CN111208183B - ECL sandwich immunosensing-based antigen detection kit - Google Patents

ECL sandwich immunosensing-based antigen detection kit Download PDF

Info

Publication number
CN111208183B
CN111208183B CN202010033170.5A CN202010033170A CN111208183B CN 111208183 B CN111208183 B CN 111208183B CN 202010033170 A CN202010033170 A CN 202010033170A CN 111208183 B CN111208183 B CN 111208183B
Authority
CN
China
Prior art keywords
antigen
electrode
concentration
fpsa
solution
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202010033170.5A
Other languages
Chinese (zh)
Other versions
CN111208183A (en
Inventor
毛俐
王彬
张飞
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Chengdu Medical College
Original Assignee
Chengdu Medical College
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Chengdu Medical College filed Critical Chengdu Medical College
Priority to CN202010033170.5A priority Critical patent/CN111208183B/en
Publication of CN111208183A publication Critical patent/CN111208183A/en
Application granted granted Critical
Publication of CN111208183B publication Critical patent/CN111208183B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
    • G01N27/3277—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction being a redox reaction, e.g. detection by cyclic voltammetry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
    • G01N27/3278—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction involving nanosized elements, e.g. nanogaps or nanoparticles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Physics & Mathematics (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Cell Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Electrochemistry (AREA)
  • Plasma & Fusion (AREA)
  • Nanotechnology (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)

Abstract

The invention provides an antigen detection kit based on Electrochemiluminescence (ECL) sandwich immunosensing, which comprises the following components: (1) a primary anti-modification electrode; (2) a silver nanoparticle-labeled secondary antibody; (3) persulfate solution. The antigen detection kit can be used for quantitatively detecting the antigen concentration, and experiments prove that the linear range of the kit for detecting fPSA antigen is 0.002-10 ng.mL ‑1 The detection limit is 0.7 pg.mL ‑1 . Compared with other methods in the prior art, the method based on ECL sandwich immunosensing has wider linear range and lower detection limit when detecting the antigen, and has good application prospect in clinical detection.

Description

ECL sandwich immunosensing-based antigen detection kit
Technical Field
The invention belongs to the field of sensors, and particularly relates to an antigen detection kit based on ECL sandwich immunosensing.
Background
Electrochemiluminescence (ECL), also called electrochemiluminescence, is a technique combining chemiluminescence and electrochemistry, and is a luminescence phenomenon that some electrically-generated intermediate substances are generated on the surface of an electrode by applying a certain voltage to perform electrochemical reaction, and then excited states are formed among the electrically-generated intermediate substances or among the electrically-generated intermediate substances and some components in a system through electron transfer, and the excited states return to the ground state. The technology integrates the advantages of high sensitivity of luminescence analysis and controllability of electrochemical potential, has wide linear range, simple instruments and equipment, convenient operation and quick analysis, and thus gradually becomes a research field of great interest for analytical chemists.
Electrochemiluminescence immunoassay is a novel luminescence immunoassay method developed in the late 80 s, and is a product combining electrochemiluminescence and immunoassay. The technology is characterized in that an electrochemical luminescent substance is used for marking an antigen or an antibody, and the change of an electrochemical luminescent signal before and after the immunoreaction of the antigen and the antibody is utilized, so that the quantitative analysis and detection of a substance to be detected are realized. Compared with other immunoassay methods, the method has the following characteristics: the electrode has no radioactive radiation hazard and no pollution, and is a specific chemiluminescence reaction initiated by electrochemistry on the surface of the electrode; the sensitivity is high; the detection speed is high, and only a few minutes or even a few seconds are needed; the stability is good; the application range is wide, and the antigen, the hapten and the antibody with different molecular sizes can be detected, and the nucleic acid probe can also be detected. Therefore, the electrochemical luminescence immunoassay has wide application prospect in the field of biological analysis and detection.
Potassium persulfate cathode electrochemical luminescence immunosensor is a new detection and analysis technology which is newly developed in recent years and combines chemistry, electrochemistry and immunosensor together. S. the 2 O 8 2- In an electrochemical reduction process, to produce SO in an oxidized state 4 ·- While dissolved oxygen is reduced to OOH · Which further reacts to form a singlet state ( 1 O 2 ) * Excess energy is released to produce optical radiation. SO 4 ·- Free radicals and OOH · The change in the concentration of the two intermediate species of the radical determines S 2 O 8 2- Of the ECL strength of (A), therefore, despite increasing SO 4 ·- Free radicals or OOH · Concentration of free radical, S 2 O 8 2- The ECL strength of (a) can be enhanced. Enhanced S production of active oxygen using enzymatic catalysis 2 O 8 2- The studies of ECL signals have been reported, however, the use of transition metal nanomaterials as co-reactants to promote SO 4 ·- Immunosensing methods to generate, enhance ECL signaling by persulfate have rarely been reported.
Prostate Specific Antigen (PSA), a specific protein secreted by the epithelial tissue of the Prostate, exists in serum in three major forms: (1) free prostate specific antigen (fPSA), accounting for 10% -30% of total prostate specific antigen (tPSA); (2) prostate specific antigen and alpha 1-anti-chylomicronComplex formation by the binding of the protease (PSA-ACT); (3) the complex formed by binding of prostate specific antigen to alpha 2-macroglobulin enzyme (PSA-. Alpha.2M). The main physiological function of the prostate specific antigen is that semen coagulation can be prevented, the prostate specific antigen has extremely high tissue organ specificity, and is the first choice marker for diagnosing prostate cancer at present. Currently, various methods for detecting fPSA antigens have been reported in the prior art (e.g., jugoslov Med Biohem)2005,24,129-134; although Journal of Immunological Methods 2011,369,74-80, the detection limit of fPSA antigen by these Methods is generally high (0.016 ng. ML) -1 ～0.08μg·L -1 ) The requirement of clinical sensitivity cannot be met. Therefore, by utilizing the advantages of potassium persulfate cathode electrochemiluminescence immunosensing, the ECL immunosensing-based detection kit with lower detection limit and wider detection range is developed, and the ECL immunosensing-based detection kit has very important significance for accurately diagnosing diseases such as prostatic cancer and the like.
Disclosure of Invention
The invention aims to provide an antigen detection kit based on ECL sandwich immunosensing and application thereof in quantitative detection of antigen concentration.
The invention provides an antigen detection kit based on ECL sandwich immunosensing, which comprises the following components:
(1) A modification-resistant electrode;
(2) A silver nanoparticle-labeled secondary antibody;
(3) A persulfate solution.
Further, the antigen detection kit also comprises a standard solution of the antigen to be detected.
Further, the primary anti-modified electrode is prepared by the following method: firstly, modifying gold particles on the surface of an electrode, then bonding primary antibody on the surface of the obtained electrode, and then blocking non-specific sites.
Further, the method for modifying the gold particles on the surface of the electrode comprises the following steps: soaking the electrode in a chloroauric acid solution for electrodeposition; the concentration of the chloroauric acid solution is preferably 0.5-2%, more preferably 1%, the voltage of the electrodeposition is preferably-0.3-0.1V, more preferably-0.2V, and the time of the electrodeposition is preferably 20-40 s, more preferably 30s; the electrode is a glassy carbon electrode, preferably a polished glassy carbon electrode;
and/or, the method for bonding the primary antibody to the surface of the obtained electrode is as follows: soaking the obtained electrode in primary antibody solution at 4 deg.C overnight; the concentration of the primary antibody solution is preferably 5 mg/mL -1 ；
And/or, the method for blocking non-specific sites comprises the following steps: and soaking the obtained electrode in bovine serum albumin, wherein the concentration of the bovine serum albumin is preferably 1%, and the soaking time is preferably 25min.
Further, the silver nanoparticle-labeled secondary antibody is prepared by the following method: mixing the secondary antibody and the silver nanoparticles, stirring under an ice bath condition, centrifuging to remove the unbound secondary antibody, and blocking the nonspecific sites with bovine serum albumin to obtain the silver nanoparticle composition; the stirring time is preferably 1 to 3 hours, and more preferably 2 hours.
Further, the silver nanoparticles are prepared by the following method: agNO is added under ice bath condition 3 The solution was added dropwise to NaBH, which was continuously stirred 4 Continuously stirring the solution until the color of the system becomes yellow green to obtain the product; the AgNO 3 The solution is preferably 1mM, the NaBH 4 The solution is preferably 2mM, and the continuous stirring time is 15min or more.
Further, the persulfate solution is potassium persulfate solution, the concentration of the persulfate solution is preferably 0.1M, and the pH is preferably 7.4.
Further, the primary antibody is fPSA primary antibody, the antigen to be detected is fPSA antigen, and the secondary antibody is fPSA secondary antibody.
The invention also provides a method for quantitatively detecting the concentration of the antigen, which is used for detecting by using the antigen detection kit based on ECL sandwich immunosensing and comprises the following specific steps:
(a) Determination of ECL intensity in standard solutions of the antigen to be tested at each concentration:
a 1 the standard solution of the antigen to be tested is configured to a gradient concentration,then the primary anti-modified electrode is added at the concentration of C i Incubating in a standard solution of the antigen to be detected to obtain an antigen modified electrode;
a 2 incubating the obtained antigen-modified electrode in a secondary antibody marked by silver nanoparticles to obtain a target electrode, and measuring the ECL intensity I of the target electrode in a persulfate solution by using an electrochemical luminescence analyzer i ；
(b) Preparing standard solution of antigen to be detected into 0 ng/mL -1 According to the method of step (a), 0 ng.mL was obtained -1 ECL Strength I of target electrode at concentration 0 ；
(c) Drawing a standard curve: by ECL intensity variation value Delta I i As ordinate, lgC i Drawing a curve for a horizontal coordinate, and performing linear regression to obtain a standard curve; wherein, delta I i ＝I i -I 0 ；
(d) Determination of the concentration of the antigen to be tested: incubating the primary anti-modified electrode in an antigen to be detected to obtain an antigen-modified electrode; incubating the obtained antigen-modified electrode in a silver nanoparticle-labeled secondary antibody to obtain a target electrode, and measuring the ECL intensity I of the target electrode in a persulfate solution by using an electrochemical luminescence analyzer x ；
Will be Delta I x Substituting into the ordinate of the standard curve obtained in the step (c), and obtaining the abscissa correspondingly, namely the lg value of the antigen concentration to be detected; wherein, delta I x ＝I x -I 0 。
Further, step (a) 1 ) Wherein the incubation temperature is room temperature, and the incubation time is 5 minutes or more, preferably 35 minutes or more, and more preferably 35 to 45 minutes;
and/or, step (a) 2 ) The incubation temperature is room temperature, and the incubation time is 5 minutes or more, preferably 25 minutes or more, and more preferably 25 to 35 minutes.
Further, in the step (a), C i 0.002-10 ng/mL -1 ；
Step (a) 2 ) And (d), the test parameters of the electrochemical luminescence analyzer are as follows: at a potential of 0V to-1.8VThe cyclic voltammetry scanning is carried out within the range, the scanning speed is 0.1V/s, and the high voltage of the photomultiplier is 600V.
Further, in step (c), the standard curve is Y =1640.27X +4883.96.
The invention also provides application of the ECL sandwich immunosensing-based antigen detection kit in quantitative detection of antigen concentration.
In the present invention, room temperature means 25. + -. 2 ℃.
In the present invention, nano-Ag is a silver nanoparticle.
The invention explains the catalytic mechanism of nano-Ag on potassium persulfate through cyclic voltammetry and Electrochemiluminescence (ECL) behaviors of nano-Ag in an aqueous solution of potassium persulfate for the first time. The catalytic mechanism is as follows:
oxidation reaction occurs during the electrochemical anode scanning process, nano-Ag is oxidized into Ag + (ii) a Reduction reaction, S, occurs during cathode scanning 2 O 8 2- Is reduced to SO 4 ·- Continue to react with HOO · The active oxygen excited state is generated by reaction and transits to the ground state to emit light, ag + During electrochemical cathode scanning, the material is electrochemically reduced to Ag 0 To promote SO in a cyclic reciprocating manner 4 ·- Catalyzes the ECL signal of persulfate.
Ag-e→Ag + (1)
S 2 O 8 2- +e→SO 4 ·- +SO 4 2- (2)
Ag + +S 2 O 8 2- →SO 4 ·- +SO 4 2- +Ag 2+ (3)
Ag + +e→Ag (4)
SO 4 ·- +H 2 O→HO · +HSO 4 - (5)
HO · →HOO · +H 2 O (6)
O 2 +H 2 O+e→HOO · +HO - (7)
Figure BDA0002365077220000041
Figure BDA0002365077220000042
The invention adopts a new solid phase coreactant nano-Ag and Ag generated in situ by electrochemical redox cycle reciprocation + Ionic catalysis of S 2 O 8 2- To SO 4 ·- Thereby sensitizing S 2 O 8 2- ECL signal of (c).
According to the invention, gold particles are modified on a Glassy Carbon Electrode (GCE) by an electrodeposition method, then a primary antibody is bonded to the surface of the electrode through an Au-S bond, BSA blocks a non-specific site, and then the primary antibody is specifically combined with fPSA antigen. And finally, specifically binding the nano-Ag labeled fPSA secondary antibody and fPSA antigen to be immobilized on the surface of the electrode, and constructing the target electrode based on solid-phase sandwich mode ECL immunosensing. In the target electrode, nano-Ag marked fPSA secondary antibody can effectively enhance the detection signal of potassium persulfate, and further enhance the sensitivity of the target electrode; meanwhile, as the concentration of fPSA antigen increases, the more nano-Ag labeled secondary antibody bound to the electrode surface, the stronger ECL intensity is detected.
Experiments prove that when the antigen detection kit disclosed by the invention is used for quantitatively detecting the concentration of the fPSA antigen, the linear range of fPSA antigen detection is 0.002-10 ng & mL -1 The detection limit is 0.7 pg.mL -1 . Compared with other methods in the prior art, the method based on ECL sandwich immunosensing has wider linear range and lower detection limit when detecting the antigen, and has good application prospect in clinical detection.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Drawings
FIG. 1: the preparation process of the target electrode is shown schematically.
FIG. 2: experimental example 1 in the electrode assembly process, each modified electrode was 0.1M S 2 O 8 2- ECL intensity in (PBS, pH 7.4): (a) bare electrode GCE, (b) DpAu/GCE, (c) anti-fPSA/DpAu/GCE, (d) BSA/anti-fPSA/DpAu/GCE, (e) fPSA/BSA/anti-fPSA/DpAu/GCE.
FIG. 3: TEM image of electrode step-by-step assembly process: (A) DpAu/GCE, (B) fPSA
BSA/anti-fPSA/DpAu/GCE, (C) a target electrode; (D) ultraviolet-visible absorption spectrum of nano-Ag sol.
FIG. 4: the electrode (a) and the target electrode (b) of the unlabeled secondary antibody were in the range of 0.1M S 2 O 8 2- ECL intensity in (PBS, pH 7.4).
FIG. 5: the incubation time of BSA/anti-fPSA/DpAu/GCE and fPSA antigen of the primary anti-modified electrode optimizes the experimental result.
FIG. 6: the incubation time of the fPSA antigen modified electrode fPSA/BSA/anti-fPSA/DpAu/GCE and the nano-Ag labeled secondary antibody optimizes the experimental result.
FIG. 7 is a schematic view of: and optimizing the ECL response experiment result of the target electrode to different concentrations of fPSA antigen.
FIG. 8: and (3) optimizing a standard curve of response of the target electrode to ECL (extracellular matrix) of fPSA antigens with different concentrations.
FIG. 9: and optimizing the stability result of the response of the target electrode to different concentrations of fPSA antigen ECL.
Detailed Description
The raw materials and equipment used in the invention are known products and are obtained by purchasing commercial products.
Wherein, the GCE electrode is a glassy carbon electrode, and phi =4mm; BSA is bovine serum albumin, >96%; fPSA is a free prostate specific antigen; the fPSA secondary antibody is labeled Anti-fPSA (paired secondary antibody) purchased from Zhengzhou Bosai bioengineering, LLC; the fPSA primary antibody is Anti-fPSA (immobilized antibody) purchased from Zheng State Bosai bioengineering, LLC.
Example 1 preparation of an ECL Sandwich-based immunosensing antigen detection kit
The antigen detection kit comprises the following components: (1) a primary anti-modification electrode; (2) a standard solution of fPSA antigen; (3) a silver nanoparticle-labeled secondary antibody; (4) persulfate solution.
First, preparing a primary anti-modified electrode:
pretreatment of the GCE electrode: using 0.5 μm and 0.05 μm Al successively for the bare electrode GCE 2 O 3 Polishing the paste, then ultrasonically washing the paste in distilled water, ethanol and distilled water for 3min in sequence, and airing the paste at room temperature for later use.
Soaking the pretreated GCE electrode in chloroauric acid (HAuCl) 4 1%) a layer of uniform gold nanoparticles was electrodeposited in the solution (electrodeposition voltage-0.2V, electrodeposition time 30 s), recorded as DpAu/GCE, rinsed with three times of water and dried at room temperature. Then, the modified electrode DpAu/GCE was immersed in 5 mg/mL -1 In the fPSA primary antibody solution, the electrode surface is modified by combining the fPSA primary antibody through Au-S bonds at 4 ℃ overnight to obtain anti-fPSA/DpAu/GCE. Blocking the redundant nano-sized Jin Weidian min with 1% BSA to obtain modified electrode BSA/anti-fPSA/DpAu/GCE, i.e., a primary anti-modified electrode.
Second, a standard solution of fPSA antigen was prepared:
taking a fPSA antigen standard, and diluting the fPSA antigen standard into standard solutions with the following gradient concentrations: 0 ng/mL -1 、0.002ng·mL -1 、0.005ng·mL -1 、0.05ng·mL -1 、0.1ng·mL -1 、0.5ng·mL -1 、1ng·mL -1 、5ng·mL -1 And 10 ng. ML -1 。
Step three, preparing a silver nanoparticle labeled secondary antibody:
(a) Synthesis of Nano-Ag
Before nano-Ag is synthesized, a container used in the synthesis process is soaked in a prepared chromic acid washing solution in advance, washed clean by tap water, washed by three times of water, and placed at room temperature to be dried for later use.
Under ice-bath conditions, 10mL of 1mM AgNO was added 3 The aqueous solution was added dropwise to 30mL2mM NaBH with constant stirring 4 And continuously stirring for 15min until the color of the sol is changed into yellow green, indicating that nano-Ag is generated, and obtaining nano-Ag sol for later use.
The obtained nano-Ag sol is tested by using an ultraviolet visible absorption spectrogram, and the result is shown in fig. 3D, so that the characteristic absorption peak position of nano-Ag is about 400nm, which is consistent with the literature report, and the result shows that the yellow-green sol is actually silver nano-particle nano-Ag.
(b) Preparation of Nano-Ag labeled Secondary antibody
20uL 5 mg. ML -1 The secondary fPSA antibody was added to 3mL of the nano-Ag sol obtained in step 1, and stirring was continued for 2h under ice bath conditions to bind the nano-Ag to the secondary fPSA antibody. Unbound fPSA secondary antibody was then removed by centrifugation at 3000rpm for 15 min.
Non-specific binding sites were blocked with BSA (200. Mu.L 1%) and nano-Ag labeled secondary antibodies were dispersed and stored at 4 ℃ until use.
Step four, preparing persulfate solution:
get K 2 S 2 O 8 A solution of 0.1M and pH 7.4 was prepared using Phosphate Buffered Saline (PBS).
The beneficial effects of the present invention are demonstrated by the following experimental examples.
Experimental example 1 ECL Signal Change 1 in electrode Assembly Process in immunoreaction of antigen detection kit, and test method
To study the change in ECL strength of each modified electrode obtained during the electrode assembly process when the antigen detection kit of the present invention is immunoreactive, the primary anti-modified electrode (BSA/anti-fPSA/DpAu/GCE) in the antigen detection kit of example 1 was immersed in 20. Mu.L of 40ng mL at 25 deg.C -1 fPSA antigen solution was incubated for 40min, and non-specifically bound antigen was washed away with PBS (pH 7.4) buffer to obtain fPSA antigen-modified electrodes (fPSA/BSA/anti-fPSA/DpAu/GCE). fPSA/BSA/anti-fPSA/DpAu/GCE was then labeled with 20. Mu.Lnano-Ag at 25 ℃The secondary antibody was incubated for 40min, and washed with PBS (pH 7.4) buffer to obtain the target electrode.
The following electrodes were taken: (a) a bare electrode GCE, (b) DpAu/GCE, (c) anti-fPSA/DpAu/GCE, (d) BSA/anti-fPSA/DpAu/GCE, (E) fPSA/BSA/anti-fPSA/DpAu/GCE, using an electrochemiluminescence analyzer (model MPI-E electrochemiluminescence analyzer, semany McJLtd.) at 0.1M K 2 S 2 O 8 In a (PBS, pH 7.4) solution, cyclic voltammetry scans were performed in a potential range of 0V to-1.8V at a scan rate of 0.1V/s and a photomultiplier high pressure of 600V, and the ECL intensities were measured, respectively.
2. Test results
The results are shown in FIG. 2. Curve a is that the GCE of the bare electrode is 0.1M S 2 O 8 2- (PBS, pH 7.4) in the presence of a relatively weak luminescence signal, S after electrodeposition of a layer of gold nanoparticles on the electrode surface 2 O 8 2- The ECL intensity of (curve b) is significantly increased, since nano-Au promotes electron transfer, increasing the number of excited states per unit time. After further incubation with fPSA primary antibody (curve c) and blocking of the excess nanogold sites with BSA (curve d), the ECL signal was successively reduced, since the S was blocked by the antibody protein and the BSA protein 2 O 8 2- Diffusion to the electrode surface. After incubation of the fPSA antigen to the electrode surface, ECL signal continues to decrease due to immune complexes (curve e). The change in ECL signal during the stepwise assembly of the target electrode is in accordance with theory, indicating successful assembly of the target electrode (the assembly scheme is shown in figure 1). Therefore, the antigen detection kit of the present invention can realize the assembly of the electrode through the immune reaction, and show different ECL signals during the assembly of the electrode.
Experimental example 2 TEM image of stepwise electrode Assembly Process in immunoreaction of antigen detection kit
1. Test method
In order to study the morphology change of each modified electrode obtained in the electrode assembly process when the antigen detection kit of the present invention undergoes an immunoreaction, a target electrode was prepared according to the same method as in experimental example 1.
Taking the following modified electrodes: the method comprises the following steps of (A) DpAu/GCE, (B) fPSA/BSA/anti-fPSA/DpAu/GCE, and (C) a target electrode, washing with a buffer solution, removing non-specific adsorption, and then, using TEM to characterize the appearance of the surface of the modified electrode.
2. Test results
The results are shown in FIGS. 3A to 3C. FIG. 3A shows the formation of gold particles (DpAu/GCE) in the form of a flower charge by electrodeposition on the surface of an electrode. FIG. 3B shows the electrode surface modified with protein composite layer fPSA/BSA/anti-fPSA/DpAu/GCE. The protein composite layer is an insulator and is not conductive under a scanning electron microscope, and a semi-transparent film is formed to cover the surface of the gold particles, so that the protein composite layer is successfully immobilized on the surface of the electrode. After the nano-Ag labeled secondary antibody is combined with fPSA antigen on fPSA/BSA/anti-fPSA/DpAu/GCE through a specific reaction, a large number of nanoparticles with the particle size of about 20nm are uniformly distributed on the surface of the electrode (figure 3C), which indicates that the nano-Ag labeled secondary antibody is successfully combined with the antigen in a specific manner, and indicates that the antigen detection kit can be used for assembling the target electrode based on ECL sandwich immunosensing.
Experimental example 3 ECL sensitization in immunoreaction with antigen detection kit
1. Test method
In order to further study the cause of the change in the electrode ECL obtained after the immunoreaction of the antigen detection kit of the present invention, a target electrode was prepared in the same manner as in experimental example 1.
In addition, with reference to the method of experimental example 1, a secondary antibody labeled with nano-Ag was replaced with a secondary antibody of fPSA (i.e., a secondary antibody of fPSA not labeled with nano-Ag), and an electrode of an unlabeled secondary antibody was prepared.
The electrode of the unlabeled secondary antibody and the target electrode were measured with an electrochemical luminescence analyzer at 0.1M K 2 S 2 O 8 In a (PBS, pH 7.4) solution, cyclic voltammetry scans were performed at a potential range of 0V to-1.8V at a scan rate of 0.1V/s and a photomultiplier tube high pressure of 600V, and the ECL intensities were measured, respectively.
2. Test results
The results are shown in FIG. 4. As is evident from the figure, the ECL intensity of the target electrode is significantly higher than that of the electrode with unlabeled secondary antibody. This is because when an unlabeled secondary antibody is specifically bound to fPSA antigen and immobilized on the electrode surface, ECL signal is reduced due to the electron blocking effect of the protein. However, when the secondary antibody labeled with nano-Ag was bound to the electrode surface, ECL intensity was significantly increased. Therefore, nano-Ag plays an obvious role in sensitizing ECL of the target electrode in potassium persulfate.
Experimental example 4 screening of conditions for immunoreaction experiment
1. Optimization of primary antibody incubation time with antigen
(1) Experimental methods
Referring to the method of experimental example 1, the incubation times of the BSA/anti-fPSA/DpAu/GCE modified primary electrode and the fPSA antigen solution are set to 0min, 5min, 10min, 15min, 20min, 25min, 30min, 35min, 40min and 45min, respectively, so as to obtain different target electrodes.
Then, the target electrodes are respectively placed at 0.1M K 2 S 2 O 8 The ECL strength was measured in (PBS, pH 7.4) solution.
(2) Results of the experiment
As shown in FIG. 5, it can be seen that S increases with the incubation time 2 O 8 2- Gradually decreases in ECL intensity. When the incubation time was extended to 35min, S 2 O 8 2- The ECL signal intensity of the primary antibody was kept substantially constant, indicating that the binding of the primary antibody to fPSA antigen on the electrode surface was saturated when the incubation time reached 35min, and therefore 35min was selected as the optimal incubation time for the primary antibody modified electrode BSA/anti-fPSA/DpAu/GCE and the fPSA antigen solution.
2. Optimization of incubation time of secondary antibody and antigen
(1) Experimental methods
Referring to the method of experimental example 1, the incubation of the fPSA antigen-modified electrode fPSA/BSA/anti-fPSA/DpAu/GCE and the nano-Ag labeled secondary antibody is set to 0min, 5min, 10min, 15min, 20min, 25min, 30min and 35min respectively, so as to obtain different target electrodes.
Then the target electrodes are respectively placed in 0.1M K 2 S 2 O 8 (PBS，pH 7.4) In the solution, cyclic voltammetry scanning is carried out in a potential range of 0V to-1.8V, the scanning speed is 0.1V/s, the high voltage of a photomultiplier is 600V, and the ECL intensity is measured.
(2) Results of the experiment
As shown in FIG. 6, it can be seen that S increases as the incubation time of the secondary antibody with the antigen increases 2 O 8 2- The ECL intensity of (a) gradually increases. However, when the incubation time was extended to 25min, S 2 O 8 2- The ECL signal intensity of (a) remained essentially constant, indicating that binding of fPSA antigen to fPSA secondary antibody was saturated when the incubation time reached 25min. Therefore, 25min was selected as the optimal incubation time for fPSA/BSA/anti-fPSA/DpAu/GCE and nano-Ag labeled secondary antibody for fPSA antigen-modified electrodes.
Experimental example 5 establishment of standard curve for ECL response to fPSA antigens at different concentrations
1. Experimental methods
In order to study the ECL response of the target electrode to different concentrations of fPSA antigen, with reference to the method of experimental example 1 and the optimized experimental conditions of experimental example 4, the incubation time of the primary anti-modified electrode BSA/anti-fPSA/DpAu/GCE and fPSA antigen solution in experimental example 1 was determined to be 35min, the incubation time of the fPSA antigen-modified electrode fPSA/BSA/anti-fPSA/DpAu/GCE and the nano-Ag labeled secondary antibody was determined to be 25min, and the concentrations of the fPSA antigen solutions were respectively set to 0ng · mL -1 、0.002ng·mL -1 、0.005ng·mL -1 、0.05ng·mL -1 、0.1ng·mL -1 、0.5ng·mL -1 、1ng·mL -1 、5ng·mL -1 And 10 ng. ML -1 And obtaining different optimized target electrodes.
Using an electrochemical luminescence analyzer at 0.1M K 2 S 2 O 8 (PBS, pH 7.4) in a potential range of 0V to-1.8V, scanning at a speed of 0.1V/s and a photomultiplier tube high pressure of 600V, and determining the ECL intensity I of the optimized target electrode i . At the same time, 0 ng. ML was incubated under the same conditions -1 The electrode from fPSA antigen was used as a blank control and the corresponding ECL intensity was recorded as I 0 . Deduction of blank informationAfter number, ECL signal responses of the optimized target electrode to different concentrations of antigen were recorded as:
ΔI i ＝I i -I 0 (i＝1，2，3…9)
2. results of the experiment
The ECL response potential versus ECL intensity plot for the optimized target electrode as a function of fPSA antigen concentration is shown in figure 7. It can be seen that as the concentration of fPSA antigen increases, the amount of nano-Ag labeled secondary antibody that is immuno-bound to the electrode surface increases, and the ECL intensity of the target electrode response increases significantly. Based on ECL catalysis of nano-Ag on persulfate, response signals can be obviously amplified. This is because as the concentration of fPSA antigen increases, the more nano-Ag labeled secondary antibody that binds to the electrode surface, the higher the intensity of ECL detected, and the further quantitative detection of fPSA antigen can be performed. The results were processed to obtain fPSA antigen concentration (c) i ) Logarithmic value of lgc i On the abscissa, the change in the electrochemiluminescence intensity Δ I measured at the fPSA antigen concentration i In ordinate, a standard curve for detection of the optimized target electrode to the fPSA antigen standard solution was obtained (as shown in fig. 8).
The linear response equation of the standard curve shown in FIG. 8 is Y =1640.27X +4883.96, and the linear correlation coefficient is R =0.9987. Wherein Y represents the change value Δ I of the electrochemiluminescence intensity, and X represents the logarithmic value lgc of the fPSA antigen concentration. The linear range of the optimized target electrode prepared by the experiment to fPSA antigen detection is 0.002-10 ng-mL -1 The detection limit is 0.7 pg.mL -1 (signal-to-noise ratio S/N = 3). As shown in table 1, the detection limit of the detection kit of the present invention for fPSA antigen is much lower than that of other analysis methods in the prior art.
TABLE 1 fPSA detection limits for the detection kits of the invention compared to other methods
Figure BDA0002365077220000091
Experimental example 6 stability test of ECL Sandwich-based immunosensing detection method of the present invention
1. Experimental methods
By adopting the method in the experimental example 5, an optimized target electrode is prepared, and ECL signal intensity of fPSA antigens with different concentrations is detected so as to evaluate the stability of the detection method based on the detection kit. The concentrations of fPSA antigen selected for stability testing were 0.005 ng-mL each -1 、0.1ng·mL -1 And 5 ng. ML -1 The ECL intensity was measured in 5 replicates per concentration under the following conditions: using an electrochemical luminescence analyzer at 0.1M K 2 S 2 O 8 (PBS, pH 7.4) in a potential range of 0V to-1.8V, the scanning speed is 0.1V/s, and the high pressure of the photomultiplier is 600V.
2. Results of the experiment
As can be seen from FIG. 9, as the antigen concentration increases, the electrochemiluminescence signal intensity also increases. The Relative Standard Deviation (RSD) of each specific concentration of 5 parallel assays is less than 2.05 percent, which indicates that the detection method has good stability and can be used for quantitative detection of fPSA antigen concentration.
Experimental example 7 Selectivity test 1 of detection method based on ECL Sandwich immunosensing according to the present invention, and Experimental method
The selectivity of the detection method based on the detection kit is inspected by adopting a standard addition method, and the specific method comprises the following steps: adding the fPSA standard antigen solution into a mixed solution of CEA, AFP and BSA proteins with the concentration twice that of the fPSA antigen to simulate the environment of human serum so that the concentration of the fPSA antigen in the final mixed solution is 0.05 ng-mL respectively -1 、0.10ng·mL -1 、1.00ng·mL -1 、10.00ng·mL -1 The recovery rate was calculated by measuring the fPSA antigen concentration under the optimized experimental conditions using the standard curve obtained under the optimized experimental conditions of experimental example 5, and the results are shown in table 2.
Recovery = Found (ng · mL) -1 )÷Add(ng·mL -1 )×100％
Where Found is the measured fPSA antigen concentration and Add is the theoretical fPSA antigen concentration in the mixed solution.
2. Results of the experiment
As can be seen from table 2, the recovery was measured between 96.8% and 108.0% at different concentrations of fPSA antigen. The detection method has good selectivity for fPSA antigen detection, and can be used for detecting actual samples.
TABLE 2 determination of the spiked recovery of fPSA antigen in the mixed solution
Figure BDA0002365077220000101
In the antigen detection kit, the silver nanoparticle labeled secondary antibody can effectively enhance the detection signal of potassium persulfate, and enhance the sensitivity of immunosensing. Experiments prove that when the antigen detection kit disclosed by the invention is used for quantitatively detecting the concentration of the fPSA antigen, the linear range of fPSA antigen detection is 0.002-10 ng & mL -1 The detection limit is 0.7 pg.mL -1 . Compared with other methods in the prior art, the method based on ECL sandwich immunosensing has wider linear range and lower detection limit when detecting the antigen, and has good application prospect in clinical detection.

Claims (13)

1. An ECL sandwich immunosensing-based antigen detection kit is characterized in that: the antigen detection kit comprises the following components:
(1) A modification-resistant electrode;
(2) A silver nanoparticle-labeled secondary antibody;
(3) A persulfate solution;
the silver nanoparticle-labeled secondary antibody is prepared by the following method: mixing the secondary antibody and the silver nanoparticles, stirring under an ice bath condition, centrifuging to remove the unbound secondary antibody, and sealing the nonspecific sites with bovine serum albumin to obtain the silver nanoparticle composition;
the silver nanoparticles are prepared by the following method: agNO is added under ice bath condition 3 The solution was added dropwise to NaBH, which was continuously stirred 4 Continuously stirring the solution until the color of the system becomes yellow green to obtain the product;
the primary anti-modification electrode is prepared by the following method: firstly, modifying gold particles on the surface of an electrode, then bonding primary antibodies on the surface of the obtained electrode, and then sealing non-specific sites;
the method for modifying the gold particles on the surface of the electrode comprises the following steps: soaking the electrode in a chloroauric acid solution for electrodeposition; the concentration of the chloroauric acid solution is 0.5-2%, the voltage of the electrodeposition is-0.3 to-0.1V, and the time of the electrodeposition is 20-40 s; the electrode is a glassy carbon electrode;
the method for bonding the primary antibody to the surface of the obtained electrode comprises the following steps: soaking the obtained electrode in primary antibody solution at 4 deg.C overnight; the concentration of the primary antibody solution is 5 mg-mL -1 ；
The method for blocking the non-specific sites comprises the following steps: soaking the obtained electrode in bovine serum albumin, wherein the concentration of the bovine serum albumin is 1%, and the soaking time is 25min;
the persulfate solution is a potassium persulfate solution, the concentration of the persulfate solution is 0.1M, and the pH value is 7.4;
the primary antibody is fPSA primary antibody, the antigen to be detected is fPSA antigen, and the secondary antibody is fPSA secondary antibody.
2. The antigen detection kit according to claim 1, characterized in that: the antigen detection kit also comprises a standard solution of the antigen to be detected.
3. The antigen detection kit according to claim 1, characterized in that: the concentration of the chloroauric acid solution is 1%, the voltage of the electrodeposition is-0.2V, and the time of the electrodeposition is 30s.
4. The antigen detection kit according to claim 1 or 2, characterized in that: in the preparation process of the silver nanoparticle marked secondary antibody, the stirring time is 1-3 hours.
5. The antigen detection kit according to claim 4, characterized in that: in the preparation process of the silver nanoparticle labeled secondary antibody, the stirring time is 2 hours.
6. The antigen detection kit according to claim 1, characterized in that: the AgNO 3 The concentration of the solution is 1mM, and the NaBH is added 4 The solution concentration is 2mM, and the continuous stirring time is more than 15 min.
7. A method for quantitatively detecting the concentration of an antigen, characterized by: the method is used for detecting by using the ECL sandwich immunosensing-based antigen detection kit of any one of claims 1 to 6, and comprises the following specific steps:
(a) Determination of ECL intensity in standard solutions of the antigens to be tested at each concentration:
a 1 the standard solution of the antigen to be detected is set to a gradient concentration, and then the primary antibody modified electrode is set to a concentration C i Incubating in a standard solution of the antigen to be detected to obtain an antigen modified electrode;
a 2 incubating the obtained antigen-modified electrode in a secondary antibody marked by silver nanoparticles to obtain a target electrode, and measuring the ECL intensity I of the target electrode in a persulfate solution by using an electrochemical luminescence analyzer i ；
(b) Preparing standard solution of antigen to be detected into 0 ng/mL -1 According to the method of step (a), 0 ng.mL was obtained -1 ECL strength of target electrode at concentrationI 0 ；
(c) Drawing a standard curve: by the ECL intensity variation value DeltaI i Is ordinate, lgC i Drawing a curve for a horizontal coordinate, and performing linear regression to obtain a standard curve; wherein, deltaI i ＝I i -I 0 ；
(d) Determination of the concentration of the antigen to be tested: incubating the primary anti-modified electrode in an antigen to be detected to obtain an antigen-modified electrode; incubating the obtained antigen-modified electrode in a silver nanoparticle-labeled secondary antibody to obtain a target electrode, and measuring the ECL (electron cyclotron luminescence) intensity of the target electrode in persulfate solution by using an electrochemical luminescence analyzerI x ；
Will be deltaI x Substituting into the ordinate of the standard curve obtained in the step (c), and obtaining the abscissa correspondingly, namely the lg value of the antigen concentration to be detected; wherein, deltaI x ＝I x -I 0 。
8. The method of claim 7, wherein: step (a) 1 ) The incubation temperature is room temperature, and the incubation time is more than 5 minutes;
step (a) 2 ) Wherein the incubation temperature is room temperature and the incubation time is 5 minutes or more.
9. The method of claim 8, wherein: step (a) 1 ) The incubation temperature is room temperature, and the incubation time is more than 35 minutes;
step (a) 2 ) Wherein the incubation temperature is room temperature and the incubation time is 25 minutes or more.
10. The method of claim 9, wherein: step (a) 1 ) The incubation temperature is room temperature, and the incubation time is 35-45 minutes;
step (a) 2 ) The incubation temperature is room temperature, and the incubation time is 25-35 minutes.
11. The method of claim 7, wherein: in the step (a), the first step of the method,C i 0.002-10 ng/mL -1 ；
Step (a) 2 ) And (d), the test parameters of the electrochemical luminescence analyzer are as follows: and performing cyclic voltammetry scanning in a potential range of 0V to-1.8V, wherein the scanning speed is 0.1V/s, and the high voltage of the photomultiplier is 600V.
12. The method of claim 7, wherein: in step (c), the standard curve is Y =1640.27X +4883.96.
13. Non-diagnostic use of the ECL sandwich immunosensing-based antigen detection kit of any one of claims 1 to 6 for the quantitative determination of antigen concentration.
CN202010033170.5A 2020-01-13 2020-01-13 ECL sandwich immunosensing-based antigen detection kit Active CN111208183B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202010033170.5A CN111208183B (en) 2020-01-13 2020-01-13 ECL sandwich immunosensing-based antigen detection kit

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202010033170.5A CN111208183B (en) 2020-01-13 2020-01-13 ECL sandwich immunosensing-based antigen detection kit

Publications (2)

Publication Number Publication Date
CN111208183A CN111208183A (en) 2020-05-29
CN111208183B true CN111208183B (en) 2023-01-31

Family

ID=70790079

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202010033170.5A Active CN111208183B (en) 2020-01-13 2020-01-13 ECL sandwich immunosensing-based antigen detection kit

Country Status (1)

Country Link
CN (1) CN111208183B (en)

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030059839A1 (en) * 2001-05-22 2003-03-27 Obiso Richard J. Method for detecting pathogens using immunoassays
CN105628931A (en) * 2016-01-19 2016-06-01 济南大学 Preparation and application of alpha fetoprotein electrochemical immunosensor based on silver deposition
CN105675697A (en) * 2016-01-19 2016-06-15 济南大学 Construction method of nanoprobe C60 based electrochemical immunosensor for carcino-embryonic antigens
CN107727714B (en) * 2017-09-10 2019-06-28 福建师范大学 One kind being based on carbon nanohorn and TiO2The preparation method of the Ratio-type electrochemical luminescence immunosensor of mesomorphic nano material
CN109142748A (en) * 2018-08-08 2019-01-04 福建医科大学 Human prostate specific antigen detection method and its kit
CN109142331A (en) * 2018-08-08 2019-01-04 福建医科大学 A kind of electrogenerated chemiluminescence method and its kit for carcinomebryonic antigen detection
CN109164090A (en) * 2018-08-08 2019-01-08 福建医科大学 The electrochemiluminescdetection detection method and its kit of tumor necrosis factor α
CN110554027A (en) * 2019-09-19 2019-12-10 济南大学 preparation method and application of immunosensor for promoting gold nanocluster electroluminescent response based on iron oxide array coreaction

Also Published As

Publication number Publication date
CN111208183A (en) 2020-05-29

Similar Documents

Publication Publication Date Title
Munge et al. Multiplex immunosensor arrays for electrochemical detection of cancer biomarker proteins
Ravalli et al. New label free CA125 detection based on gold nanostructured screen-printed electrode
Wu et al. A disposable electrochemical immunosensor for flow injection immunoassay of carcinoembryonic antigen
CN103116023B (en) ECL (electrochemiluminescence) immunosensor for detecting tumor markers and preparation method and applications thereof
Zhou et al. The sandwich-type electrochemiluminescence immunosensor for α-fetoprotein based on enrichment by Fe3O4-Au magnetic nano probes and signal amplification by CdS-Au composite nanoparticles labeled anti-AFP
Lai et al. Electrochemical stripping analysis of nanogold label-induced silver deposition for ultrasensitive multiplexed detection of tumor markers
Liu et al. Core–shell Fe3O4–Au magnetic nanoparticles based nonenzymatic ultrasensitive electrochemiluminescence immunosensor using quantum dots functionalized graphene sheet as labels
CN102967706B (en) Preparation method and application of flow injection chemiluminiscence immuno sensor for detecting tumor marker
Han et al. Gold nanoparticles enhanced electrochemiluminescence of graphite-like carbon nitride for the detection of Nuclear Matrix Protein 22
Hassani et al. A sensitive aptamer-based biosensor for electrochemical quantification of PSA as a specific diagnostic marker of prostate cancer
Ge et al. Disposable electrochemical immunosensor for simultaneous assay of a panel of breast cancer tumor markers
Cao et al. Electrochemical immunosensor based on binary nanoparticles decorated rGO-TEPA as magnetic capture and Au@ PtNPs as probe for CEA detection
Qin et al. Aggregation-induced electrochemiluminescence resonance energy transfer with dual quenchers for the sensitive detection of prostate-specific antigen
CN109613244B (en) Preparation method and application of Ag @ Pt-CuS labeled immunosensor
Rashidiani et al. Ultrahigh sensitive enhanced-electrochemiluminescence detection of cancer biomarkers using silica NPs/graphene oxide: a comparative study
CN108896638B (en) Preparation method and application of immunosensor based on titanium dioxide doped graphene loaded sea cucumber-like gold-palladium core-shell nanoparticles
CN110907511A (en) Gold-curcumin nanoparticle quenched CdS hybrid TiO2Electrochemical luminescence sensor for detecting insulin by nanobelt
Cheng et al. Integrated electrochemical lateral flow immunoassays (eLFIAs): recent advances
Feng et al. A novel strategy for multiplexed immunoassay of tumor markers based on electrochemiluminescence coupled with cyclic voltammetry using graphene-polymer nanotags
Cui et al. Novel graphene/Au-CdS: Eu composite-based electrochemiluminescence immunosensor for cancer biomarker detection by coupling resonance energy transfer and enzyme catalytic reaction
Ge et al. Ultra-sensitive magnetic immunoassay of HE4 based on surface enhanced Raman spectroscopy
CN106093396A (en) A kind of preparation method and application of immunosensor based on Au GQD@PtPd
Li et al. Electrochemical sensors applied for in vitro diagnosis
CN111208183B (en) ECL sandwich immunosensing-based antigen detection kit
Mao et al. Potential controlling highly-efficient catalysis of wheat-like silver particles for electrochemiluminescence immunosensor labeled by nano-Pt@ Ru and multi-sites biotin/streptavidin affinity

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant