CN112300779A - Polymerization-enhanced electrochemical luminescence probe and preparation method and application thereof - Google Patents
Polymerization-enhanced electrochemical luminescence probe and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a polymerization enhanced electrochemiluminescence probe and a preparation method and application thereof. The polymerization enhanced electrochemiluminescence probe is formed by covalently connecting a polymerization active group and an electrochemiluminescence group. The polymerization enhanced electrochemiluminescence probe can be efficiently polymerized onto peroxidase and adjacent protein molecules under the catalysis of the peroxidase, plays a role in signal amplification, has the advantages of simplicity, high signal enhancement efficiency, strong compatibility with the existing molecules and an immunoassay system and the like, and can be used for high-sensitivity detection of nucleic acid and protein.
Description
Technical Field
The invention relates to the technical field of biochemical analysis, in particular to a polymerization enhanced electrochemiluminescence probe and a preparation method and application thereof.
Background
With the continuous improvement of the modern scientific and medical technology level, people have realized that the level of nucleic acid and protein molecules in human body has great relevance to the occurrence and development of diseases, and the early monitoring of the disease markers has great significance for the prevention and early treatment of diseases, so that the accurate and high-sensitivity detection of functional biomolecules such as protein, nucleic acid and the like has great significance for the early diagnosis of diseases. Protein molecules have been widely used as disease markers in clinical diagnosis of tumors, heart diseases, infectious diseases, and the like. microRNA can be used as a regulatory factor to participate in complex biological processes in vivo, is confirmed to be related to disease pathogenesis (cancer, neuropathy and the like), and is a potential tumor early screening marker. Furthermore, in recent years, the discovery of circulating tumor DNA has also opened new avenues for early in vitro diagnosis of tumors. Therefore, the development of a sensitive and efficient method for detecting biomolecules has important application value.
The electrochemiluminescence immunoassay is an existing commercialized method for detecting protein molecules in vitro, a sandwich structure is formed on the basis of a pair of antibodies and target protein molecules, and the detection antibodies are marked with terpyridyl ruthenium, so that the level of the target molecules can be judged through electrochemiluminescence signals. However, this approach is limited in the limited number of ruthenium terpyridyl labels on the antibody and the limited ability to detect low abundance targets of physiological significance currently identified and potentially to be discovered. Most of the existing nucleic acid detection technologies rely on early nucleic acid amplification means, such as Polymerase Chain Reaction (PCR), loop-mediated isothermal amplification (LAMP), nucleic acid sequence-dependent amplification (NASBA), and the like, to finally realize highly sensitive nucleic acid detection, but these methods have risks of pollution and amplification errors in the nucleic acid amplification process, and the accuracy is inferior to that of direct detection methods.
Disclosure of Invention
The present invention is directed to overcoming the disadvantages and drawbacks of the prior art, and the first object of the present invention is to provide a polymerization-enhanced electrochemiluminescence probe, the second object of the present invention is to provide a method for preparing the polymerization-enhanced electrochemiluminescence probe, and the use of the polymerization-enhanced electrochemiluminescence probe in molecular detection.
In order to realize the first object, the invention is realized by the following technical scheme:
a polymerization enhanced electrochemical luminescence probe is formed by covalently connecting a polymerization active group and an electrochemical luminescence group.
Further, the polymerization active group is tyramine or dopamine.
Further, the electrochemiluminescence group is a terpyridyl ruthenium group.
In order to achieve the second purpose, the invention is realized by the following technical scheme:
the preparation method of the polymerization enhanced electrochemiluminescence probe comprises the following three methods: firstly, preparing active terpyridyl ruthenium-NHS ester, and then, covalently connecting the active terpyridyl ruthenium-NHS ester with a polymerization active group to obtain the polymerization enhanced electrochemiluminescence probe; or the polymerization active group reacts with terpyridyl ruthenium, and the polymerization enhanced electrochemiluminescence probe is obtained by taking NHS and DCC as catalysts; or the polymerization active group reacts with terpyridyl ruthenium, and the polymerization enhanced electrochemiluminescence probe is obtained by taking NHS and EDC as catalysts.
For the first preparation method, the specific steps are as follows:
(1) preparation of ruthenium terpyridyl-NHS activated ester (Ru (bpy)3 2+-NHS)；
(2) Tribipyridine ruthenium-NHS activated ester (Ru (bpy)3 2+-NHS) covalently linked to a polymeric activating group to provide the polymeric enhanced electrochemiluminescence probe of the invention.
The step (1) specifically comprises: cis-bis (2, 2-Bipyridine) ruthenium (II) dichloride hydrate (cis-Dichlorobis (2,2 ' -Bipyridine) ruthenam (II)) and 2,2 ' -Bipyridine-4,4 ' -dicarboxylic acid (2,2 ' -Bipyridine-4,4 ' -dicarboxylic acid) are heated and reacted in a mixed solution of methanol and water to synthesize carboxylated ruthenium terpyridine, and then sodium hexafluorophosphate solution is added into the reaction mixture to obtain precipitated solid ruthenium terpyridine solid (Ru (bpy))2(dcbpy)(PF6)2Ruthenium-bis (2,2 ' -bipyridine) (2,2 ' -bipyridine-4,4 ' -dicarboxylic acid) bis (hexafluorophosphate); then, solid ruthenium terpyridyl, dicyclohexylcarbodiimide and N-hydroxysuccinimide were added to anhydrous N, N-Dimethylformamide (N, N-Dimethylformamide) to prepare ruthenium terpyridyl-NHS activated ester (Ru (bpy))3 2+-NHS)。
The step (2) specifically comprises: adding the terpyridyl ruthenium-NHS activated ester (Ru (bpy)32+ -NHS) and Triethylamine (Triethlyamine) prepared in the step 1) and polymerization active molecules into anhydrous N, N-Dimethylformamide (N, N-Dimethylformamide), carrying out oscillation reaction at room temperature, and carrying out covalent connection to obtain the polymerization enhanced electrochemiluminescence probe.
In said step (2), the active terpyridyl ruthenium-NHS ester is covalently linked to the amino group of the polymeric active molecule. The NHS group of the terpyridyl ruthenium-NHS activated ester can perform condensation reaction with amino on a polymerization active molecule, so that the polymerization active group is polymerized with adjacent protein to realize accumulation of an electrochemiluminescence group and amplification of an electrochemiluminescence signal.
For the second preparation method, the specific steps are as follows:
(1) preparation of ruthenium terpyridyl solid (Ru (bpy)2(dcbpy)(PF6)2；
(2) Terpyridyl ruthenium solid (Ru (bpy)2(dcbpy)(PF6)2Covalently linked to a polymeric active group to obtain the polymeric enhanced electrochemiluminescence probe of the invention.
The step (1) specifically comprises: cis-bis (2, 2-Bipyridine) ruthenium (II) dichloride hydrate (cis-Dichlorobis (2,2 ' -Bipyridine) ruthenam (II)) and 2,2 ' -Bipyridine-4,4 ' -dicarboxylic acid (2,2 ' -Bipyridine-4,4 ' -dicarboxylic acid) are heated and reacted in a mixed solution of methanol and water to synthesize carboxylated ruthenium terpyridine, and then sodium hexafluorophosphate solution is added into the reaction mixture to obtain precipitated solid ruthenium terpyridine solid (Ru (bpy))2(dcbpy)(PF6)2Namely ruthenium-bis (2,2 ' -bipyridine) (2,2 ' -bipyridine-4,4 ' -dicarboxylic acid) bis (hexafluorophosphate).
Then, solid ruthenium terpyridyl, dicyclohexylcarbodiimide and N-hydroxysuccinimide were added to anhydrous N, N-Dimethylformamide (N, N-Dimethylformamide) to prepare ruthenium terpyridyl-NHS activated ester (Ru (bpy))3 2+-NHS)。
The step (2) specifically comprises: adding the solid terpyridyl ruthenium (Ru (bpy) prepared in the step 1) into anhydrous N, N-Dimethylformamide (N, N-dimethyl formamide)2(dcbpy)(PF6)2NHS, DCC, Triethylamine (Triethylamine) and polymerized active molecules, oscillating and reacting at room temperature, and covalently connecting to obtain the polymerized reinforced electrochemical luminescence probe.
For the third preparation method, the specific steps are as follows:
(1) preparation of ruthenium terpyridyl solid (Ru (bpy)2(dcbpy)(PF6)2；
(2) Terpyridyl ruthenium solid (Ru (bpy)2(dcbpy)(PF6)2Covalently linked to a polymeric active group to obtain the polymeric enhanced electrochemiluminescence probe of the invention.
The step (1) specifically comprises: cis-bis (2, 2-Bipyridine) ruthenium (II) dichloride hydrate (cis-Dichlorobis (2,2 ' -Bipyridine) ruthenam (II)) and 2,2 ' -Bipyridine-4,4 ' -dicarboxylic acid (2,2 ' -Bipyridine-4,4 ' -dicarboxylic acid) are heated and reacted in a mixed solution of methanol and water to synthesize carboxylated ruthenium terpyridine, and then sodium hexafluorophosphate solution is added into the reaction mixture to obtain precipitated solid ruthenium terpyridine solid (Ru (bpy))2(dcbpy)(PF6)2Namely ruthenium-bis (2,2 ' -bipyridine) (2,2 ' -bipyridine-4,4 ' -dicarboxylic acid) bis (hexafluorophosphate).
Adding solid ruthenium terpyridyl, dicyclohexylcarbodiimide and N-hydroxysuccinimide into anhydrous N, N-Dimethylformamide (N, N-Dimethylformamide) to prepare ruthenium terpyridyl-NHS activated ester (Ru (bpy)3 2+-NHS)。
The step (2) specifically comprises: adding the terpyridyl ruthenium solid prepared in the step 1) into anhydrous N, N-Dimethylformamide (N, N-dimethyl formamide)Body (Ru (bpy)2(dcbpy)(PF6)2NHS, EDC, Triethylamine (Triethylamine) and polymerized active molecules, oscillating and reacting at room temperature, and covalently connecting to obtain the polymerized enhanced electrochemical luminescence probe.
In order to achieve the third object, the invention is realized by the following technical scheme:
the invention relates to an application of a polymerization enhanced electrochemical luminescence probe in protein molecule detection, wherein the polymerization enhanced electrochemical luminescence probe, a capture probe marked with a functional group and a detection probe coupled with peroxidase jointly form a detection system.
Further, the capture probe is a biotin-modified antibody or aptamer molecule, the detection probe is a peroxidase-modified detection antibody or aptamer-antibody composition, and the aptamer-antibody composition is composed of a digoxin-modified aptamer molecule and a peroxidase-modified anti-digoxin antibody.
Further, the peroxidase is horseradish peroxidase HRP or ascorbic acid peroxidase APEX.
Further, the specific process for detecting the protein molecule is as follows: the capture probe, the target protein molecule and the detection probe are combined to form a sandwich structure, after microspheres/particles coated by streptavidin or avidin are captured, unbound antibodies and impurity molecules are washed away, then a polymerization enhanced electrochemiluminescence probe and hydrogen peroxide are added into a system for polymerization reaction, the microsphere/particle-capture antibody-target protein molecule-detection antibody compound is added into an electrochemiluminescence reaction tank, an electrochemiluminescence signal value is detected, and the detection of the target protein molecule is realized through the analysis of electrochemiluminescence intensity.
The invention relates to an application of a polymerization enhanced electrochemical luminescence probe in nucleic acid molecule detection, wherein the polymerization enhanced electrochemical luminescence probe, capture probe DNA marked with a functional group, antigen marked detection probe DNA and an antibody coupled with peroxidase form a detection system, and the antigen and the antibody can be specifically combined.
Further, the specific process for detecting nucleic acid molecules is as follows: capturing the microspheres/particles which can be firmly combined with functional groups of the capture probes, washing out unbound probes and impurity molecules, adding the antibody into the system to enable the antibody to be combined with antigens on the microsphere/particle compound, washing out the unbound antibodies, finally adding a polymerization enhanced electrochemiluminescence probe and hydrogen peroxide into the system to carry out polymerization reaction, adding the microsphere/particle-capture probe-target nucleic acid molecule-detection probe compound into an electrochemiluminescence reaction tank, detecting an electrochemiluminescence signal value, and realizing the detection of target nucleic acid molecules by analyzing the electrochemiluminescence intensity.
The basic principle of polymerization of the probe is shown in fig. 1, in the presence of hydrogen peroxide, peroxidase can convert terpyridyl ruthenium-tyramine (or dopamine) into an intermediate state with transient activity, then activated substrate molecules rapidly and stably undergo covalent reaction with electron-rich regions (tyrosine residues) of adjacent protein molecules, and since adjacent proteins such as horseradish peroxidase, antibodies, antigens and the like all contain a large number of tyrosine binding sites, a large amount of terpyridyl ruthenium-dopamine is enriched, and finally an electrochemiluminescence signal is efficiently amplified. For the dopamine-terpyridyl ruthenium probe, the signal amplification principle is similar to that of the tyramine-ruthenium probe, except that dopamine can be subjected to polymerization reaction with groups including phenolic hydroxyl, amino and sulfhydryl.
The detection principle of the probe is shown in figure 2, the magnetic beads coated with streptavidin can specifically capture and separate the horseradish peroxidase labeled with biotin to form a streptavidin-coated magnetic bead-biotin-horseradish peroxidase complex, the streptavidin-coated magnetic beads and the horseradish peroxidase in the complex are protein molecules and can be used as substrates for polymerization reaction of the terpyridyl ruthenium-tyramine probe, and the terpyridyl ruthenium-tyramine probe is catalyzed by the horseradish peroxidase to be polymerized onto the horseradish peroxidase molecules and adjacent streptavidin molecules in the presence of hydrogen peroxide.
The polymerization enhanced electrochemiluminescence probe can be efficiently polymerized onto peroxidase and adjacent protein molecules under the catalysis of the peroxidase, plays a role in signal amplification, has the advantages of simplicity, high signal enhancement efficiency, strong compatibility with the existing molecules and an immunoassay system and the like, and can be used for high-sensitivity detection of nucleic acid and protein.
For a better understanding and practice, the invention is described in detail below with reference to the accompanying drawings.
Drawings
FIG. 1 is a schematic diagram of the polymerization reaction principle of a polymerization-enhanced electrochemiluminescence probe.
FIG. 2 is a functional verification schematic diagram of a polymerization enhanced electrochemiluminescence probe system.
FIG. 3 is a synthetic scheme of the polymerization-enhanced electrochemiluminescence probe of example 1.
FIG. 4 is a spectral representation of a polymerization enhanced electrochemiluminescence probe of example 1.
FIG. 5 is a functional verification of the polymerization-active terpyridyl ruthenium probe system in example 2.
FIG. 6 is a schematic diagram of the detection method of the polymerization-enhanced electrochemiluminescence probe for detecting protein molecules in example 3.
FIG. 7 is a schematic diagram of the detection method of the polymerization-enhanced electrochemiluminescence probe for detecting nucleic acid molecules in example 4.
Detailed Description
All materials, reagents and equipment selected for use in the present invention are well known in the art, but do not limit the practice of the invention, and other reagents and equipment well known in the art may be suitable for use in the practice of the following embodiments of the invention.
Example 1
Synthesis of polymerization enhanced electrochemiluminescence probe
1. Synthesis of terpyridyl ruthenium solid (Ru (bpy)2(dcbpy)(PF6)2
See fig. 3. 200mg of cis-bis (2, 2-bipyridine) ruthenium (II) dichloride hydrate, 150mg of 2,2 '-bipyridine-4, 4' -dicarboxylic acid and 200mg of sodium hydrogen carbonateThe mixture was added to a three-necked flask containing 32mL of methanol and 8mL of water, and the mixture was heated to 80 ℃ and refluxed for 10 hours, whereby the reaction solution was changed from brown to orange-red. After the reaction is finished, cooling to room temperature, adjusting the pH of the reaction solution to 4.4 by using concentrated sulfuric acid, keeping out of the sun and carrying out ice bath for 2 hours to precipitate unreacted 2,2 '-bipyridyl-4, 4' -dicarboxylic acid, filtering by using filter paper, and collecting filtrate to obtain a carboxylated ruthenium terpyridyl solution. 12.5mL of a 200mg/mL sodium hexafluorophosphate solution was added to the above carboxylated ruthenium terpyridyl solution, and after stirring for 5 minutes, the mixture was transferred to an ice bath for 2 hours to cause formation of chestnut crystals. Transferring the mixture to a centrifuge tube, centrifuging at 5000g in a centrifuge at 4 deg.C for 5 min, discarding the supernatant, and freeze-drying the precipitate to obtain ruthenium terpyridyl solid (Ru (bpy)2(dcbpy)(PF6)2。
2. Preparation of ruthenium terpyridyl-NHS activated ester (Ru (bpy)3 2+-NHS)
See fig. 3. 230mg of dicyclohexylcarbodiimide and 120mg of N-hydroxysuccinimide were dissolved in 2mL of anhydrous N, N-Dimethylformamide (N, N-Dimethylformamide) under stirring and ice-bath conditions, and 190mg of ruthenium terpyridyl solid was added thereto, followed by stirring and reaction at 0 ℃ for 30 minutes under ice-bath conditions and then stirring and reaction at room temperature for 5 hours. After the reaction is finished, centrifuging the reaction solution for 5 minutes at the temperature of 4 ℃ at 5000g, collecting supernatant, and subpackaging and freezing the supernatant in a refrigerator at the temperature of 80 ℃ below zero for later use.
3. Preparation of a polymerization-active ruthenium terpyridyl Probe
See fig. 3. Diluting the active terpyridyl ruthenium-NHS ester with anhydrous N, N-dimethylformamide to a final concentration of 10 mM; 984.363 μ L of anhydrous DMF was added with 15.63mg of tyramine (or 13.786mg of dopamine) and 15.637 μ L of triethylamine at 90mM and 112.5mM, respectively; 50 mu L of 10mM terpyridyl ruthenium-NHS activated ester and 5 mu L of tyramine (or dopamine)/triethylamine mixed solution are added into 45 mu L of anhydrous DMF until the final concentrations of the terpyridyl ruthenium-NHS activated ester, the tyramine (or dopamine) and the triethylamine in the system are respectively 5mM, 4.5mM and 5.625mM, after shaking reaction for 3.5 hours at room temperature, a polymerization activity terpyridyl ruthenium probe is obtained, and is frozen and stored at 20 ℃ below zero after subpackaging, and the probe concentration is 4.5 mM.
The fluorescence spectrum of the polymerized active ruthenium terpyridyl is shown in FIG. 4, wherein, the graph A is Ru (byp)3 2+-fluorescence spectrum of tyramine; FIG. B is Ru (byp)3 2+Fluorescence spectrum of dopamine. The results show that, with Ru (byp)3 2+Comparison of fluorescence spectra of Ru (byp)3 2+-tyramine and Ru (byp)3 2+The fluorescence emission peaks of dopamine all produced a red shift of about 20nm, indicating that tyramine or dopamine molecules are covalently linked to Ru (byp)3 2+On the molecule.
Example 2
Functional verification of polymerization activity terpyridyl ruthenium probe system
1. Construction of magnetic bead-horseradish peroxidase System
The biotin-labeled horseradish peroxidase was diluted with PBS to various concentrations (40ng/mL, 20ng/mL, 5ng/mL, 2.5ng/mL, 1ng/mL, 0.5 ng/mL), 100. mu.L of streptavidin-coated magnetic beads was taken, the supernatant was discarded, the magnetic beads were resuspended in 50. mu.L of biotin-horseradish peroxidase solution, incubated at 37 ℃ for 15 minutes, and washed once with PBS buffer.
2. Polymerization of electrochemiluminescent probes
Diluting the polymerized active terpyridyl ruthenium probe to the concentration of 3 mu M by PBS, adding 100 mu L of polymerized active terpyridyl ruthenium probe solution into the magnetic bead capture complex, after resuspending the magnetic bead complex, adding 100 mu L of hydrogen peroxide PBS solution with the concentration of 10mM, rapidly mixing uniformly, reacting for 5 minutes at 37 ℃, enriching the magnetic bead complex to the bottom of a centrifuge tube, discarding the supernatant, washing once by 100 mu LPBS, finally resuspending the magnetic bead complex by 100 mu LPBS buffer solution, adding 20 mu L of the magnetic bead complex into an electrochemical luminescence pool, then adding tripropylamine buffer solution for electrochemical luminescence detection, and reading the electrochemical luminescence signal value.
The results are shown in fig. 5, where the electrochemiluminescence intensity is proportional to the concentration of biotin-horseradish peroxidase, indicating that the polymerization-active electrochemiluminescence probe can be catalytically polymerized, and the degree of polymerization is proportional to the horseradish peroxidase activity.
Example 3
Application of polymerization enhanced electrochemiluminescence probe in detecting protein molecules
In this example, the detection method of prostate specific antigen using the polymerization-enhanced electrochemiluminescence probe of the present invention is shown in FIG. 6.
1. Formation of capture antibody-target protein molecule-detection antibody "sandwich" -like antigen-antibody complexes
20. mu.L of a biotin-labeled PSA monoclonal antibody having a concentration of 2mg/mL, a 20. mu.L of a PSA sample having a concentration of 1mg/mL, and 20. mu.L of an HRP-labeled PSA monoclonal antibody having a concentration of 1mg/mL were mixed and incubated at 37 ℃ for 15 minutes, then 100. mu.L of a streptavidin-coated magnetic bead having a concentration of 0.72mg/mL was added thereto, and incubated at 37 ℃ for 15 minutes, and then unbound antibody and protein molecule impurities were washed off.
2. Polymerization of electrochemiluminescent probes
Diluting the polymerized active terpyridyl ruthenium probe to the concentration of 3 mu M by PBS, adding 100 mu L of polymerized active terpyridyl ruthenium probe solution into the magnetic bead capture complex, after resuspending the magnetic bead complex, adding 100 mu L of hydrogen peroxide PBS solution with the concentration of 10mM, rapidly mixing uniformly, reacting for 5 minutes at 37 ℃, enriching the magnetic bead complex to the bottom of a centrifuge tube, discarding the supernatant, washing once by 100 mu LPBS, finally resuspending the magnetic bead complex by 100 mu LPBS buffer solution, adding 20 mu L of the magnetic bead complex into an electrochemical luminescence pool, then adding tripropylamine buffer solution for electrochemical luminescence detection, and reading the electrochemical luminescence signal value.
Example 4
Application of polymerization-enhanced electrochemiluminescence probe in detection of nucleic acid molecules
In this embodiment, the polymerization-enhanced electrochemiluminescence probe of the present invention is used to detect a nucleic acid sequence of listeria monocytogenes 16sRNA, and the detection method is shown in fig. 7.
1. Construction of capture and detection probes for detection of nucleic acids
Designing a capture probe 5 '-biotin-AAAAAAGTCCGTGGTAGGGCAGGTTGGGGTGACT-3' (SEQ ID NO:1) according to the nucleic acid sequence of 16sRNA of the Listeria monocytogenes; designing a detection probe 5 '-GGTTGGTGTGGTTGGAAAAAA (SEQ ID NO:2) -digoxin-3'; both probes were diluted separately with PBS to a final concentration of 100 nM.
2. Capture of target nucleic acid molecules
And respectively taking 10 mu L of capture probe, 10 mu L of detection probe and 10 mu L of Listeria monocytogenes 16sRNA sample, adding the samples into 20 mu L of LPBS buffer solution, hybridizing at 70 ℃ for 30 minutes, recovering to 30 ℃, adding 100 mu L of streptavidin-coated magnetic beads into the reaction system, incubating at 37 ℃ for 15 minutes, and washing out unbound probe and nucleic acid molecule impurities.
3. Polymerization of electrochemiluminescent probes
Diluting the polymerized active terpyridyl ruthenium probe to the concentration of 3 mu M by PBS, adding 100 mu L of polymerized active terpyridyl ruthenium probe solution into the magnetic bead capture complex, after resuspending the magnetic bead complex, adding 100 mu L of hydrogen peroxide PBS solution with the concentration of 10mM, rapidly mixing uniformly, reacting for 5 minutes at 37 ℃, enriching the magnetic bead complex to the bottom of a centrifuge tube, discarding the supernatant, washing once by 100 mu LPBS, finally resuspending the magnetic bead complex by 100 mu LPBS buffer solution, adding 20 mu L of the magnetic bead complex into an electrochemical luminescence pool, then adding tripropylamine buffer solution for electrochemical luminescence detection, and reading the electrochemical luminescence signal value.
The polymerization-enhanced electrochemiluminescence probe can be efficiently polymerized onto horseradish peroxidase and adjacent protein molecules under the catalysis of the horseradish peroxidase to play a role in signal amplification, has the advantages of simplicity, high signal enhancement efficiency, strong compatibility with the existing molecules and an immunoassay system and the like, and can be used for high-sensitivity detection of nucleic acid and protein.
The present invention is not limited to the above-described embodiments, and various modifications and variations of the present invention are intended to be included within the scope of the claims and the equivalent technology of the present invention if they do not depart from the spirit and scope of the present invention.
Sequence listing
SEQUENCE LISTING
<110> university of south China
<120> polymerization enhanced electrochemical luminescence probe and preparation method and application thereof
<160> 13
<170>PatentIn version 3.1
<210>1
<211>34
<212> DNA
<213> Artificial sequence
<220>
<223> Capture Probe sequence in example 4
<400>1
aaaaaagtcc gtggtagggc aggttggggt gact 34
<210>2
<211>21
<212> DNA
<213> Artificial sequence
<220>
<223> detection Probe sequence in example 4
<400>2
ggttggtgtg gttggaaaaa a 21
Claims (10)
1. The polymerization-enhanced electrochemiluminescence probe is characterized by being formed by covalently connecting a polymerization active group and an electrochemiluminescence group.
2. The polymerically enhanced electrochemiluminescence probe of claim 1, wherein the polymeric active group is tyramine or dopamine.
3. The polymerization-enhanced electrochemiluminescence probe of claim 2, wherein the electrochemiluminescence group is a ruthenium terpyridyl group.
4. The method for preparing the polymerization-enhanced electrochemiluminescence probe as claimed in claim 3, which is characterized by comprising the following steps: firstly, preparing active terpyridyl ruthenium-NHS ester, and then, covalently connecting the active terpyridyl ruthenium-NHS ester with a polymerization active group to obtain the polymerization enhanced electrochemiluminescence probe; or the polymerization active group reacts with terpyridyl ruthenium, and the polymerization enhanced electrochemiluminescence probe is obtained by taking NHS and DCC as catalysts; or the polymerization active group reacts with terpyridyl ruthenium, and the polymerization enhanced electrochemiluminescence probe is obtained by taking NHS and EDC as catalysts.
5. The use of the polymerization-enhanced electrochemiluminescence probe of claim 3 in protein molecule detection, wherein the polymerization-enhanced electrochemiluminescence probe, a capture probe labeled with a functional group, and a detection probe coupled with peroxidase constitute a detection system.
6. The use of the polymerization-enhanced electrochemiluminescence probe of claim 5 in protein molecule detection, wherein the capture probe is a biotin-modified antibody or aptamer molecule, the detection probe is a peroxidase-modified detection antibody or aptamer-antibody composition, and the aptamer-antibody composition comprises a digoxigenin-modified aptamer molecule and a peroxidase-modified anti-digoxigenin antibody.
7. The use of the polymerization-enhanced electrochemiluminescence probe of claim 5 in protein molecule detection, wherein the peroxidase is horseradish peroxidase (HRP) or ascorbic Acid Peroxidase (APEX).
8. The use of the polymerized enhanced electrochemiluminescence probe according to claim 5 in protein molecule detection, wherein the specific process for detecting protein molecules is as follows: the capture probe, the target protein molecule and the detection probe are combined to form a sandwich structure, after microspheres/particles coated by streptavidin or avidin are captured, unbound antibodies and impurity molecules are washed away, then a polymerization enhanced electrochemiluminescence probe and hydrogen peroxide are added into a system for polymerization reaction, the microsphere/particle-capture antibody-target protein molecule-detection antibody compound is added into an electrochemiluminescence reaction tank, an electrochemiluminescence signal value is detected, and the detection of the target protein molecule is realized through the analysis of electrochemiluminescence intensity.
9. The use of the poly-enhanced electrochemiluminescence probe of claim 3 in nucleic acid detection, wherein the poly-enhanced electrochemiluminescence probe, capture probe DNA labeled with a functional group, detection probe DNA labeled with an antigen, and an antibody coupled with peroxidase constitute a detection system, and the antigen and the antibody can specifically bind to each other.
10. The use of the polymerization-enhanced electrochemiluminescence probe according to claim 9 for detecting nucleic acid, wherein the specific process for detecting nucleic acid molecules is as follows: capturing the probe DNA, the target single-chain nucleic acid and the detection probe DNA to form a sandwich structure, washing away unbound probes and impurity molecules after capturing microspheres/particles coated by streptavidin or avidin, then adding the antibody into the system to enable the antibody to be bound to antigens on a microsphere/particle composite, washing away the unbound antibodies, finally adding a polymerization enhanced electrochemiluminescence probe and hydrogen peroxide into the system to carry out polymerization reaction, adding the microsphere/particle-capture probe-target nucleic acid molecule-detection probe composite into an electrochemiluminescence reaction pool, detecting an electrochemiluminescence signal value, and realizing the detection of target nucleic acid molecules by analyzing the electrochemiluminescence intensity.
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| CN117288941A (en) * | 2023-08-18 | 2023-12-26 | 华南师范大学 | Method for rapidly synthesizing self-enhanced electrochemiluminescence probe in water phase based on lysine and application thereof |
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