescence application mode at present, and the high-concentration co-reactant (mostly alkylamino) is a prerequisite condition for obtaining high-intensity electrochemiluminescence and a limitation condition for further popularization and application of the electrochemiluminescence technology; the coreactant-free electrochemical luminescence system has important value and wide application prospect with related technology research and development.
At this stage, the academia generally adopts the mode of coupling the co-reactant with the electrochemical luminescent substance through covalent bond or embedding the co-reactant in the luminescent substance carrier to realize the electrochemical luminescent effect without external co-reactant. For example, JU adopts a mode of covalently coupling a coreactant triethylamine and a luminescent substance polymer quantum dot to shorten an electron transfer path and reduce a transmission distance of a radical intermediate, thereby realizing the application of electrochemiluminescence sensing without an additional coreactant (Angew. chem. int. Ed.2021,60, 197-201). There is no application technology that only relies on anodic oxygen or cathodic reduction to realize co-reactant-free electrochemiluminescence.
Disclosure of Invention
Aiming at the defects of the prior art, particularly the situation that the electrochemical luminescence immunosensor needs co-reactants, the invention provides a preparation method of a co-reactant-free electrochemical luminescence immunosensor by taking nano indium phosphide/zinc sulfide nano-crystals as markers.
Description of terms:
antigen: the antigen (Ag) of the invention is a conventional antigen such as an alpha fetoprotein antigen, a carcinoembryonic antigen, a carbohydrate antigen 125, a prostate specific antigen and the like.
A first antibody: the primary antibody (Ab) of the present invention1) The invention refers to the antibody correspondingly produced to the antigen, and the invention has better effect on the monoclonal antibody corresponding to the antigen.
Secondary antibody: the secondary antibody of the invention refers to a secondary antibody which is generated correspondingly to the antigen and the primary antibody.
The technical scheme of the invention is as follows:
a preparation method of a coreactant-free electrochemical luminescence immunosensor comprises the following steps:
(1) preparing an oil-soluble indium phosphide nanocrystalline (InP NCs) dispersion solution under anhydrous and oxygen-free conditions by taking indium bromide as an indium source, tris (dimethylamino) phosphine as a phosphine source, zinc bromide as a stabilizer and oleylamine as a solvent and a ligand;
(2) preparing oil-soluble indium phosphide/zinc sulfide nanocrystals (InP/ZnS NCs) by using oil-soluble indium phosphide nanocrystals as nanocrystal cores, zinc acetate as a zinc source and n-dodecyl mercaptan as a sulfur source under anhydrous and oxygen-free conditions;
(3) dissolving oil-soluble indium phosphide/zinc sulfide nanocrystals in dichloromethane, and sequentially adding N, N-dimethylformamide, mercaptopropionic acid and thiomalic acid to prepare a water-soluble indium phosphide/zinc sulfide nanocrystal solution;
(4) preparing a water-soluble indium phosphide/zinc sulfide nanocrystalline labeled secondary antibody, namely: InP/ZnS | Ab2；
(5) InP/ZnS | Ab in the form of a sandwich immune complex formed on the surface of the working electrode2Grafting and fixing the material on the surface of a working electrode to prepare the co-reactant-free electrochemical luminescence immunosensor.
According to the present invention, preferably, the oil-soluble indium phosphide nanocrystal dispersion solution in step (1) is prepared as follows:
(a) mixing indium bromide (InBr)3) Zinc bromide (ZnBr)2) Mixing with oleylamine, introducing nitrogen, and stirring at 110-130 ℃ for 0.5-1.5 h until the solution is clear;
(b) and continuously heating to 200-210 ℃, adding tris (dimethylamino) phosphine and oleylamine, and reacting for 10-30 min until the solution turns to red from colorless, thereby obtaining the oil-soluble indium phosphide nano-crystalline (InP NCs) dispersion solution.
According to the present invention, in the preparation of the oil-soluble indium phosphide nanocrystal (InP NCs) dispersion solution of step (1), preferred conditions are as follows:
indium bromide in step (a): zinc bromide: the mass ratio of oleylamine ═ 17: 110: 750.
in the step (b), the concentration of the tri (dimethylamino) phosphine is 2-2.5 mmol/mL, and the concentration of the tri (dimethylamino) phosphine is as follows: the volume ratio of oleylamine is 1: (1-1.2).
According to the present invention, most preferably, the oil-soluble indium phosphide nanocrystal dispersion solution in step (1) is prepared as follows:
putting 0.34mmol of indium bromide, 2.2mmol of zinc bromide and 5mL of 3mmol/L oleylamine into a three-neck flask, mixing in a temperature-controlled heating magnetic stirrer, introducing nitrogen, stirring at 120 ℃ for 1h, and removing water and oxygen in the mixed solution until the solution is clear; and continuously heating to 200 ℃, adding 1mL of 2.4mmol/mL tris (dimethylamino) phosphine and 1mL of oleylamine, reacting for 20min until the solution turns red from colorless, and stopping heating to obtain the oil-soluble indium phosphide nanocrystalline dispersion solution.
According to the present invention, preferably, the oil-soluble indium phosphide/zinc sulfide nanocrystals (InP/ZnS NCs) in step (2) are prepared as follows:
adding zinc acetate into the oil-soluble indium phosphide nanocrystalline (InP NCs) dispersion solution prepared in the step (1), introducing nitrogen, and stirring at 110-130 ℃ for 0.5-1.5 h; continuously heating to 220-240 ℃, adding n-dodecyl mercaptan, and reacting for 0.5-1.5 h; and then centrifugally separating the obtained solution, and washing the obtained precipitate with ethanol or dichloromethane to obtain the oil-soluble indium phosphide/zinc sulfide nanocrystals (InP/ZnS NCs).
According to the present invention, it is preferable that the ratio of zinc acetate: mass ratio of n-dodecyl mercaptan of 5: 2.
according to the present invention, it is preferable that the ratio of the amounts of the zinc acetate in step (2) and the indium bromide in step (1) is 50: 17.
according to the present invention, it is preferable that the water-soluble indium phosphide/zinc sulfide nanocrystals (InP/ZnS NCs) in step (3) are prepared as follows:
dissolving the oil-soluble indium phosphide/zinc sulfide nanocrystals (InP/ZnS NCs) prepared in the step (2) in dichloromethane, then adding N, N-dimethylformamide, mercaptopropionic acid and thiomalic acid, introducing nitrogen, heating at 120-140 ℃ for 10-30 min for ligand exchange, washing the quantum dots subjected to ligand exchange with methanol for three times to obtain water-soluble indium phosphide/zinc sulfide nanocrystals, and preparing a 4mg/mL water-soluble indium phosphide/zinc sulfide nanocrystal solution and storing in a refrigerator at 4 ℃ for later use.
According to the present invention, preferably, the dichloromethane in the step (3): n, N-dimethylformamide: the volume ratio of mercaptopropionic acid is 4: 6: 1.
according to the present invention, it is preferable that the ratio of the amounts of the thiomalic acid in step (3) and the indium bromide in step (1) is 25: 17.
according to the present invention, it is preferable that the volume-to-mass ratio of the dichloromethane in step (3) to the indium bromide in step (1) is 50: 3, unit: mL/g.
According to the present invention, it is preferable that a water-soluble InP/ZnS | Ab-labeled secondary antibody of indium phosphide/zinc sulfide nanocrystal is prepared in the step (4)2The process of (2) is as follows:
adding 1-ethyl- (3-dimethyl amino propionic acid) carbodiimide hydrochloride (EDC) and hydroxysuccinimide (NHS) into the water-soluble indium phosphide/zinc sulfide nanocrystalline solution (InP/ZnS NCs) prepared in the step (3) for activation for half an hourRe-dissolving the precipitate in Phosphate Buffer Solution (PBS), adding corresponding secondary antibody, incubating at 37 deg.C for 3 hr, and blocking with bovine serum albumin to obtain InP/ZnS | Ab2(ii) a InP/ZnS | Ab2Reconstituted in PBS and stored in a 4 degree freezer.
Preferably according to the invention, the 1-ethyl- (3-dimethylaminopropionic acid) carbodiimide hydrochloride in step (4): the volume ratio of the hydroxysuccinimide is 1: 1.
according to the present invention, it is preferable that the 1-ethyl- (3-dimethylaminopropionic acid) carbodiimide hydrochloride in the step (4): the volume ratio of the water-soluble indium phosphide/zinc sulfide nanocrystalline solution is (1-2): 100.
according to the present invention, it is preferable that the process of preparing the co-reactant-free type electrochemiluminescence immunosensor in the step (5) is as follows:
(i) the method comprises the following steps Polishing a Glassy Carbon Electrode (GCE) by using alumina, and then cleaning the polished glassy carbon electrode by using alcohol and ultrapure water; scanning GCE in 1.0mM para aminobenzoic acid (ABA) aqueous solution for 4 sections and 2 circles at a scanning speed of 10mV/S, wherein the scanning potential range is 0.4-1.2V, so that ABA is electropolymerized to the surface of the GCE, washing the electrode with 10mM PBS (pH 7.4), and removing unreacted ABA;
(ii) the method comprises the following steps (ii) dropwise adding ethyl- (3-dimethylaminopropionic acid) carbodiimide hydrochloride (EDC) and a hydroxysuccinimide (NHC) solution to the surface of the GCE modified electrode obtained in the step (i), activating for half an hour, washing the electrode with 10mM PBS (pH 7.4), and removing unreacted EDC and NHC;
(iii) the method comprises the following steps Anti-primary antibody (Ab)1) Dropwise adding the solution on the surface of the activated electrode obtained in the step (ii), incubating for 3h, sealing unreacted active sites of the electrode by bovine serum albumin, and cleaning the electrode;
(iv) the method comprises the following steps (iv) adding antigen (Ag) dropwise to the surface of the electrode treated in the step (iii), and incubating at room temperature for 90 min; cleaning the electrode, and adding InP/ZnS | Ab2Dripping the solution on the surface of an electrode for incubation for 1 h; InP/ZnS | Ab based on form of immune complex formation2Grafted and fixed on the surface of the working electrode to realize the preparation of the co-reactant-free electrochemical luminescence immunosensor.
According to the present invention, it is preferred that the bovine serum albumin in step (iii) has a volume fraction of 2%.
According to the present invention, it is preferred that the washing solution used for washing the electrode in steps (iii) and (iv) is a PBST solution, and the indexes of the solution are as follows: 0.01mol/L PBS contains 0.025mol/L Tween 20, and pH is 7.4.
According to the present invention, preferably, the antigen (Ag) in step (iv) is an alpha-fetoprotein antigen, a carcinoembryonic antigen, a carbohydrate antigen or a prostate specific antigen.
The invention also provides a method for carrying out immunodetection by using the coreactant-free electrochemical luminescence immunosensor, which comprises the following steps:
i: driving the indium phosphide nanocrystalline Inp/ZnSNCs fixed on the surface of the GCE by adopting a cyclic voltammetry method in a 100mM Carbonate Buffer Solution (CBS) with the pH value of 9 to generate electrochemiluminescence, wherein the GCE with the immune complex and the indium phosphide nanocrystals (Inp/ZnSNCs) modified on the surface is used as a working electrode, a platinum electrode is used as a counter electrode, and an Ag/AgCl electrode is used as a reference electrode;
II: drawing a working curve according to the relation between the maximum light intensity signal of the electrochemiluminescence and the concentration of the standard solution of the antigen to be detected;
III: and (3) carrying out electrochemiluminescence test on each sample solution to be tested according to the methods in the steps I and II, and detecting the antigen concentration in the sample solution to be tested according to the maximum light intensity signal and the working curve on the obtained electrochemiluminescence curve.
The principle of the invention is as follows:
the invention adopts indium phosphide/zinc oxide nano-crystals coated by mercaptopropionic acid and thiomalic acid as markers; carboxyl on the surface of the indium phosphide/zinc sulfide can be grafted with amino on the surface of the second antibody after being activated by EDC and NHS, so that the second antibody can be labeled.
The invention adopts a mode of forming carbon-nitrogen bonds under electropolymerization conditions to graft para aminobenzoic acid (ABA) to the surface of the working electrode GCE, and completes grafting of a first antibody by adopting EDC and NHS to further activate carboxyl of the ABA on the surface of the GCE.
The invention relates to a hardware device for collecting electrochemiluminescence spectrum, in particular to an electrochemiluminescence spectrum collecting system constructed in ZL2016203006983 which is a detection system capable of accurately collecting electrochemiluminescence spectrum information. The system realizes spectrum collection by combining a Versa STAT 3 type electrochemical analyzer with an Acton SP-2300 type CCD grating spectrometer. The invention relates to a spectrum collection method of electrochemiluminescence immunoassay, which is constructed in electrochemiluminescence immunoassay, namely electrochemiluminescence multi-component immunoassay method based on spectral resolution principle, ZL 201675805, and the method adopts a potential window of 0-1.6V, a scanning speed of 50 millivolts/second, an initial potential of 0V and a positive initial scanning direction. The electrochemical luminescence light intensity acquisition device is an MPI-EII type multifunctional electrochemical luminescence instrument produced by Sienna Ruimai analysis instruments Limited.
The invention has the beneficial effects that:
1. the invention provides a co-reactant-free electrochemical luminescence immunosensor, which is modified with indium phosphide/zinc phosphide nanocrystals coated with mercaptopropionic acid and thiomalic acid, has high electrochemical luminescence efficiency under the condition of no additional co-reactant, is an electrochemical luminophor with excellent performance, and is suitable for being widely applied to the fields of physical analysis, environmental monitoring, clinical diagnosis and the like.
2. The method has high selectivity. The electrochemical luminescence immunosensor without the co-reactant is constructed based on specific recognition and combination between the antigen and the antibody, so that interference protein in a liquid to be detected cannot be combined with the first antibody and the second antibody of the antigen, and the detection system is not interfered.
3. The method adopts an electrochemiluminescence immunosensing system without a co-reactant, does not need to add the co-reactant in the preparation process, avoids the damage to biological substances and cells caused by the co-reactant, is beneficial to simplifying detection steps and procedures, and provides experience for the subsequent simplified automatic cancer detection.
Drawings
FIG. 1 is a fluorescent UV spectrum of the oil-soluble indium phosphide nanocrystal prepared in example 1.
FIG. 2 is a UV spectrum of the oil-soluble indium phosphide/zinc sulfide nanocrystals prepared in example 1.
FIG. 3 is a UV spectrum of the water-soluble InP/ZnS nanocrystals prepared in example 1.
FIG. 4 is an electrochemiluminescence spectrum of the immunoreaction reagent-free immunosensor prepared in example 1, in which the water-soluble indium phosphide/zinc sulfide nanocrystals with a prostate-specific antigen concentration of 1pg/mL are used as markers.
FIG. 5 is a high power transmission electron micrograph of the water-soluble indium phosphide/zinc sulfide nanocrystals prepared in example 2.
FIG. 6 is an X-ray diffraction pattern of water-soluble indium phosphide/zinc sulfide nanocrystals in example 3.
FIG. 7 is the X-ray energy spectrum analysis spectrum of the water-soluble indium phosphide/zinc sulfide nano-crystal in example 4.
FIG. 8 is an electrochemiluminescence spectrum of water-soluble indium phosphide/zinc sulfide nanocrystals in example 5.
FIG. 9 is an electrochemiluminescence intensity diagram of the immunoreaction reagent-free immunosensor prepared in example 6, in which the concentration of prostate specific antigen is 0.5pg/mL and the water-soluble indium phosphide/zinc sulfide nanocrystals are used as markers.
FIG. 10 is an electrochemiluminescence intensity diagram of the immunoreaction reagent-free immunosensor prepared in example 7, in which the concentration of prostate specific antigen is 0.1pg/mL and the water-soluble indium phosphide/zinc sulfide nanocrystals are used as markers.
FIG. 11 is an electrochemiluminescence intensity diagram of the immunoreaction reagent-free immunosensor prepared in example 8, in which the concentration of prostate specific antigen is 0.05pg/mL and the water-soluble indium phosphide/zinc sulfide nanocrystals are used as markers.
FIG. 12 is an electrochemiluminescence intensity chart of the immunoreaction reagent-free immunosensor prepared in example 9, in which the concentration of prostate specific antigen is 0.01pg/mL and the water-soluble indium phosphide/zinc sulfide nanocrystals are used as markers.
FIG. 13 is the electrochemiluminescence intensity of the non-co-reactant type immunosensor prepared in example 10, in which the water-soluble InP/ZnS nanocrystals having a concentration of prostate specific antigen of 0.005pg/mL were used as the markers.
FIG. 14 is an electrochemiluminescence intensity chart of the immunoreaction reagent-free immunosensor prepared in example 11, in which the concentration of prostate specific antigen is 0.0004pg/mL and the water-soluble indium phosphide/zinc sulfide nanocrystals are used as markers.
FIG. 15 is an electrochemiluminescence intensity chart of the immunoreaction reagent-free immunosensor prepared in example 12, in which the water-soluble indium phosphide/zinc sulfide nanocrystals with the prostate-specific antigen concentration of 0pg/mL are used as markers.
FIG. 16 is a graph showing the operation of a co-reagent-free type immunosensor in which the water-soluble indium phosphide/zinc sulfide nanocrystals of prostate-specific antigen prepared in example 13 were used as a marker.
FIG. 17 shows the selectivity of the immunoreactive reagent-free immunosensor in which the water-soluble indium phosphide/zinc sulfide nanocrystals of prostate-specific antigen prepared in example 14 were used as the marker.
FIG. 18 is an electrochemiluminescence intensity diagram of an immunosensor in which N-N-butyldiethanolamine is a co-reactant, the immunosensor being a water-soluble indium phosphide/zinc sulfide nanocrystal of the prostate specific antigen prepared in the comparative example.
Detailed Description
The present invention will be further described with reference to the following examples and drawings, but is not limited thereto.
Example 1
Preparation of oil-soluble indium phosphide nanocrystals (InP NCs): putting 0.34mmol of indium bromide, 2.2mmol of zinc bromide and 5mL of 3mmol/L oleylamine into a three-neck flask, mixing in a temperature-controlled heating magnetic stirrer, introducing nitrogen, stirring at 120 ℃ for 1h, and removing water and oxygen in the mixed solution until the solution is clear; continuing to heat to 200 ℃, adding 1mL of tris (dimethylamino) phosphine with the concentration of 2.4mmol/mL and 1mL of oleylamine, reacting for 20min until the solution turns red from colorless, and stopping heating to obtain an oil-soluble indium phosphide nanocrystal dispersion solution; and then, centrifugally separating the obtained dispersion solution, and washing the obtained precipitate with ethanol to obtain the oil-soluble indium phosphide nanocrystal.
And (3) carrying out fluorescence ultraviolet spectrum test on the oil-soluble indium phosphide nanocrystal, wherein the fluorescence ultraviolet spectrum is shown in figure 1.
Preparation of oil-soluble indium phosphide/zinc sulfide nanocrystals (InP/ZnS NCs): adding 1mmol of zinc acetate into oil-soluble indium phosphide nanocrystalline (InP NCs) dispersion solution, introducing nitrogen, and stirring at 120 ℃ for 1 h; continuously heating to 230 ℃, adding 0.1mL of n-dodecyl mercaptan with the concentration of 4mmol/mL, and reacting for 1 h; and then, centrifugally separating the obtained solution, and washing the obtained precipitate with ethanol to obtain the oil-soluble indium phosphide/zinc sulfide nanocrystals (InP/ZnS NCs).
And (3) performing fluorescence ultraviolet spectrum test on the oil-soluble indium phosphide/zinc sulfide nano-crystal, wherein the fluorescence ultraviolet spectrum is shown in figure 2.
Preparation of water-soluble indium phosphide/zinc sulfide nanocrystals (InP/ZnS NCs):
dissolving 30mg of oil-soluble indium phosphide/zinc sulfide nanocrystals (InP/ZnS NCs) in 2mL of dichloromethane, then adding 3mL of N, N-dimethylformamide, 0.5mL of mercaptopropionic acid and 0.5mmol of thiomalic acid, introducing nitrogen, heating at 130 ℃ for 20min for ligand exchange, washing the ligand-exchanged quantum dots with methanol for three times to obtain water-soluble indium phosphide/zinc sulfide nanocrystals, and preparing a 4mg/mL water-soluble indium phosphide/zinc sulfide nanocrystal solution and storing in a refrigerator at 4 ℃ for later use.
And (3) carrying out fluorescence ultraviolet spectrum test on the water-soluble indium phosphide/zinc sulfide nano-crystal, wherein the fluorescence ultraviolet spectrum is shown in figure 3.
Water-soluble indium phosphide/zinc sulfide nanocrystalline-labeled secondary InP/ZnS | Ab2The preparation of (1): adding 10 mu L of 1-ethyl- (3-dimethyl amino propionic acid) carbodiimide hydrochloride (EDC) with the concentration of 100mg/mL and 10 mu L of hydroxysuccinimide (NHS) with the concentration of 100mg/mL into 1mL of 4mg/mL water-soluble indium phosphide/zinc sulfide nanocrystals (InP/ZnS NCs), activating for half an hour, re-dissolving the obtained precipitate after centrifugation in Phosphate Buffer Solution (PBS), adding corresponding secondary antibody, incubating for 3 hours at 37 ℃, and then blocking with bovine serum albumin to obtain the InP/ZnS | Ab2(ii) a InP/ZnS | Ab2Reconstituted in PBS and stored in a 4 degree freezer.
Preparing a co-reactant-free electrochemical luminescence immunosensor by using water-soluble indium phosphide/zinc sulfide nano-crystals as markers:
(i) the method comprises the following steps Polishing a Glassy Carbon Electrode (GCE) by using alumina, and then cleaning the polished glassy carbon electrode by using alcohol and ultrapure water; scanning GCE in 1.0mM para aminobenzoic acid (ABA) aqueous solution for 4 sections and 2 circles at a scanning speed of 10mV/S, wherein the scanning potential range is 0.4-1.2V, so that ABA is electropolymerized to the surface of the GCE, washing the electrode with 10mM PBS (pH 7.4), and removing unreacted ABA;
(ii) the method comprises the following steps (ii) dropwise adding ethyl- (3-dimethylaminopropionic acid) carbodiimide hydrochloride (EDC) and a hydroxysuccinimide (NHC) solution to the surface of the GCE modified electrode obtained in the step (i), activating for half an hour, washing the electrode with 10mM PBS (pH 7.4), and removing unreacted EDC and NHC;
(iii) the method comprises the following steps Anti-primary antibody (Ab)1) (iii) dropwise adding the solution onto the surface of the activated electrode obtained in the step (ii), incubating for 3h, blocking unreacted active sites of the electrode by bovine serum albumin with the volume fraction of 2%, and washing the electrode by using a PBST solution;
(iv) the method comprises the following steps (iv) dropping 10 μ l of 1pg/mL prostate specific antigen onto the treated electrode surface of (iii) and incubating at room temperature for 90 min; the electrode was cleaned with PBST solution and InP/ZnS | Ab2Dripping the solution on the surface of an electrode for incubation for 1 h; InP/ZnS | Ab based on form of immune complex formation2Grafted and fixed on the surface of the working electrode to realize the preparation of the co-reactant-free electrochemical luminescence immunosensor.
The indices of the PBST solutions used in steps (iii), (iv) are as follows: 0.01mol/L PBS contains 0.025mol/L Tween 20, and pH is 7.4.
The GCE modified with immune complex and indium phosphide nanocrystalline Inp/ZnS NCs on the surface prepared in this example was used as a working electrode, a platinum electrode was used as a counter electrode, and an Ag/AgCl electrode was used as a reference electrode, and the indium phosphide nanocrystalline Inp/ZnS NCs immobilized on the surface of the GCE was driven by cyclic voltammetry in a 100mM Carbonate Buffer Solution (CBS) with a pH of 9 to generate electrochemiluminescence, and the electrochemiluminescence spectrum is shown in fig. 4.
Example 2
The procedure is as in example 1, except that the prepared indium phosphide/zinc sulfide nanocrystalline solution is dropped on a copper mesh to observe the morphology. The high-power transmission electron microscope photo of the prepared water-soluble indium phosphide/zinc sulfide nano-crystal is tested and shown in figure 5.
Example 3
The procedure is the same as in example 1, except that the prepared indium phosphide/zinc sulfide nanocrystalline solution is dried in a drop manner to prepare powder, and the X-ray diffraction spectrum of the prepared water-soluble indium phosphide/zinc sulfide nanocrystalline is shown in FIG. 6.
Example 4
The procedure is the same as in example 1, except that the prepared indium phosphide/zinc sulfide nanocrystalline solution is dried in a drop manner to prepare powder, and the X-ray energy spectrum analysis spectrogram of the water-soluble indium phosphide/zinc sulfide nanocrystalline prepared by the powder test is shown in FIG. 7.
Example 5
The procedure is as in example 1, except that the prepared indium phosphide/zinc sulfide nanocrystal solution is dropped on the surface of the electrode to test the prepared water-soluble indium phosphide/zinc sulfide nanocrystal electrochemiluminescence spectrogram as shown in FIG. 8
Example 6
The procedure is as in example 1, except that: the antigen added in step (iv) is 10 microliters to 0.5pg/mL, and the electrochemiluminescence intensity of the coreactant-free electrochemiluminescence immunosensor is shown in fig. 9.
Example 7
The procedure is as in example 1, except that: the antigen added in the step (iv) is 10 microliters and 0.1pg/mL, and the electrochemiluminescence intensity diagram of the coreactant-free electrochemiluminescence immunosensor is shown in FIG. 10.
Example 8
The procedure is as in example 1, except that: the antigen added in step (iv) is 10 μ l and 0.05pg/mL, and the electrochemiluminescence intensity of the coreactant-free electrochemiluminescence immunosensor is shown in FIG. 11.
Example 9
The procedure is as in example 1, except that: the antigen added in the step (iv) is 10 microliters and 0.01pg/mL, and the electrochemiluminescence intensity diagram of the coreactant-free electrochemiluminescence immunosensor is shown in FIG. 12.
Example 10
The procedure is as in example 1, except that: the antigen added in step (iv) is 10 μ l and 0.005pg/mL, and the electrochemiluminescence intensity of the coreactant-free electrochemiluminescence immunosensor is shown in FIG. 13.
Example 11
The procedure is as in example 1, except that: the antigen added in step (iv) is 10 microliters 0.0004pg/mL, and the electrochemiluminescence intensity of the coreactant-free electrochemiluminescence immunosensor is shown in fig. 14.
Example 12
The procedure is as in example 5, except that: the antigen added in step (iv) is 10. mu.l 0pg/mL, and the electrochemiluminescence intensity of the coreactant-free electrochemiluminescence immunosensor is shown in FIG. 15.
Example 13
The electrochemical luminescence immunoassay method without the co-reactant comprises the following steps:
i: the method comprises the steps that a GCE with an immune compound and indium phosphide nanocrystalline Inp/ZnSNCs modified on the surface is used as a working electrode, a platinum electrode is used as a counter electrode, an Ag/AgCl electrode is used as a reference electrode, and the indium phosphide nanocrystalline Inp/ZnSNCs fixed on the surface of the GCE is driven to generate electrochemiluminescence by adopting a cyclic voltammetry method in a 100mM Carbonate Buffer Solution (CBS) with the pH value of 9;
II: drawing a working curve according to the relation between the electrochemical luminescence maximum light intensity signal and the concentration of the antigen standard solution to be detected;
III: and (3) carrying out electrochemiluminescence test on each sample solution to be tested according to the methods in the steps I and II, and detecting the antigen concentration in the sample solution to be tested according to the maximum light intensity signal and the working curve on the obtained electrochemiluminescence curve.
The operation curve of the immunoreactive reagent-free immunosensor of this example is shown in FIG. 16.
Example 14
The procedure is as in example 1, except that: the antigens in the step (4) are respectively carbohydrate antigen 125, alpha fetoprotein antigen, carcinoembryonic antigen, prostate specific antigen and a mixture of four detection substances.
The selectivity spectrum of the sensor in this example is shown in fig. 17. As shown in fig. 17, the sensor prepared in this example has good selectivity for carbohydrate antigen 125, and other antigen proteins do not interfere with the sensing detection of the target antigen of the present invention.
Comparative example
The procedure is as in example 1 except that in step (iv) the electrochemiluminescence intensity of the electrochemiluminescence immunosensor containing N-N-butyldiethanolamine as a co-reactant is shown in FIG. 18.
As can be seen from the comparative example and example 1, the electrochemical luminescence intensities of the electrochemical luminescence immunosensor without the co-reactant prepared by the method of the present invention and the electrochemical luminescence immunosensor with N-N-butyldiethanolamine as the co-reactant are almost the same, which indicates that the method of the present invention still has high electrochemical luminescence efficiency without adding the co-reactant.

Claims (10)

1. A preparation method of a coreactant-free electrochemical luminescence immunosensor comprises the following steps:
(1) preparing an oil-soluble indium phosphide nanocrystalline (InP NCs) dispersion solution under anhydrous and oxygen-free conditions by taking indium bromide as an indium source, tris (dimethylamino) phosphine as a phosphine source, zinc bromide as a stabilizer and oleylamine as a solvent and a ligand;
(2) preparing oil-soluble indium phosphide/zinc sulfide nanocrystals (InP/ZnS NCs) by using oil-soluble indium phosphide nanocrystals as nanocrystal cores, zinc acetate as a zinc source and n-dodecyl mercaptan as a sulfur source under anhydrous and oxygen-free conditions;
wherein, the preparation process of the oil-soluble indium phosphide/zinc sulfide nanocrystalline comprises the following steps:
adding zinc acetate into the oil-soluble indium phosphide nanocrystal dispersion solution prepared in the step (1), introducing nitrogen, and stirring at 110-130 ℃ for 0.5-1.5 h; continuously heating to 220-240 ℃, adding n-dodecyl mercaptan, and reacting for 0.5-1.5 h; then centrifugally separating the obtained solution, and washing the obtained precipitate with ethanol or dichloromethane to obtain oil-soluble indium phosphide/zinc sulfide nanocrystals;
(3) dissolving oil-soluble indium phosphide/zinc sulfide nanocrystals in dichloromethane, and sequentially adding N, N-dimethylformamide, mercaptopropionic acid and thiomalic acid to prepare a water-soluble indium phosphide/zinc sulfide nanocrystal solution;
(4) preparing a water-soluble indium phosphide/zinc sulfide nanocrystalline labeled secondary antibody, namely: InP/ZnS | Ab2；
(5) InP/ZnS | Ab in the form of a sandwich immune complex formed on the surface of the working electrode2Grafting and fixing the material on the surface of a working electrode to prepare the co-reactant-free electrochemical luminescence immunosensor.
2. The method for preparing an immunosensor according to claim 1, wherein the oil-soluble indium phosphide nanocrystal dispersion solution in step (1) is prepared as follows:
(a) mixing indium bromide (InBr)3) Zinc bromide (ZnBr)2) Mixing with oleylamine, introducing nitrogen, and stirring at 110-130 ℃ for 0.5-1.5 h until the solution is clear;
(b) and continuously heating to 200-210 ℃, adding tris (dimethylamino) phosphine and oleylamine, and reacting for 10-30 min until the solution turns to red from colorless, thereby obtaining the oil-soluble indium phosphide nano-crystalline (InP NCs) dispersion solution.
3. The method of preparing an immunosensor according to claim 2, wherein the oil-soluble indium phosphide nanocrystal (InP NCs) dispersion solution of step (1) is prepared under the following conditions:
indium bromide in step (a): zinc bromide: the mass ratio of oleylamine = 17: 110: 750;
in the step (b), the concentration of the tri (dimethylamino) phosphine is 2-2.5 mmol/mL, and the concentration of the tri (dimethylamino) phosphine is as follows: oleylamine volume ratio = 1: (1-1.2).
4. The method of claim 2, wherein the oil-soluble indium phosphide nanocrystal dispersion solution in the step (1) is prepared as follows:
putting 0.34mmol of indium bromide, 2.2mmol of zinc bromide and 5mL of 3mmol/L oleylamine into a three-neck flask, mixing in a temperature-controlled heating magnetic stirrer, introducing nitrogen, stirring at 120 ℃ for 1h, and removing water and oxygen in the mixed solution until the solution is clear; and continuously heating to 200 ℃, adding 1mL of 2.4mmol/mL tris (dimethylamino) phosphine and 1mL of oleylamine, reacting for 20min until the solution turns red from colorless, and stopping heating to obtain the oil-soluble indium phosphide nanocrystal dispersion solution.
5. The method for preparing an immunosensor according to claim 1, wherein the ratio of zinc acetate: mass ratio of n-dodecyl mercaptan = 5: 2; the mass ratio of the zinc acetate to the indium bromide in step (1) is = 50: 17.
6. the method of claim 1, wherein the water-soluble indium phosphide/zinc sulfide nanocrystals (InP/ZnS NCs) in step (3) are prepared as follows:
dissolving the oil-soluble indium phosphide/zinc sulfide nanocrystals (InP/ZnS NCs) prepared in the step (2) in dichloromethane, then adding N, N-dimethylformamide, mercaptopropionic acid and thiomalic acid, introducing nitrogen, heating at 120-140 ℃ for 10-30 min for ligand exchange, washing the quantum dots subjected to ligand exchange with methanol for three times to obtain water-soluble indium phosphide/zinc sulfide nanocrystals, and preparing a 4mg/mL water-soluble indium phosphide/zinc sulfide nanocrystal solution and storing in a refrigerator at 4 ℃ for later use;
the dichloromethane ratio is as follows: n, N-dimethylformamide: mercaptopropionic acid at a volume ratio of = 4: 6: 1;
the mass ratio of thiomalic acid to indium bromide in step (1) is = 25: 17;
the volume mass ratio of the dichloromethane to the indium bromide in the step (1) is 50: 3, unit: mL/g.
7. The method of preparing the immunosensor according to claim 1, wherein the water-soluble indium phosphide/zinc sulfide nanocrystal labeled secondary antibody (InP/ZnS | Ab 2) in step (4) is prepared as follows:
adding 1-ethyl- (3-dimethyl amino propionic acid) carbodiimide hydrochloride (EDC) and hydroxysuccinimide (NHS) into the water-soluble indium phosphide/zinc sulfide nanocrystalline solution (InP/ZnS NCs) prepared in the step (3) for activation for half an hour, re-dissolving the precipitate obtained after centrifugation into Phosphate Buffer Solution (PBS), adding corresponding secondary antibody for incubation for 3 hours at 37 ℃, and then sealing by bovine serum albumin to obtain a substance, namely InP/ZnS | Ab2(ii) a InP/ZnS | Ab2Re-dissolving in PBS, and storing in 4 degree refrigerator;
the 1-ethyl- (3-dimethylaminopropionic acid) carbodiimide hydrochloride: hydroxysuccinimide volume ratio = 1: 1;
the 1-ethyl- (3-dimethylaminopropionic acid) carbodiimide hydrochloride: the volume ratio of the water-soluble indium phosphide/zinc sulfide nanocrystalline solution is = (1-2): 100.
8. the method for preparing an immunosensor according to claim 1, wherein the co-reagent-free electrochemiluminescence immunosensor is prepared by the following process in step (5):
(i) the method comprises the following steps Polishing a Glassy Carbon Electrode (GCE) by using alumina, and then cleaning the polished glassy carbon electrode by using alcohol and ultrapure water; scanning GCE in 1.0mM para aminobenzoic acid (ABA) aqueous solution for 4 sections and 2 circles at a scanning speed of 10mV/S, wherein the scanning potential range is 0.4-1.2V, so that ABA is electropolymerized to the surface of the GCE, washing an electrode by PBS (phosphate buffer solution) with the pH value of 7.4 and 10mM, and removing unreacted ABA;
(ii) the method comprises the following steps (ii) dropwise adding ethyl- (3-dimethylaminopropionic acid) carbodiimide hydrochloride (EDC) and a hydroxysuccinimide (NHC) solution to the surface of the GCE modified electrode obtained in the step (i), activating for half an hour, washing the electrode with 10mM PBS (pH 7.4), and removing unreacted EDC and NHC;
(iii) the method comprises the following steps Anti-primary antibody (Ab)1) (iii) dropwise adding the solution onto the surface of the activated electrode obtained in the step (ii), incubating for 3h, blocking unreacted active sites of the electrode by using bovine serum albumin, and cleaning the electrode;
(iv) the method comprises the following steps (iv) adding antigen (Ag) dropwise to the treated electricity of (iii)Incubating the surface of the sample for 90min at room temperature; cleaning the electrode, and adding InP/ZnS | Ab2Dripping the solution on the surface of an electrode for incubation for 1 h; InP/ZnS | Ab based on form of immune complex formation2Grafted and fixed on the surface of the working electrode to realize the preparation of the co-reactant-free electrochemical luminescence immunosensor.
9. The method of claim 8, wherein the bovine serum albumin in step (iii) is 2% by volume; the washing liquid used for washing the electrode in the steps (iii) and (iv) is a PBST solution, and indexes of the solution are as follows: 0.01mol/L PBS contains 0.025mol/L Tween 20, and the pH is = 7.4; the antigen (Ag) in the step (iv) is alpha fetoprotein antigen, carcinoembryonic antigen, carbohydrate antigen or prostate specific antigen.
10. A method for detecting a coreactant-free electrochemiluminescence immunosensor comprising the sandwich immunosensor prepared by the method of any one of claims 1-9, comprising the steps of:
i: driving indium phosphide nanocrystals (Inp/ZnS NCs) immobilized on the surface of a GCE by cyclic voltammetry in a 100mM Carbonate Buffer Solution (CBS) with pH =9 to generate electrochemiluminescence, using the GCE modified with an immunocomplex and indium phosphide nanocrystals (Inp/ZnS NCs) prepared by any one of claims 1-9 as a working electrode, a platinum electrode as a counter electrode, and an Ag/AgCl electrode as a reference electrode;
II: drawing a working curve according to the relation between the electrochemical luminescence maximum light intensity signal and the concentration of the antigen standard solution to be detected;
III: and (3) carrying out electrochemiluminescence test on each sample solution to be tested according to the methods in the steps I and II, and detecting the antigen concentration in the sample solution to be tested according to the maximum light intensity signal and the working curve on the obtained electrochemiluminescence curve.
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