CN117169494A - ECL detection method for nucleic acid specific site modification, ECL nano beacon, preparation method thereof and kit - Google Patents
ECL detection method for nucleic acid specific site modification, ECL nano beacon, preparation method thereof and kit Download PDFInfo
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Abstract
The invention relates to an Electrochemiluminescence (ECL) detection method based on nucleic acid specific site modification of a nano-beacon, an Electrochemiluminescence (ECL) nano-beacon, a preparation method thereof and a kit. The electrochemiluminescence nano-beacon prepared by the method is a metal doped inorganic oxide nano-particle modified by a secondary antibody, and the compound containing the electrochemiluminescence nano-beacon and target nucleic acid is placed on the surface of an electrode to perform electrochemiluminescence detection, so that the modification of the target nucleic acid is qualitatively and quantitatively analyzed according to the existence and the intensity of an electrochemiluminescence signal. By the ECL detection method of the present invention, nucleic acid specific site modifications, such as methylation sites of DNA, can be detected simply, sensitively, rapidly and universally.
Description
Technical Field
The invention relates to an electrochemical luminescence (ECL) detection method based on nucleic acid specific site modification of a nano beacon, an ECL nano beacon used in the ECL detection method, a preparation method thereof and a kit used in the ECL detection method.
Background
There are numerous chemical modifications in nucleic acids, such as DNA and RNA, which dynamically regulate gene expression as a regulatory mechanism without altering the nucleic acid sequence.
DNA methylation (DNA methylation) is a type of apparent modification which occurs at the carbon atom at position 5 of cytosine and is stably inherited, and is widely found in animal and plant genomes. DNA methylation is widely involved in a variety of physiological processes during mammalian growth and development, including gene silencing, gene imprinting (genomic imprinting), X-chromosome inactivation, and the occurrence of diseases.
Part of the tumor is accompanied by the hypermethylation event of a specific gene, and the local hypermethylation event of the gene occurs earlier than the malignant hyperplasia of cells, so that the detection of the methylation level of the specific gene can be used as one of important bases for early prediction and diagnosis of the tumor. For example, early colorectal cancer (CRC) screening detection is based on epigenetic biomarkers, and FDA has approved early screening and auxiliary diagnosis of CRC by detecting an increase in CpG methylation of a specific gene promoter (see non-patent document 1). In addition, cancer suppressor genes which undergo hypermethylation are different from one tumor type, and for example, cancer suppressor genes RASSF1A, BRCA, APC, CDKN2A and the like are in a hypermethylated state in ovarian cancer, and cancer suppressor genes PCDHB15, WBSCR17, IGF1, GYPC and the like are in a hypermethylated state in breast cancer (see non-patent document 2). By detecting the methylation level of the specific cancer suppressor genes, not only can the screening and diagnosis of specific tumors be enhanced, but also the evaluation of the targeted treatment effect of the tumors and the prognosis observation can be facilitated.
The standard method of DNA methylation detection is currently bisulfite treatment, specifically, the conversion of cytosine (C) in DNA to uracil (U) is achieved by treating the DNA with bisulfite, e.g., sodium bisulfite, but the methylation of 5-methylcytosine (5-mC) remains unchanged, whereby 5-mC is distinguishable from C by subsequent PCR or sequencing, thereby allowing detection of methylated DNA.
However, bisulfite treatment requires nucleic acid pretreatment under stringent chemical and temperature conditions, and the PCR or sequencing detection operation is complicated, requiring specialized technicians and specialized equipment, making the detection method inefficient, time consuming, and costly. Thus, there is an urgent need to develop simple, rapid, sensitive methods for detecting methylated DNA.
Recently, a methylated DNA detection method based on specific antibody recognition of a methylation site has attracted attention. Such detection methods do not require nucleic acid pretreatment, and can be used for electrochemical (for example, non-patent document 3), fluorescence, etc. detection by labeling different beacons with specific antibodies.
ECL technology developed in recent years has been widely used in bioassays, such as tumor protein marker detection (for example, non-patent document 4). ECL technology is a method of initiating specific luminescence by electrochemical reaction to produce excited states on the surface of an electrode using electrochemical principles. Because ECL is electroluminescence, compared with fluorescence, the ECL does not need to be externally added with an excitation light source and has no influence of photobleaching, light interference and the like, and therefore, the ECL has the advantages of simple equipment, low cost, low background signal, high detection sensitivity and the like.
However, to date, there is no viable and satisfactory ECL detection method for nucleic acid-specific site modification detection in the prior art.
Prior art literature
Non-patent literature
Non-patent document 1: yvette N Lamb et al, epi proColon2.0 CE：A Blood-Based Screening Test for Colorectal Cancer，Mol Diagn Ther.2017 Apr；21(2):225-232
Non-patent document 2: hong Tingting specific detection of DNA epigenetic modification, university of Wuhan, 2017, doctor's treatises
Non-patent document 3: eloy povidano et al Amperometric Bioplatforms To Detect Regional DNA Methylation with Single-Base Sensitivity, anal chem 2020, 92, 5604-12
Non-patent document 4: xiaoming methou et al, synthesis, labeling and bioanal ytical applications of a tris (2, 2' -bipyridyl) Ruthenium (II) -based electroc hemiluminescence probe, nat Protoc 2014 May;9 (5)
Disclosure of Invention
Problems to be solved by the invention
The invention aims to provide a simple, sensitive, rapid and universal electrochemiluminescence detection method for modifying specific sites of nucleic acid. In addition, the invention also provides an electrochemiluminescence nano beacon used in the detection method, a preparation method thereof and a kit used in the detection method.
Means for solving the problems
As a result of intensive and thorough research into the above-described problems, the present inventors have found that a simple, sensitive, rapid and versatile electrochemiluminescence detection of specific site modification of nucleic acids can be achieved by synthesizing an electrochemiluminescence nanobeacon (hereinafter also referred to as ECL nanobeacon) and combining a specific antibody to recognize a methylation site.
Specifically, the application provides the following technical scheme.
[1] A method for preparing an electrochemiluminescence nano-beacon, which comprises the following steps:
doping metal complex ions onto the inorganic oxide nano particles to obtain metal doped inorganic oxide nano particles; and
and binding the secondary antibody to the metal-doped inorganic oxide nano-particles to obtain the metal-doped inorganic oxide nano-particles modified by the secondary antibody, wherein the secondary antibody recognizes the specific antibody modified by the specific site of the nucleic acid.
[2] The production method according to the above [1], wherein the inorganic oxide nanoparticle is a silica nanoparticle, a titania nanoparticle, a zinc oxide nanoparticle, or an iron oxide nanoparticle, or a nanoparticle coated with silica, titania, zinc oxide, or iron oxide.
[3] The production method according to the above [1] or [2], wherein the inorganic oxide nanoparticle is a silica nanoparticle.
[4] The production method according to any one of the above [1] to [3], wherein the secondary antibody is a protein recognizing against a general moiety of the specific antibody.
[5]According to [1] above]～[4]The method according to any one of the preceding claims, wherein the metal complex ion is a tris (bipyridine) ruthenium (II) complex ion (Ru (bpy) 3 2+ )。
[6] An electrochemiluminescence nanobeacon prepared by the preparation method described in the above [1] to [5 ].
[7] An electrochemiluminescence detection method for specific site modification of nucleic acid based on nano beacons comprises the following steps:
step 1: mixing magnetic particles modified with capture nucleic acid serving as a reaction reagent 1 with a sample to be detected, and identifying and capturing target nucleic acid modification in the sample;
step 2: capturing and marking the specific site modification of the target nucleic acid by taking the specific antibody modified by the specific site of the nucleic acid as a reaction reagent 2;
and step 3: the method of (2) labeling the magnetic particle-capturing nucleic acid-target nucleic acid-specific antibody complex obtained in step (2) with the electrochemical luminescence nanobeacon of (6) as a reagent 3; and
And 4, step 4: and (3) placing the magnetic particle-capture nucleic acid-target nucleic acid-specific antibody-nano beacon compound obtained in the step (3) on the surface of an electrode, adding a co-reactant, performing electrochemiluminescence detection, and performing qualitative and quantitative analysis on the modification of the target nucleic acid according to the existence and intensity of an electrochemiluminescence signal.
[8] The detection method according to the above [7], wherein the nucleic acid is DNA or RNA.
[9] The detection method according to the above [7] or [8], wherein the specific site modification is methylation modification, methylolation modification, or pseudouracil (pseudo) modification.
[10] The detection method according to any one of [7] to [9], wherein biotin is bonded to the end of the capture nucleic acid, and the capture nucleic acid is modified on the magnetic microparticles by biotin-streptavidin reaction.
[11] The detection method according to any one of the above [7] to [10], wherein the coreactant is tripropylamine.
[12] The detection method according to any one of [7] to [11], wherein the electrode is one selected from a glassy carbon electrode, an ITO electrode, and a screen-printed electrode.
[13] The detection method according to any one of [7] to [12], wherein the pH of the electrochemiluminescence electrolyte used in the step 4 is 6.5 or more.
[14] The detection method according to any one of [7] to [13], wherein the concentration of the coreactant is 10mM or more.
[15] The detection method according to any one of the above [7] to [14], wherein the concentration of the specific antibody is 1. Mu.g/mL or more
[16] The detection method according to any one of [7] to [15], wherein the incubation time after the addition of the specific antibody is 10 minutes or longer.
[17] The detection method according to any one of [7] to [16], wherein the concentration of the electrochemiluminescence nanobeacon is 4. Mu.g/mL or more.
[18] The detection method according to any one of [7] to [17], wherein the incubation time after addition of the electrochemiluminescence nanobeacon is 15 minutes or longer.
[19] A kit for an antibody-based electrochemiluminescence detection method comprising the electrochemiluminescence nanobeacon of [6] above.
[20] A nanoparticle for an electrochemiluminescence nanobeacon doped with a metal complex ion.
[21] An electrochemiluminescent nanobeacon comprising an inorganic oxide nanoparticle doped with metal complex ions and a secondary antibody bound to the inorganic oxide nanoparticle.
Effects of the invention
(1) The detection of the specific site of the nucleic acid is realized by nucleic acid hybridization capture and specific antibody recognition marks of modified sites, such as methylation sites, so that the traditional nucleic acid pretreatment and PCR amplification process in bisulfite treatment are not needed, the method is simple, and the detection efficiency is high.
(2) The electrochemiluminescence detection method based on the magnetic particles is low in cost and easy to automate, integrate and make into a kit.
(3) The ECL nano-beacon is utilized to amplify signals, so that the detection sensitivity of electrochemiluminescence is improved, and the detection limit of methylated DNA can even reach fM level.
(4) The invention has universality for detecting specific site modification of nucleic acid, and can detect different target specific site modification of nucleic acid by designing different capture nucleic acids and utilizing different specific antibodies.
Drawings
Fig. 1 is a schematic diagram showing a detection flow of an electrochemiluminescence detection method according to an embodiment of the present invention.
FIG. 2 is a schematic diagram showing a method for producing the reaction reagent 1 used in the electrochemiluminescence detection method of the present invention.
FIG. 3 is a schematic diagram showing a method for producing the reagent 3 used in the electrochemiluminescence detection method of the present invention.
FIG. 4 is a graph showing the results of characterization of ECL nanobeacons prepared in example 1, wherein FIG. 4 (A) shows COOH-Ru@SiO obtained in example 1 2 FIG. 4 (B) shows a TEM photograph of COOH-Ru@SiO obtained by DLS 2 (a) And Ab2-Ru@SiO 2 (b) Particle size distribution of (3).
FIG. 5 is a graph showing the characterization result of ECL nanobeacons prepared in example 1, wherein FIG. 5 (A) shows Ab2-Ru@SiO 2 The ultraviolet visible spectrum of the nanobeacon, FIG. 5 (B) shows [ Rubyp ] 3 2+ ]Concentration standard curve at 457nm, and FIG. 5 (C) shows concentration standard curve of BSA standard at 620 nm.
Fig. 6 is a graph showing the relationship between the storage time and ECL intensity of ECL nanobeacons prepared in example 1.
Fig. 7 is a graph showing measurement results of the ECL sensing system prepared in example 3, in which fig. 7 (a) is a Cyclic Voltammogram (CV) diagram showing gradual modification of the ECL sensing system and fig. 7 (B) is an Electrochemical Impedance Spectroscopy (EIS) diagram showing gradual modification of the ECL sensing system. Wherein a represents a bare GCE electrode, B represents S-MBs/GCE, c represents B-Cap/S-MBs/GCE, d represents BSA/B-Cap/S-MBs/GCE, e represents T/BSA/B-Cap/S-MBs/GCE, f represents Ab-5mC/T/BSA/B-Cap/S-MBs/GCE, g represents Ab2-Ru@SiO 2 /Ab-5mC/T/BSA/B-Cap/S-MBs/GCE。
FIG. 8 is a schematic diagram showing Ru@SiO prepared in example 3 2 Graph of ECL response under different ECL detection conditions. Fig. 8 (a) shows a graph of the relationship between the concentration of different TPrA and the ECL intensity, and fig. 8 (B) shows a graph of the relationship between the pH of different ECL detection electrolytes and the ECL intensity.
FIG. 9 is a graph showing the response of the ECL sensing system prepared in example 3 to ECL under different detection conditions. FIG. 9 (A) is a graph showing the relationship between the concentration of Ab-5mC and the ECL intensity. FIG. 9 (B) is a graph showing the relationship between incubation time of different Ab-5mC and ECL intensity. FIG. 9 (C) shows a different Ab2-Ru@SiO 2 Graph of concentration versus ECL intensity. FIG. 9 (D) shows a different Ab2-Ru@SiO 2 Graph of incubation time versus ECL intensity.
FIG. 10 is a fluorescent verification chart showing the detection principle of the ECL sensor system prepared in example 3 on a target nucleic acid, wherein a, b and c represent a background solution (no T) and a negative control sequence (T) respectively when Cy5-Pro nucleic acid detection probes are used 0 ) And a target DNA sequence (T) 5mC ) Is a fluorescent imaging image of (2); d. e and F represent the background solution (without T) using the fluorescence labeled secondary antibody (Ab 2-Alexa F647), the negative control sequence (T) 0 ) And a target DNA sequence (T) 5mC ) Is a fluorescent imaging image of (3).
FIG. 11 is a graph showing the ECL response of the ECL sensing system prepared in example 3 at different concentrations of methylated DNA. Wherein FIG. 11 (A) shows ECL response curves of methylated DNA at different concentrations, wherein a-i represent 0.5pM, 1pM, 10pM, 50pM, 100pM, 500pM, 10nM and 50nM, respectively; fig. 11 (B) shows a standard curve for detecting methylated DNA at different concentrations, n=3.
Fig. 12 is a graph showing the result of evaluating the selectivity of the ECL detection method of the present invention.
Fig. 13 is a graph showing the results of evaluation of the stability of the ECL detection method of the present invention. Fig. 13 (a) is a graph showing the result of evaluation of signal stability in the ECL detection method of the present invention, and fig. 13 (B) is a graph showing the result of evaluation of stability in long-term detection in the ECL detection method of the present invention.
Detailed Description
Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. The following description of the embodiments is merely illustrative of the inventive concept of the present invention, and is not intended to limit the present invention.
The invention provides an electrochemiluminescence detection method based on specific site modification of nucleic acid of a nano beacon, the electrochemiluminescence nano beacon used in the detection method, a preparation method thereof, a kit used in the detection method and the like.
One embodiment of the present invention relates to an electrochemical luminescence detection method (hereinafter, also referred to as ECL detection method) based on specific site modification of nucleic acid by a nanobeacon, comprising the steps of:
step 1: mixing magnetic particles modified with capture nucleic acid serving as a reaction reagent 1 with a sample to be detected, and identifying and capturing target nucleic acid modification in the sample;
Step 2: capturing and marking the specific site modification of the target nucleic acid by taking the specific antibody modified by the specific site of the nucleic acid as a reaction reagent 2;
and step 3: the method of (2) labeling the magnetic particle-capturing nucleic acid-target nucleic acid-specific antibody complex obtained in step (2) with the electrochemical luminescence nanobeacon of (6) as a reagent 3; and
and 4, step 4: and (3) placing the magnetic particle-capture nucleic acid-target nucleic acid-specific antibody-nano beacon compound obtained in the step (3) on the surface of an electrode, adding a co-reactant, performing electrochemiluminescence detection, and performing qualitative and quantitative analysis on the modification of the target nucleic acid according to the existence and intensity of an electrochemiluminescence signal.
The invention uses electrochemiluminescence to detect specific site modification of nucleic acids, such as DNA or RNA. The nucleic acid-specific site modification is not particularly limited, and may be, for example, methylation modification of DNA (for example, methylation of 5-methylcytosine, hereinafter also referred to as 5 mC), hydroxymethylation modification (for example, hydroxymethylation of 5-methylcytosine, hereinafter also referred to as 5 hmC), pseudouracil modification of RNA (for example, methylation of N at position 6 on adenine, hereinafter also referred to as m 6A), or the like.
Electrochemiluminescence systems can be classified into two main categories according to luminescence reagents: (1) a metal complex electrochemiluminescence system; (2) Organic compound electrochemiluminescence system comprises polycyclic aromatic hydrocarbon and hydrazide. Among the commonly used electrochemiluminescence metal complexes are those of metal ions such as Ru, os, re, ir, cr, pd, al, cd, pt, mo, tb, eu, wherein Ru, ir, os, re is of great interest for having good electrochemiluminescence properties.
Of the Ru metal complexes, tris (bipyridine) ruthenium (II) complex ions (Ru (bpy) 3 2+ ) The method is widely applied due to the characteristics of good water solubility, stable chemical property, reversible oxidation-reduction, high luminous efficiency, wide pH range of application, electrochemical regeneration, long service life of excited state and the like. Wherein Ru (bpy) 3 2+ Reaction with the co-reactant Tripropylamine (TPrA) enables ECL detection at low potential, ru (bpy) 3 2+ The reaction equation with tripropylamine TPrA is as follows:
Tripropylamine→Tripropylamine·+e- (1)
Ru(bpy) 3 2+ →Ru(bpy) 3 3+ +e- (2)
Ru(bpy) 3 3+ +Tripropylamine·→[Ru(bpy) 3 2+ ]*+products (3)
[Ru(bpy) 3 2+ ]*→Ru(bpy) 3 2+ +hv (4)
the electrochemiluminescence detection method of the invention is preferably carried out by means of a ruthenium (II) complex ion system, but can also be carried out using other metal complex ion systems, for example iridium (III) complex ion systems and the like.
FIG. 1 is a schematic diagram showing a detection flow of the electrochemiluminescence detection method of the present invention. As shown in fig. 1, the ECL detection method of the present invention uses a reagent 1, a reagent 2, and a reagent 3.
Wherein the reaction reagent 1 is a magnetic particle modified with a capture nucleic acid designed for a target nucleic acid having a nucleic acid sequence complementary to the target nucleic acid, both of which are capable of stable specific hybridization. The capture nucleic acid can be synthesized by a method conventional in the art, or can be directly used as a commercially available product.
FIG. 2 is a schematic diagram showing a method for producing the reagent 1 used in the present invention. As shown in fig. 2, the capture nucleic acid sequence may be modified at the end with biotin, and the magnetic particles may be bound with streptavidin, and the capture nucleic acid may be modified on the magnetic particles by a biotin-streptavidin reaction. The streptavidin-conjugated magnetic microparticles (hereinafter also referred to as S-MBs) can be synthesized by a method conventional in the art, or can be directly used as a commercially available product. The biotin-streptavidin binding method is the most commonly used method in the art, but the binding method of the capture nucleic acid to the magnetic microparticles is not limited to this, and any method capable of binding the capture nucleic acid to the magnetic microparticles may be used.
The reagent 2 is a specific antibody (hereinafter also referred to as a primary antibody) modified at a specific site with respect to the target nucleic acid, and the antibody can be prepared by a conventional antibody preparation method in the art according to the specific site modification, or can be used as it is. The primary antibody is, for example, an antibody against methylated cytosine (5 mC) or hydroxymethylcytosine (5 hmC) of DNA.
The reactant 3 is the electrochemiluminescence nano beacon prepared in the invention, namely the metal doped inorganic oxide nano particle modified by the secondary antibody.
One embodiment of the present invention relates to a method for producing an electrochemiluminescence nanobeacon (hereinafter also referred to as ECL nanobeacon), comprising the steps of:
doping metal complex ions onto the inorganic oxide nano particles to obtain metal doped inorganic oxide nano particles; and
and binding the secondary antibody to the metal-doped inorganic oxide nano-particles to obtain the metal-doped inorganic oxide nano-particles modified by the secondary antibody, wherein the secondary antibody recognizes the specific antibody modified by the specific site of the nucleic acid.
The ECL nano beacon is prepared by using a nano material as a carrier, and the nano material has a huge surface area or a porous structure, so that a large number of luminophores can be loaded for hypersensitive detection. The nanomaterial used in the present invention is preferably an inorganic oxide nanoparticle from the viewpoints of high stability, low cost, and easy modification. Specific examples of the inorganic oxide nanoparticles include silica nanoparticles, titania nanoparticles, zinc oxide nanoparticles, ferrite nanoparticles, and nanoparticles having surfaces coated with silica, titania, zinc oxide, ferrite, or the like, and among these, silica nanoparticles are more preferable.
The preparation method of the ECL nano beacon comprises the step of doping metal complex ions onto inorganic oxide nano particles. As the doping method, a doping method commonly used in the art, for example, an electrochemical method, a sol-gel method, an ion exchange method, a hydrolysis precipitation method, or the like can be used, but is not limited thereto, and any method capable of doping metal complex ions to inorganic oxide nanoparticles can be used.
The preparation method of the ECL nano beacon further comprises the step of combining the secondary antibody to the metal-doped inorganic oxide nano particles to obtain the metal-doped inorganic oxide nano particles modified by the secondary antibody. The binding of the secondary antibody to the metal-doped inorganic oxide nanoparticle is preferably performed by covalent binding, but other methods such as electrostatic adsorption and the like may be used. Specific examples of the bonding method constituting the covalent bond include carboxyl-amino bonding and aldehyde-amino bonding. The reaction for carrying out covalent bonding is not particularly limited, and a covalent reaction that can react at room temperature and is rapid and efficient in bonding is preferable.
FIG. 3 is a schematic diagram showing a method for producing the reactant 3 of the present invention. In FIG. 3, the ion (Ru (bpy) is represented as a tris (bipyridine) ruthenium (II) complex 3 2+ ) For example, the binding of the secondary antibody (hereinafter also referred to as Ab 2) to the metal-doped inorganic oxide nanoparticles was performed using the EDC/NHS system (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride)/N-hydroxysuccinimide system). Specifically, ru (bpy) 3 2+ Doped on the silica nanoparticles to obtain ruthenium doped silica nanoparticles (hereinafter also referred to as Ru@SiO 2 ) The method comprises the steps of carrying out a first treatment on the surface of the Then Ru@SiO 2 Carboxylation to form ruthenium carboxylation doped silica nanoparticles (also referred to as COOH-Ru@SiO hereinafter 2 ). Then, the secondary antibody was bound to COOH-Ru@SiO using EDC/NHS system 2 The second antibody-modified ruthenium-doped silica nanoparticle (hereinafter also referred to as Ab 2-Ru@SiO) 2 )。
In the ECL nano beacon, a large amount of metal complex ions can be combined on every 1 nano particle, so that an electrochemical signal can be remarkably amplified, and the sensitivity of ECL detection can be greatly improved.
In addition, in the ECL nano-beacon, a plurality of secondary antibody molecules can be combined on each 1 nano-particle, so that the combination efficiency of the nano-beacon and the primary antibody can be improved.
Therefore, by using the ECL nano-beacon provided by the invention, the sensitivity of ECL detection can be greatly improved, and the detection of nucleic acid specific site modification can even reach fM level.
The secondary antibodies of the present invention recognize the nucleic acid-specific site-modified primary antibody, which is preferably not designed for the specific portion of the primary antibody to which the nucleic acid-specific site modification binds, but for the universal portion of the primary antibody. That is, the secondary antibody of the present invention is preferably a protein recognizing against a general moiety of the primary antibody, and thus the present invention can provide a general ECL nanobeacon for various antibody-based ECL detection methods. The secondary antibody is not particularly limited, and may be prepared by a method of preparing an antibody which is conventional in the art, or may be used as it is.
Thus, an embodiment of the present invention also provides an ECL nanobeacon prepared by the above-described method for preparing an ECL nanobeacon of the present invention, wherein preferably the ECL nanobeacon of the present invention is a universal ECL nanobeacon of an antibody-based ECL detection method.
The ECL nanobeacons prepared in the present invention have excellent storage stability, and can be stored for 10 days or more under normal temperature storage conditions, for example, and therefore, the ECL nanobeacons of the present invention can be used as they are after preparation or can be used as needed after storage for a suitable period of time.
Thus, one embodiment of the present invention also provides a kit for an antibody-based electrochemiluminescence detection method comprising the ECL nanobeacon of the invention described above.
The kit may contain various reagents required for the purpose of use, for example, reagents 1 and 2, instructions for use, and the like, in addition to the ECL nanobeacon of the present invention.
Specifically, as shown in fig. 1, the ECL detection method of the present invention may include the following steps 1 to 4.
Step 1: the reaction reagent 1 (for example, S-MBs/B-Cap) is mixed with a sample to be detected (for example, target DNA) to perform recognition and capture on target nucleic acid in the sample. The sequence of the capture nucleic acid (e.g., capture DNA) bound on the B-Cap is designed to be complementary to the target DNA sequence, so that it can bind to the target nucleic acid by hybridization after the sample to be tested is added, thereby capturing the target DNA to the magnetic particles.
Wherein, after the target DNA is identified by S-MBs/B-Cap, the reaction system can be blocked to prevent nonspecific adsorption of immunological reagents, and the blocking can be performed by using heterologous proteins or detergents, such as Tween-20, BSA, animal serum, skimmed milk powder, etc., preferably BSA.
Step 2: the target DNA is subjected to capture labelling by adding a reagent 2 (e.g., ab-5 mC). The primary antibody as the reaction reagent 2 is designed for a specific antibody of a specific site modification of a nucleic acid, and is capable of recognizing the specific site modification on a target nucleic acid, thereby enabling capture labeling of the specific site modification of the target nucleic acid to form a magnetic particle-capture DNA-target DNA-Ab-5mC complex, for example.
Among them, from the viewpoint of obtaining a higher ECL signal intensity, the primary antibody concentration, the incubation time after the primary antibody addition, and the like are preferably optimized. The primary antibody concentration is, for example, 1.0. Mu.g/mL or more, specifically 2.0. Mu.g/mL or more, 2.5. Mu.g/mL or more, 5. Mu.g/mL or more, 10. Mu.g/mL or more, 20. Mu.g/mL or more, and the like, and among these, 2.5. Mu.g/mL to 20. Mu.g/mL is preferable. The incubation time for the primary antibody is, for example, 5 to 80 minutes, specifically 5, 10, 20, 30, 40, 50, 60, 70, 80 minutes, and preferably 20 to 40 minutes.
And step 3: adding reagent 3 (e.g. Ab2-Ru@SiO 2 ) The target DNA is labeled with a detection signal. The secondary antibody bound to the reagent 3 can specifically bind to the primary antibody in the complex formed in the step 2, thereby labeling the complex with a detection signal to form, for example, magnetic microparticles-capturing DNA-target DNa-Ab-5mC-Ab2-Ru@SiO 2 Is a complex of (a) and (b).
Among them, from the viewpoint of obtaining a higher ECL signal intensity, optimization of ECL nanobeacon concentration, incubation time after ECL nanobeacon addition, and the like is preferable. The concentration of the ECL nanobeacon is, for example, 1. Mu.g/mL or more, specifically 3. Mu.g/mL or more, 4. Mu.g/mL or more, 6. Mu.g/mL or more, 9. Mu.g/mL or more, 12. Mu.g/mL or more, 15. Mu.g/mL or more, 18. Mu.g/mL or more, and preferably 4. Mu.g/mL to 15. Mu.g/mL. The ECL nanobeacon incubation time is, for example, 5 to 80 minutes, specifically 5, 10, 20, 30, 40, 50, 60, 70, 80 minutes, and preferably 20 to 40 minutes.
And 4, step 4: and (3) placing the compound obtained in the step (3) on the surface of an electrode, and performing electrochemiluminescence detection. Among them, from the viewpoint of obtaining a higher ECL signal intensity, it is preferable to optimize the pH value of the ECL detection electrolyte and the concentration of the coreactant (e.g., tprA). The pH of the ECL detection electrolyte is, for example, 5.0 to 10.0, specifically 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 10.0, preferably 6.5 or more, and more preferably 7.4. The concentration of the coreactant is, for example, 5.0 to 160.0mM, specifically 10.0mM or more, 20.0mM or more, 40.0mM or more, 80.0mM or more, preferably 20.0mM to 80.0mM, more preferably 40.0mM to 80.0mM.
Wherein the complex obtained in the step 3 is a complex formed of magnetic particles, capture DNA, target DNA, primary antibody and ECL nanobeacons, which is capable of generating an electrochemiluminescence signal when the complex is placed on the electrode surface, as shown on the right side of fig. 1.
According to the presence or absence and the intensity of ECL signal, the nucleic acid specific site modification of the sample to be detected can be qualitatively and quantitatively analyzed, for example, when ECL signal is not detected in a reaction system, it can be judged that the sample to be detected does not contain nucleic acid specific site modification, and therefore the complex is not formed and electrochemical luminescence signal cannot be generated. In addition, as demonstrated in the examples of the present invention, the intensity of electrochemiluminescence is linearly dependent on the logarithm of the concentration of methylated DNA, so that quantitative analysis of methylated DNA can be performed based on the intensity of electrochemiluminescence.
In the present invention, the detection of the electrochemiluminescence signal may be performed using an electrochemical chemical analysis system commonly used in the art, and the electrode used may be one of commonly used electrodes, such as a glassy carbon electrode, an ITO electrode, or a screen-printed electrode. A standard three electrode system is preferably used in the present invention, which is configured with a platinum wire counter electrode, an Ag/AgCl reference electrode, and a glassy carbon working electrode (GCE).
The ECL detection method of the present invention is not limited to the above-described step, and the magnetic particle-capturing nucleic acid-target nucleic acid-primary antibody-ECL nanobeacon complex can be formed by binding the primary antibody to the hybridization product of the target nucleic acid-capturing nucleic acid and then adding the ECL nanobeacon of the present invention to form the primary-secondary antibody complex, but the magnetic particle-capturing nucleic acid-target nucleic acid-primary antibody-ECL nanobeacon complex can also be formed by binding the primary antibody to the hybridization product of the target nucleic acid-capturing nucleic acid after the primary antibody and the secondary antibody-modified metal-doped inorganic oxide nanoparticle are formed into the primary-secondary antibody complex. Furthermore, in the invention, the primary antibody can be directly modified on the metal doped electrodeless oxide nano particles modified by the secondary antibody, and the whole can be used as an ECL nano beacon for detection.
Examples
The present invention will be specifically described below by way of examples, but the present invention is not limited to these examples.
All reagents used in this example were of analytical grade.
Wherein the meanings of the abbreviations used are as follows:
Ru(bpy) 3 2+ : tris (2, 2' -bipyridine) dichloro ruthenium (II) hexahydrate
TEOS: tetraethoxysilane (TEOS)
NH 4 OH: ammonia water
Ru@SiO 2 : ruthenium doped silica nanoparticles
Ab2-Ru@SiO 2 : ruthenium doped silicon dioxide nano particle modified by secondary antibody
CTES: carboxyethyl silanetriol sodium salt
COOH-Ru@SiO 2 : ruthenium carboxylated doped silica nanoparticles
DI water: deionized water
EDC: n- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride
NHS: n-hydroxysuccinimide
MES: 2- (N-morpholino) ethanol sulfonic acid
PBS: phosphate buffer
BSA: bovine serum albumin
S-MBs: streptavidin magnetic particles
B-Cap: biotin-capturing DNA
T: DNA sample
Ab-5mC: anti-5-methylcytosine antibodies
Example 1: preparation of reactant 3 (ECL nanobeacons)
(1)Ru@SiO 2 Is prepared from
(1) 7.5mL of cyclohexane (from Allatin (China, shanghai)), 1.8mL of n-hexanol (from Boschniakia (China, shanghai)), 1.77mL of Triton X-100 (from Sigma (China, shanghai)) were mixed for 15 minutes;
(2) 340 mu L Ru (bpy) is added 3 2+ (Sigma (China, shanghai)) (40 mM) in water,
mixing for 30 minutes;
(3) 100 μl TEOS (from Sigma (China, shanghai)) was added and mixed for 30 minutes;
(4) 60 mu L of NH was added 4 OH (purchased from Michelia (China, shanghai)) and mixing for 24 hours in the dark to obtain Ru@SiOj 2 ；
(2)Ab2-Ru@SiO 2 Is prepared from
(1) Adding 50 μl of CTES (purchased from carbofuran (China, shanghai)), mixing in dark for 7h to obtain COOH-Ru@SiO 2 ；
(2) Sequentially washing with 15mL of acetone, ethanol and DI water, centrifuging at 10000rpm for 10min, and dispersing in DI water;
(3) 10 mu L of 50mg/mL COOH-Ru@SiO is taken 2 Activation with 500. Mu.L EDC/NHS (50 mg/mL each in 25mM MES (pH 5.5), EDC, NHS both purchased from carbofuran (China, shanghai)) for 35min; followed by washing with 10mM PBS (pH 7.4) at 12000rpm for 5min and dispersing in 1mL of 10mM PBS;
(4) 20. Mu.L of 2mg/mL Ab2 (rabbit anti-mouse IgG antibody (trade name: ab6709, purchased from Ai Bokang (China, shanghai)) was added and shaken at 60rpm for 4 hours;
(5) Washing with 25mM MES buffer and 10mM PBS at 12000rpm for 5min to obtain Ab2-Ru@SiO 2 ；
(6) Finally, the obtained Ab2-Ru@SiO 2 Stored in 0.1% BSA (purchased from Kaiki's organism (China, jiangsu))/5 mM PBS buffer at 4℃until use.
Example 2: characterization of ECL nanobeacons
A: TEM-based COOH-Ru@SiO 2 Confirmation of formation
For COOH-Ru@SiO obtained in example 1 2 Nanoparticles were photographed using a JEM-2100 transmission electron microscope (JEOL, japan). The obtained Transmission Electron Micrograph (TEM) is shown in fig. 4 (a).
As can be seen from FIG. 4 (A), the obtained COOH-Ru@SiO 2 The nanoparticles were uniformly dispersed and uniformly sized and were spherical particles of about 30nm in diameter.
B: ab2-Ru@SiO based on Dynamic Light Scattering (DLS) method 2 Confirmation of formation
Dynamic Light Scattering (DLS) method for measuring COOH-Ru@SiO respectively using a 90Plus/BI-MAS instrument (Brookhaven, U.S.) 2 And Ab2-Ru@SiO 2 Is a particle size distribution of (a). The results are shown in fig. 4 (B).
As can be seen from FIG. 4 (B), COOH-Ru@SiO 2 (a) The hydration particle size of the catalyst is distributed around 50nm, the particle size distribution is narrow, the size is uniform, and the catalyst has monodispersity; ab2-Ru@SiO 2 (b) The hydration particle diameter of (C) is about 70nm and slightly larger than COOH-Ru@SiO 2 Indicating Ab2 and Ru@SiO 2 The coupling is successful, the dispersibility is good, and the aggregation phenomenon does not occur.
C：Ab2-Ru@SiO 2 Confirmation of the number of ruthenium molecules bound and the number of antibodies bound
To obtain a single Ru@SiO 2 The number of ruthenium molecules bound thereto was determined by using Ab2-Ru@SiO obtained in example 1 2 Quantitative analysis of the ultraviolet visible spectrum (UV) was performed using a Nanodrop-2000C ultraviolet-visible spectrophotometer (Thermo, usa). The results are shown in fig. 5 (a).
As shown in figure 5 (a),Ru@SiO of 100. Mu.g/mL 2 Absorbance A at 457nm was 0.26, according to [ Ru (bpy) of FIG. 5 (B) 3 ] 2+ The concentration standard curve is 100 mug/mL Ru@SiO 2 Middle [ Ru (bpy) 3 ] 2+ The concentration of (C) was 12.53. Mu.g/mL. Thus, 1mL of Ru@SiO 2 Middle [ Ru (bpy) 3 ] 2+ The number of molecules was estimated to be N [Ru(bpy)3] 2+ ＝N A ·n＝N A ·m Ru /M Ru ＝1.01×10 16 The method comprises the steps of carrying out a first treatment on the surface of the Thereby obtaining single Ru@SiO 2 Middle-wrapped [ Ru (bpy) 3 ] 2+ Number of molecules=n [Ru(bpy)3] 2+ /N Ru@SiO2 ＝3.1×10 4 。
Protein quantification kit (purchased from Bradford method by recording microplate reader(China, shanghai)) determination of Ab2-Ru@SiO obtained in example 1 2 Ab2 absorbance at 620nm, ab2-Ru@SiO 2 Ab2 of (B) had an absorbance A620 of 0.63.
Ab2-Ru@SiO was obtained from the BSA protein standard curve of FIG. 5 (C) 2 The concentration of Ab2 in the solution was 73.11. Mu.g/mL, and the concentration was determined by the molar mass MAb2=150KD=1.5X10 of IgG-Ab2 5 g/mol, 1mL 73.11. Mu.g/mL Ab2 of Ab2 number NAb2=NA.n=NA.mAb2/MAb2=2.94×10 14 。
According to 2mg Ru@SiO 2 Middle Ru@SiO 2 1mL of 1mg/mL Ru@SiO was obtained 2 Middle Ru@SiO 2 Number N of (2) 2 Is 3.22 multiplied by 10 13 From this, a single Ru@SiO can be derived 2 Number of antibodies bound thereon = NAb2/N 2 ＝2.94210 14 /3.22×10 13 ≈9。
D：Ab2-Ru@SiO 2 Is stored in a container of a container
Ab2-Ru@SiO obtained in example 1 2 ECL signals were measured in aqueous solution containing 1% bsa at 4 ℃ and stored in the dark, once every 5 days, and the results are shown in fig. 6.
In the examples section, ECL was measured using an MPI-a multifunctional electrochemiluminescence analysis system (sienna, china) in an electrolytic cell of a standard three-electrode system configured with a platinum wire counter electrode, an Ag/AgCl reference electrode and a glassy carbon working electrode (GCE) of 5mm diameter.
Detection of electrolyte in ECL using a three electrode system (0.1M PBS,0.1M KNO 3 ECL response was recorded in 40mm tpra, ph 7.4) using electrochemical Cyclic Voltammetry (CV) (experiments were performed at CHI 630D electrochemical workstation (china, shanghai cinnabar) with continuous potential scans from 0 to +1.25v at a scan rate of 100mV/s and a photomultiplier tube (PMT) voltage set at 600V. In addition, all ECL assays were performed at room temperature, as follows.
As can be seen from FIG. 6, ab2-Ru@SiO 2 The ECL strength of the probe is not changed basically within 20 days, and the probe has better storage stability in an aqueous solution containing 1% BSA.
Example 3: ECL detection method
The DNA sequences used in this example were synthesized and purified by Shanghai Bioengineering Co. The specific cases of the sequences used are shown in Table 1.
TABLE 1
(1) Preparation of DNA samples
A DNA sample (T) containing the following sequence was prepared:
negative control sequence: 0X 5mC-RASSF1A (T) 0 ) (sequence number 2)
Target DNA sequence: 7X 5mC-RASSF1A (T) 5mC ) (sequence number 3)
Non-target DNA sequence: 7X 5mC-PCDHGB7 (T) P ) (sequence number 5)
(2) Preparation of reactant 1 (S-MBs/B-Cap):
(1) 2 mu L S-MBs (purchased from Baimei Biotechnology (Jiangsu, tin-free)) were washed with TE buffer (B & W buffer with 2M NaCl, pH 7.5);
(2) mix with 25. Mu.L of 0.1. Mu. M B-Cap (in pH7.5 TE buffer) and incubate at 37℃for 15 min;
(3) washing with 10mM PBS to remove redundant B-caps and obtain S-MBs/B-caps;
(3) Identification of target DNA
(1) Mu. L T (in 10mM PBS) was added and incubated at 37℃for 30 min;
(2) then washed with 10mM PBS to wash off unreacted T.
(4) Ab-5mC recognition of 5mC
(1) mu.L of 2% BSA (in 10mM PBS) was added and incubated at 37℃for 30 min
(2) 50. Mu.L of Ab-5mC (trade name: ab10805, available from Ai Bokang (China, shanghai), 2.5. Mu.g/mL) was added, incubated at 37℃for 20 minutes,
(3) the unreacted Ab-5mC was then washed off with 10mM PBS.
(5) Tagging with ECL nanobeacons
(1) 50 mu L of Ab2-Ru@SiO are added 2 (6. Mu.g/mL) incubated at 37℃for 20 min;
(2) then washed with 10mM PBS and dispersed in 65. Mu.L of 10mM PBS.
(6) ECL detection
(1) The S-MBs/B-Cap-T-Ab-5mC-Ab2-Ru@SiO 2 The solution of the complex (hereinafter also simply referred to as complex) was dropped onto the GCE electrode, dried, and immersed in ECL test electrolyte (0.1M PBS,0.1M KNO) 3 40mM TPrA, pH 7.4).
(2) ECL was collected with PMT 600 by potential scanning from 0 to 1.25V.
Example 4: characterization of ECL sensing systems
Hereinafter, the GCE electrode detection system comprising the above-described complex obtained in example 3 is also referred to as ECL sensing system of the present invention.
(1) Measurement of ECL sensing systems of the invention
Electrochemical Cyclic Voltammetry (CV) and Electrochemical Impedance Spectroscopy (EIS) tests were performed on each step of the ECL sensing system prepared in example 3, wherein the CV was performed at the CHI 630D electrochemical workstation (china, shanghai-china), and the EIS test was performed on DH 7000 (china, jiangsu-dong-china). The results are shown in FIG. 7.
In FIG. 7, a represents a bare GCE electrode, B represents S-MBs/GCE, c represents B-Cap/S-MBs/GCE, d represents BSA/B-Cap/S-MBs/GCE, e represents T/BSA/B-Cap/S-MBs/GCE, f represents Ab-5mC/T/BSA/B-Cap/S-MBs/GCE, g represents Ab2-Ru@SiO2/Ab 5mC/T/BSA/B-Cap/S-MBs/GCE.
As can be seen from FIG. 7, the magnetic glassy carbon electrode bare electrode (a) or modified with S-MB and different complexes (B-g) has a gradual increase in electrode surface resistance due to poor conductivity of the magnetic particles and complexes after being added to GCE, and thus the electron transfer efficiency is reduced, the corresponding redox current is gradually reduced (FIG. 7A: a-g), and the EIS resistance is gradually increased (FIG. 7B: a-g).
(2) Optimization of electrochemical parameters of ECL sensing systems of the present invention
The pH and TprA concentration of the ECL test electrolyte used in example 3 were changed so that the pH was varied in the range of 6.0 to 8.5 and the TprA concentration was varied in the range of 10mM to 80 mM. The results are shown in fig. 8.
As can be seen from fig. 8, good ECL strength can be obtained when the ECL detection electrolyte has a pH value of 6.5 or more, wherein the ECL signal strength is highest at a pH value of 7.4, and is the optimal pH; in addition, good results were obtained at TprA concentrations above 10mM, where the ECL signal intensity was highest at 40mM, the optimal TprA concentration.
Modification of Ab-5mC concentration, ab-5mC incubation time, ab2-Ru@SiO in example 3 2 Concentration Ab2-Ru@SiO 2 The incubation time is changed to ensure that the concentration of Ab-5mC is changed within the range of 0-20 mug/mL, the incubation time of Ab-5mC is changed within the range of 10-40 minutes, and Ab2-Ru@SiO 2 The concentration is changed within the range of 3-15 mug/mL, so that Ab2-Ru@SiO 2 The incubation time varied from 10 to 30 minutes and the results are shown in FIG. 9.
As can be seen from FIG. 9, good ECL strength was obtained at a concentration of 1. Mu.g/mL or more of Ab-5mC, and the ECL signal strength was smoothed at a concentration of 2.5. Mu.g/mL or more of Ab-5mC, which is the optimal concentration of Ab-5 mC; good ECL strength can be obtained when Ab-5mC incubation time is more than 10 min, wherein Ab-5mC incubation time is 20 min or moreWhen the ECL signal intensity reaches stability, the optimal Ab-5mC incubation time is reached; ab2-Ru@SiO 2 Good ECL strength can be obtained at a concentration of more than 4 mug/mL, wherein Ab2-Ru@SiO 2 The concentration is more than 6 mug/mL, the ECL signal intensity is stable or higher, and the ECL signal intensity is the optimal Ab2-Ru@SiO 2 Concentration; ab2-Ru@SiO 2 Good ECL strength can be obtained when the incubation time is more than 15 minutes, wherein Ab2-Ru@SiO 2 And when the incubation time is more than 20 minutes, the ECL signal intensity is stable, and the optimal Ab-5mC incubation time is achieved.
Example 5: feasibility verification of ECL detection method of the invention
Let T in example 3 be T respectively 0 、T 5mC The procedure of example 3 was performed without T as a background solution to obtain a complex.
The complex was dispersed in PBS, 3. Mu.L of the complex was placed on ITO conductive glass, excitation wavelength was given at 620nm, and the resulting fluorescence micrograph was taken with a fluorescence microscope (Leica DMi8 inverted microscope, camera Photometrics Prime B was set up) and shown in FIGS. 10 a, B and c.
As shown in FIGS. 10 a, B and c, S-MB/B-Cap, to which no nucleic acid is bound, does not see fluorescence, and T is bound 0 、T 5mC After that, fluorescence can be generated on the magnetic particles, and the result shows that the B-Cap can capture T 0 、T 5mC The methylation site does not affect the complementary pairing of double-stranded bases. In addition, B-Cap was also demonstrated to bind to S-MB.
Ab2-Alexa F647-Ru@SiO was obtained by using a fluorescent-labeled secondary antibody (Ab 2-Alexa F647) capable of specifically binding to Ab-5mC instead of Ab2 in example 1 2 For example 3. The complex obtained in example 3 was photographed by a fluorescence microscope in the same manner as described above. The results are shown in fig. 10 d, e, f.
As shown in d, e and f of FIG. 10, there are no binding T and binding T 0 None of S-MB/B-Cap of (C) is fluorescent, combined with T 5mC The magnetic particles of (2) can see significant fluorescence, indicating that Ab-5mC can bind to the methylation site on the target DNA strand. From the above results, it is clear that the ECL detection method of the present invention is feasibleA kind of electronic device.
(3) Detection limit measurement of ECL sensing system of the present invention
The concentration of the methylated DNA in T in example 3 was changed so as to vary in the concentration range of 0.5pM to 50,000pM, and the results are shown in FIG. 11.
As can be seen from fig. 11, the intensity (I) of ECL increases with increasing concentration of methylated DNA (fig. 11 (a)), and the intensity (I) of ECL is linearly related to the logarithm of the concentration of methylated DNA (fig. 11 (B)), with a correlation coefficient of 0.9915. Furthermore, the minimum detection Limit (LOD) of methylated DNA calculated at a triple signal-to-noise ratio was 0.13pM.
(3) Selective verification of the ECL detection method of the present invention
Each pair of ECL sensor systems prepared in example 3 was used to detect the presence of T 0 、T 5mC And background solution were measured and the results are shown in fig. 12.
As shown in fig. 12, T 0 And background solution hardly generates ECL signal, while T 5mC Resulting in a higher ECL strength. Thus, the ECL sensing system of the present invention was demonstrated to have good selectivity.
(4) Verification of stability of ECL detection method of the present invention
T-containing in 0-250 seconds for the ECL sensing system of example 3 5mC The results of applying a scan potential of 0V to +1.25V 10 times in succession are shown in fig. 13 (a).
As shown in fig. 13 (a), the ECL signal was stable, and thus, it was found that the ECL signal in the ECL detection method of the present invention was excellent in stability.
The prepared reagent 1 (B-Cap/S-MB) was stored in PBS and stored at 4℃and ECL detection was performed on a sample containing 500pM target DNA sequence (RASSF 1A) every seven days, and the results are shown in FIG. 13 (B).
As shown in FIG. 13 (B), the detection method of the present invention has good detection stability within 28 days, and shows good long-term detection stability.
From the experimental results of the above embodiments, the electrochemiluminescence detection method of the present invention is simple and rapid, has high detection efficiency, low cost, and can be used as a kit, and has high detection sensitivity, and the detection limit for nucleic acid specific site modification can even reach fM level.
The ECL detection method of the present invention, the ECL nanobeacons used in the detection method, the method for producing the ECL nanobeacons, and the like have been described above based on the embodiments, but the present invention is not limited to these. Other modes of combining and constructing some of the constituent elements of the embodiments, which are obtained by implementing various modifications of the embodiments that will be apparent to those skilled in the art, are also included in the scope of the present invention as long as the gist of the present invention is not departing.
Sequence list
<110> Canon medical systems Co Ltd
NANJING University
<120> ECL detection method for nucleic acid specific site modification, ECL nano-beacon, preparation method thereof and kit
<130> 98G81002333-CN-A/CMC21B012
<160> 5
<170> PatentIn version 3.3
<210> 1
<211> 28
<212> DNA
<213> artificial sequence
<220>
<223> B-Cap
<400> 1
ctccttcgtc ccctcctcac accccacc 28
<210> 2
<211> 60
<212> DNA
<213> artificial sequence
<220>
<223> 0×5mC-RASSF1A（T0）
<400> 2
gctttgcggt cgccgtcgtt gtggccgtcc ggggtggggt gtgaggaggg gacgaaggag 60
<210> 3
<211> 60
<212> DNA
<213> artificial sequence
<220>
<223> 7×5mC-RASSF1A（T5mC）
<220>
<221> modified base
<222> (7)..(7)
<223> m5c
<220>
<221> modified base
<222> (11)..(11)
<223> m5c
<220>
<221> modified base
<222> (14)..(14)
<223> m5c
<220>
<221> modified base
<222> (17)..(17)
<223> m5c
<220>
<221> modified base
<222> (26)..(26)
<223> m5c
<220>
<221> modified base
<222> (30)..(30)
<223> m5c
<220>
<221> modified base
<222> (53)..(53)
<223> m5c
<400> 3
gctttgcggt cgccgtcgtt gtggccgtcc ggggtggggt gtgaggaggg gacgaaggag 60
<210> 4
<211> 22
<212> DNA
<213> artificial sequence
<220>
<223> Cy5-Pro
<400> 4
acaacgacgg cgaccgcaaa gc 22
<210> 5
<211> 59
<212> DNA
<213> artificial sequence
<220>
<223> 7×5mC-PCDHGB7（TP）
<220>
<221> modified base
<222> (6)..(6)
<223> m5c
<220>
<221> modified base
<222> (8)..(8)
<223> m5c
<220>
<221> modified base
<222> (16)..(16)
<223> m5c
<220>
<221> modified base
<222> (19)..(19)
<223> m5c
<220>
<221> modified base
<222> (24)..(24)
<223> m5c
<220>
<221> modified base
<222> (29)..(29)
<223> m5c
<220>
<221> modified base
<222> (31)..(31)
<223> m5c
<400> 5
agctgcgcgc agaggcgccg ggccggcccg cggcaggtac tatttccttt gctgctgct 59
Claims (21)
1. A method for preparing an electrochemiluminescence nano-beacon, which comprises the following steps:
doping metal complex ions onto the inorganic oxide nano particles to obtain metal doped inorganic oxide nano particles; and
and binding the secondary antibody to the metal-doped inorganic oxide nano-particles to obtain the metal-doped inorganic oxide nano-particles modified by the secondary antibody, wherein the secondary antibody recognizes the specific antibody modified by the specific site of the nucleic acid.
2. The preparation method according to claim 1, wherein the inorganic oxide nanoparticle is a silica nanoparticle, a titania nanoparticle, a zinc oxide nanoparticle, or an iron oxide nanoparticle, or a nanoparticle coated with silica, titania, zinc oxide, or iron oxide.
3. The production method according to claim 1 or 2, wherein the inorganic oxide nanoparticle is a silica nanoparticle.
4. A method of preparation according to any one of claims 1 to 3, wherein the secondary antibody is a protein which recognizes against a generic part of the specific antibody.
5. The process according to any one of claims 1 to 4, wherein the metal complex ion is a tris (bipyridine) ruthenium (II) complex ion (Ru (bpy) 3 2+ )。
6. An electrochemiluminescence nanobeacon prepared by the preparation method of claims 1 to 5.
7. An electrochemiluminescence detection method for specific site modification of nucleic acid based on nano beacons comprises the following steps:
step 1: mixing magnetic particles modified with capture nucleic acid serving as a reaction reagent 1 with a sample to be detected, and identifying and capturing target nucleic acid modification in the sample;
step 2: capturing and marking the specific site modification of the target nucleic acid by taking the specific antibody modified by the specific site of the nucleic acid as a reaction reagent 2;
and step 3: the detection signal of the magnetic particle-capturing nucleic acid-target nucleic acid-specific antibody complex obtained in the step 2 is labeled by using the electrochemiluminescence nano-beacon as a reaction reagent 3; and
And 4, step 4: and (3) placing the magnetic particle-capture nucleic acid-target nucleic acid-specific antibody-nano beacon compound obtained in the step (3) on the surface of an electrode, adding a co-reactant, performing electrochemiluminescence detection, and performing qualitative and quantitative analysis on the target nucleic acid according to the existence and intensity of electrochemiluminescence signals.
8. The method according to claim 7, wherein the nucleic acid is DNA or RNA.
9. The detection method according to claim 7 or 8, wherein the specific site modification is a methylation modification, a methylolation modification, or a pseudouracil modification.
10. The detection method according to any one of claims 7 to 9, wherein biotin is bound to the end of the capture nucleic acid, and the capture nucleic acid is modified on the magnetic microparticles by biotin-streptavidin reaction.
11. The detection method according to any one of claims 7 to 10, wherein the coreactant is tripropylamine.
12. The detection method according to any one of claims 7 to 11, wherein the electrode is one selected from a glassy carbon electrode, an ITO electrode, and a screen-printed electrode.
13. The detection method according to any one of claims 7 to 12, wherein the pH of the electrochemiluminescence electrolyte used in the step 4 is 6.5 or more.
14. The detection method according to any one of claims 7 to 13, wherein the concentration of the coreactant is 10mM or more.
15. The detection method according to any one of claims 7 to 14, wherein the concentration of the specific antibody is 1 μg/mL or more
16. The detection method according to any one of claims 7 to 15, wherein the incubation time after the addition of the specific antibody is 10 minutes or longer.
17. The detection method according to any one of claims 7 to 16, wherein the concentration of the electrochemiluminescence nano-beacon is 4 μg/mL or more.
18. The detection method according to any one of claims 7 to 17, wherein the incubation time after addition of the electrochemiluminescent nanobeacons is 15 minutes or more.
19. A kit for use in the detection method of any one of claims 7 to 18, comprising the electrochemiluminescent nanobeacon of claim 6.
20. A nanoparticle for an electrochemiluminescence nanobeacon doped with a metal complex ion.
21. An electrochemiluminescent nanobeacon comprising an inorganic oxide nanoparticle doped with metal complex ions and a secondary antibody bound to the inorganic oxide nanoparticle.
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| CN202210592343.6A CN117169494A (en) | 2022-05-27 | 2022-05-27 | ECL detection method for nucleic acid specific site modification, ECL nano beacon, preparation method thereof and kit |
| JP2023087169A JP2023174612A (en) | 2022-05-27 | 2023-05-26 | Electrochemiluminescent nanoprobe, preparation method thereof, electrochemiluminescence detection method for nucleic acid-specific site modification, kit for electrochemiluminescence detection method using antibody, and nanoparticle for electrochemiluminescent nanoprobe |
| US18/325,445 US20230384300A1 (en) | 2022-05-27 | 2023-05-30 | Electrochemiluminescent nanoprobe, preparation method thereof, electrochemiluminescence detection method for nucleic acid specific site modification, kit for electrochemiluminescence detection method using antibody, and nanoparticle for electrochemiluminescent nanoprobe |
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