Electrochemical luminescence probe, electrochemical luminescence sensor and application thereof

Abstract

The invention provides an electrochemical luminescence probe, an electrochemical luminescence sensor and application thereof.The electrochemical luminescence probe comprises an electrochemical luminescence part, a nucleic acid aptamer and a light absorption part which are connected in sequence, and the luminescence spectrum of the electrochemical luminescence part and the light absorption spectrum of the light absorption part are at least partially overlapped; the nucleic acid aptamer is used for being specifically combined with a biomarker to be detected; wherein under the condition that the nucleic acid aptamer is specifically combined with the biomarker, the nucleic acid aptamer is separated from the electrochemical luminescence part. According to the electrochemical luminescence probe, the electrochemical luminescence sensor and the application of the electrochemical luminescence probe, the detection accuracy is improved, and the lowest detection limit is increased.

Description

Technical Field

[0001] The present application relates to the field of biological detection, and in particular to an electrochemiluminescent probe, an electrochemiluminescent sensor and applications thereof. Background Art

[0002] Biomarker refers to a biochemical indicator that can mark changes or possible changes in the structure or function of a system, organ, tissue, cell, or subcellular structure. In the related art, a variety of methods have been developed for the detection of biomarkers, such as chromatography, electrochemistry, fluorescence, colorimetry, chemiluminescence, etc. Among them, electrochemiluminescence (ECL) is a combination of electrochemistry and optics. Compared with other optical-based detection methods, electrochemiluminescence does not require an external light source, has almost no background signal, and has good spatiotemporal controllability. However, when the concentration of the biomarker to be tested in the biological sample is low, the detection signal generated by electrochemiluminescence is easily interfered, reducing the accuracy of the detection.

[0003] Therefore, the accuracy of electrochemiluminescence detection used to detect biomarkers in the related art needs to be improved. Summary of the invention

[0004] The present application provides an electrochemiluminescent probe, an electrochemiluminescent sensor and applications thereof, aiming to provide an electrochemiluminescent probe with high accuracy.

[0005] In a first aspect, the present application provides an electrochemiluminescent probe, which comprises an electrochemiluminescent portion, a nucleic acid aptamer and a light absorbing portion connected in sequence, wherein the luminescence spectrum of the electrochemiluminescent portion at least partially overlaps with the light absorbing spectrum of the light absorbing portion, and the nucleic acid aptamer is used to specifically bind to a biomarker to be detected;

[0006] When the aptamer specifically binds to the biomarker, the aptamer is separated from the electrochemiluminescent part.

[0007] In some embodiments, the nucleic acid aptamer is a single-stranded polynucleic acid, and the nucleic acid aptamer includes one or more of deoxyribonucleic acid, ribonucleic acid, and nucleic acid analogs.

[0008] In some embodiments, the biomarker is one of a nucleic acid and a protein.

[0009] In some embodiments, the nucleic acid aptamer meets any of the following requirements:

[0010] (1) The nucleic acid aptamer is a CD63 nucleic acid aptamer, and the sequence of the CD63 nucleic acid aptamer is shown in SEQ ID NO.1;

[0011] (2) the nucleic acid aptamer is a CD9 nucleic acid aptamer, and the sequence of the CD9 nucleic acid aptamer is shown in SEQ ID NO.2;

[0012] (3) The nucleic acid aptamer is a CD81 nucleic acid aptamer, and the sequence of the CD81 nucleic acid aptamer is shown in SEQ ID NO.3.

[0013] In some embodiments, the light absorbing part includes one or more of methylene blue, ascorbic acid, diphenyl ether, ferrocene, trivalent iron ions, and divalent copper ions.

[0014] In some embodiments, the electrochemical luminescence portion is a single-atom electrochemical luminescence portion, which includes a MXenes structure with atomic vacancies and a luminescent metal element nucleated at the atomic vacancies, and the nucleic acid aptamer is connected to the MXenes structure.

[0015] In some embodiments, the electrochemical luminescent moiety is represented by the chemical formula M' a -R z -M n+1-x X n T y express,

[0016] Wherein, M is a transition metal element, X is selected from carbon or nitrogen, n is 1 or 2, x>0, and M and X form a MXenes structure containing M atomic vacancies;

[0017] M' is one or more luminescent metal elements selected from Pt, Ru, Au, Ag, Rh, Pd, M' occupies the vacancy of M atom, 0<a≤x;

[0018] R is an organic ligand group that binds to M', z>0;

[0019] T is an oxygen-containing or F-containing functional group bound to the surface of the MXenes structure, and y>0.

[0020] In some embodiments, the transition metal is selected from one or more of Sc, Ti, Zr, V, Nb, Cr, Mo, and Hf.

[0021] In some embodiments, the electrochemical luminescent portion is composed of the chemical formula Pt a -R z -Ti 3-x C 2 T y express,

[0022] Wherein, R is an organic ligand group that binds to Pt, and z>0;

[0023] T is an oxygen-containing or F-containing functional group bound to the surface of the MXenes structure, and y>0.

[0024] In a second aspect, the present application provides an electrochemiluminescent sensor, comprising:

[0025] The electrochemiluminescent probe provided according to the first aspect;

[0026] Glassy carbon electrode loaded with electrochemiluminescent probe.

[0027] In a third aspect, the present application provides an application of the electrochemiluminescence sensor provided in the second aspect in detecting biomarkers carried by exosomes, wherein the biomarkers are polynucleotides or proteins.

[0028] In the electrochemiluminescent probe, electrochemiluminescent sensor and application thereof provided in the present application, by setting the connection between the nucleic acid aptamer and the electrochemiluminescent part, when the nucleic acid aptamer specifically binds to the biomarker to be detected, the configuration of the nucleic acid aptamer itself will change accordingly, thereby detaching from the electrochemiluminescent part, so that the electrochemiluminescent probe can produce structural changes for the biomarker to be detected and identify the biomarker to be detected; by setting the luminescence spectrum of the electrochemiluminescent part and the absorption spectrum of the light absorption part to at least partially overlap, when the electrochemiluminescent part, the nucleic acid aptamer and the light absorption part are connected in sequence, the light absorption part can absorb the ECL signal generated by the electrochemiluminescent part to form ECL signal quenching, and when the electrochemiluminescent part and the nucleic acid aptamer are detached, the ECL signal generated by the electrochemiluminescent part is restored, so that the ECL signal can be detected to achieve the detection of the biomarker to be detected; when the nucleic acid aptamer specifically binds to the biomarker to be detected, the ECL signal generated by the electrochemiluminescent part is restored, thereby improving the recognition specificity of the electrochemical probe for the biomarker, avoiding or reducing the interference of other substances other than the biomarker on the ECL signal, and improving the accuracy of detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present application and, together with the description, serve to explain the principles of the present application.

[0030] Figure 1 This is an ECL signal intensity-potential curve of the glassy carbon electrode carrying different substances in the embodiment of the present application.

[0031] Figure 2 The absorption spectra of MB molecules and Pt-Cp-Ti 3-x C 2 T y Luminescence spectra of MXenes.

[0032] Figure 3 This is an ECL signal intensity-potential curve diagram of the glassy carbon electrode carrying different substances in the embodiment of the present application;

[0033] Figure 4 This is a curve diagram of the ECL quenching degree of MB-CD63 aptamers at different concentrations in the examples of this application;

[0034] Figure 5 It is a line graph of the ECL signal intensity of exosome samples with the same concentration and different incubation times in the examples of the present application;

[0035] Figure 6 The ECL signal intensity-potential curve of exosome samples with different concentrations in the embodiment of the present application;

[0036] Figure 7 This is a linear regression graph of the ECL signal intensity of exosome samples with different concentrations in the examples of this application;

[0037] Figure 8 This is a bar graph of the ECL signal intensity of exosome samples extracted from different cell lines in the examples of this application.

[0038] The above drawings have shown clear embodiments of the present application, which will be described in more detail later. These drawings and text descriptions are not intended to limit the scope of the present application in any way, but to illustrate the concept of the present application to those skilled in the art by referring to specific embodiments. DETAILED DESCRIPTION

[0039] The various embodiments or implementation schemes in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments.

[0040] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "illustrative embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials, or characteristics described in conjunction with the implementation or examples are included in at least one implementation or example of the present application. In this specification, the schematic representation of the above terms does not necessarily refer to the same implementation or example. Moreover, the specific features, structures, materials, or characteristics described may be combined in any one or more implementations or examples in a suitable manner.

[0041] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In the description of this application, the meaning of "plurality" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

[0042] In the related art, electrochemiluminescence (ECL) is used to detect biomarkers. When the concentration of the biomarker to be detected in the biological sample is low, the detection signal is easily interfered. For example, exosomes are a type of tiny extracellular vesicles that carry biomarkers from cells. When using ECL technology to detect biomarkers in exosomes, there is a problem that the detection limit is not low enough, especially when the concentration of exosomes in the biological sample is low, the signal is easily interfered.

[0043] Therefore, the inventors have developed a new electrochemiluminescent probe to solve the technical problem that the detection signal is easily disturbed when using ECL technology to detect biomarkers with low concentrations. The electrochemiluminescent probe, electrochemiluminescent sensor and their applications provided by the present application are described in detail below.

[0044] In a first aspect, an electrochemiluminescent probe is provided, which includes an electrochemiluminescent part, a nucleic acid aptamer and a light absorbing part connected in sequence, the luminescence spectrum of the electrochemiluminescent part at least partially overlaps with the light absorbing spectrum of the light absorbing part, and the nucleic acid aptamer is used to specifically bind to a biomarker to be detected; wherein, when the nucleic acid aptamer specifically binds to the biomarker, the nucleic acid aptamer is separated from the electrochemiluminescent part.

[0045] The electrochemiluminescent part is the part of the electrochemiluminescent probe that can emit light based on ECL. By applying a certain electrical signal to the chemical system containing the electrochemiluminescent part, the electrochemiluminescent part produces a new substance, which can react with the chemical substances existing in the system or undergo a decomposition reaction itself. The reaction not only provides sufficient energy, but also produces a suitable luminophore and accepts the released energy of the reaction to form an excited state luminophore. When the unstable excited state returns to the ground state, it emits emission light consistent with the properties of the luminophore. The luminescence spectrum or luminescence intensity can be measured by ordinary optical means to perform trace analysis of the substance.

[0046] Nucleic acid aptamers can bind specifically and selectively to the biomarker to be tested. When the nucleic acid aptamer binds specifically to the target substance, the configuration of the nucleic acid aptamer itself will change accordingly. The nucleic acid aptamer can be connected and fixed to the electrochemical luminescent part through hydrogen bonding, chelation, adsorption, etc. When the nucleic acid aptamer binds specifically to the biomarker to be tested, the configuration of the nucleic acid aptamer itself will change accordingly, thereby detaching from the electrochemical luminescent part.

[0047] The light-absorbing part can absorb light waves of a specific wavelength. The luminescence spectrum of the electrochemiluminescent part overlaps at least partially with the light-absorbing spectrum of the light-absorbing part, that is, when the electrochemiluminescent part emits a light wave, the light-absorbing part can absorb all or part of the light wave, thereby generating an ECL signal in which the light-absorbing part quenches the electrochemiluminescent part. When the biomarker to be tested is present in the biological sample, the electrochemiluminescent probe recognizes the biomarker, the nucleic acid aptamer binds to the biomarker to be tested and desorbs from the electrochemiluminescent part, causing the ECL signal to recover; when the biomarker to be tested is not present in the biological sample, the nucleic acid aptamer remains connected to the electrochemiluminescent part, and the light-absorbing part causes the ECL signal to be quenched.

[0048] In the present application scheme, by setting the connection between the nucleic acid aptamer and the electrochemical luminescent part, when the nucleic acid aptamer specifically binds to the biomarker to be detected, the configuration of the nucleic acid aptamer itself will change accordingly, thereby detaching from the electrochemical luminescent part, so that the electrochemical luminescent probe can produce structural changes for the biomarker to be detected and identify the biomarker to be detected; by setting the luminescence spectrum of the electrochemical luminescent part and the absorption spectrum of the light absorption part to at least partially overlap, when the electrochemical luminescent part, the nucleic acid aptamer and the light absorption part are connected in sequence, the light absorption part can absorb the ECL signal generated by the electrochemical luminescent part to form ECL signal quenching, and when the electrochemical luminescent part and the nucleic acid aptamer are detached, the ECL signal generated by the electrochemical luminescent part is restored, so that the ECL signal can be detected to achieve the detection of the biomarker to be detected; when the nucleic acid aptamer specifically binds to the biomarker to be detected, the ECL signal generated by the electrochemical luminescent part is restored, thereby improving the recognition specificity of the electrochemical probe for the biomarker, avoiding or reducing the interference of other substances other than the biomarker on the ECL signal, and improving the accuracy of detection.

[0049] In some embodiments, the nucleic acid aptamer is a single-stranded polynucleic acid, and the nucleic acid aptamer includes one or more of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and nucleic acid analogs.

[0050] Nucleic acid aptamers are single-stranded polynucleic acids. Nucleic acid aptamers may include phosphate groups, and can selectively adsorb to the electrochemical luminescent portion through the phosphate groups, so that the nucleic acid aptamer is connected to the electrochemical luminescent portion. Single-stranded polynucleic acids include multiple bases arranged along the required sequence. The base sequence in the nucleic acid aptamer can be designed according to the type of biomarker to be detected, the structure of the peptide chain, the amino acid sequence, the base sequence, etc., so that the nucleic acid aptamer can specifically bind to the biomarker to be identified.

[0051] Nucleic acid analogs include, but are not limited to, one or more of arabino nucleic acids (ANA), bridged nucleic acids (BNA), cyclohexenyl nucleic acids (CeNA), 2'-fluoroarabino nucleic acids (FANA), glycol nucleic acids (GNA), hexose nucleic acids (HNA), locked nucleic acids (LNA), morpholino nucleic acids (Morpholino), peptide nucleic acids (PNA), and threose nucleic acids (TNA). Nucleic acid analogs retain bases, so that nucleic acid analogs can recognize the biomarker to be tested through a specific base sequence and achieve specific binding with the biomarker to be tested. Compared with DNA and RNA, nucleic acid analogs replace phosphate groups and ribose groups, so that nucleic acid aptamers prepared using nucleic acid analogs have different physical and chemical properties, which is conducive to improving the connection between nucleic acid aptamers and electrochemical luminescent parts and light absorption parts.

[0052] The specificity of nucleic acid aptamers is related to the base sequence, while the connection between nucleic acid aptamers and electrochemical luminescent parts is independent of the base sequence, so the same electrochemical luminescent part can be connected to nucleic acid aptamers with different base sequences, and nucleic acid aptamers with different base sequences can be specifically identified with different biomarkers. Those skilled in the art can design nucleic acid aptamers with specificity according to the biomarkers to be detected, and then connect nucleic acid aptamers with different base sequences to electrochemical luminescent parts with the same structure, thereby obtaining electrochemical luminescent probes for detecting different biomarkers.

[0053] In some embodiments, the biomarker is one of a nucleic acid and a protein.

[0054] When the biomarker is a nucleic acid, a base-complementary aptamer can be designed according to the base sequence of the biomarker, and the biomarker and the aptamer combine to form a double-stranded structure. When the biomarker is a protein, a aptamer can be designed according to the amino acid sequence and protein structure of the biomarker, and the biomarker and the aptamer combine to form a protein-nucleic acid complex.

[0055] In some embodiments, the nucleic acid aptamer meets any of the following requirements:

[0056] (1) The nucleic acid aptamer is a CD63 nucleic acid aptamer, and the sequence of the CD63 nucleic acid aptamer is shown in SEQ ID NO.1:

[0057] 5′-CAC CCC ACC TCG CTC CCG TGA CAC TAA TGC TA-3′;

[0058] (2) The nucleic acid aptamer is a CD9 nucleic acid aptamer, and the sequence of the CD9 nucleic acid aptamer is shown in SEQ ID NO.2:

[0059] 5′-GAC AAC AAG ATG AGA TTG CAC ATC ATC CGG ACC ATG TTT GAC-3′;

[0060] (3) The nucleic acid aptamer is a CD81 nucleic acid aptamer, and the sequence of the CD81 nucleic acid aptamer is shown in SEQ ID NO.3:

[0061] 5′-TAG GGA AGAGAA GGA CAT ATG AT-40N-TTG ACT AGT ACA TGA CCA CTT GA-3′.

[0062] CD63 nucleic acid aptamers are used to specifically recognize CD63 proteins in exosomes. CD63 protein is a key factor in regulating exosome production and endosomal cargo sorting. The CD63 gene encoding CD63 protein is located on human chromosome 12q13 and encodes a protein containing 237 amino acids. CD63 protein can be used as a biomarker for non-invasive detection and real-time monitoring of early cancer. The detection of CD63 in exosomes of patients with viral infection can also monitor viral load and assist in the early diagnosis of viral infection.

[0063] CD9 nucleic acid aptamers are used to specifically recognize CD9 proteins in exosomes. CD9 proteins play an important regulatory role in cell adhesion, cell movement, activation, differentiation, etc. It is an important protein molecule that mediates sperm-egg plasma membrane fusion. CD9 proteins are closely related to the formation, release and function of exosomes. CD9 proteins can be used as biomarkers for the detection of breast cancer, prostate cancer, colorectal cancer, lung cancer and other cancers.

[0064] CD81 nucleic acid aptamers are used to specifically recognize CD81 proteins in exosomes. CD81 is a potential antiviral and anticancer drug target. It is a co-receptor for some viruses, such as hepatitis C virus (HCV) and human immunodeficiency virus (HIV). It also plays an important role in the occurrence and metastasis of tumors. CD81 protein can be used as a biomarker for hepatitis C virus, B cell lymphoma, other hematological diseases and non-hematological malignant tumor cells.

[0065] In some embodiments, the light absorbing part includes methylene blue (MB), ascorbic acid, diphenyl ether, ferrocene, trivalent iron ion (Fe +3 ), divalent copper ion (Cu +2 )

[0066] In the electrochemiluminescent probe used to detect the same biomarker, each nucleic acid aptamer can be connected to different types of light absorbing parts, and can also be connected to at least two biomarkers at the same time. For example, methylene blue and ascorbic acid are simultaneously connected to the CD63 nucleic acid aptamer.

[0067] Exemplarily, the light absorbing part is methylene blue, and the methylene blue is connected to the 3' end of the nucleic acid aptamer.

[0068] Optionally, the luminescence spectrum of the electrochemical luminescence part is located within the absorption spectrum of the light absorption part, so that light of a specific wavelength emitted by the electrochemical luminescence part under the action of an electrical signal can be absorbed by the light absorption part.

[0069] In some embodiments, the electrochemical luminescent portion is a single-atom electrochemical luminescent portion, which includes a MXenes structure with atomic vacancies and a luminescent metal element nucleated in the atomic vacancies, and the nucleic acid aptamer is connected to the MXenes structure.

[0070] MXenes are two-dimensional nanostructures composed of transition metal carbides, nitrides, and carbonitrides. The structure is a two-dimensional nanolayer with a thickness of several atoms. It mainly uses MAX phase as the main precursor. By etching the A element, the MXenes structure with atomic vacancies is obtained. It has a good specific surface area with good conductivity and is a good carrier in the field of electrochemiluminescence sensing.

[0071] In the present application, by loading luminescent metal atoms on a MXenes structure with atomic vacancies, the luminescent metal atoms occupy the atomic vacancies on the MXenes structure, which can effectively avoid the agglomeration of the luminescent metal atoms. The MXenes structure as a carrier has good conductivity and a large specific surface area, and can be applied to electrochemiluminescence sensors. The excited free radicals on the electrochemiluminescence probe are not prone to self-quenching, which enhances the electrochemiluminescence intensity and enables sensitive detection of biomarkers.

[0072] In some embodiments, the electrochemical luminescent moiety is represented by the chemical formula M' a -R z -M n+1-x X n T y express,

[0073] Wherein, M is a transition metal element, X is selected from carbon or nitrogen, n is 1 or 2, x>0, and M and X form a MXenes structure containing M atomic vacancies;

[0074] M' is one or more luminescent metal elements selected from Pt, Ru, Au, Ag, Rh, Pd, M' occupies the vacancy of M atom, 0<a≤x;

[0075] R is an organic ligand group that binds to M', z>0;

[0076] T is an oxygen-containing or F-containing functional group bound to the surface of the MXenes structure, and y>0.

[0077] In this application, M in the chemical formula n+1-x X n T y It represents the MXenes structure containing M atomic vacancies. The material is synthesized with MAX phase as the main precursor, where M is a transition metal, A is any element of group IIIA or IVA, and X is carbon or nitrogen. By etching the A element in the MAX phase, a chemical formula of M is obtained. n+1-x X n T y MXenes structure.

[0078] Among them, x can be expressed as the concentration of M atom vacancies in the MXenes structure. During the etching of element A, some MA bonds are damaged, resulting in some M atoms falling off during the etching process, thus containing a certain concentration of M atom vacancies in the MXenes structure. It is understandable that the concentration of M atom vacancies is difficult to measure directly, so the specific range of x is not easy to determine, and x>0 indicates that there are M atom vacancies in the MXenes structure.

[0079] T represents oxygen-containing or F-containing functional groups bound to the surface of the MXenes structure. The specific type of the functional group is related to the etchant. Oxygen-containing or F-containing functional groups can make the MXenes structure have good hydrophilicity and are more useful in the field of electrochemiluminescent sensors. y represents the content of oxygen-containing or F-containing functional groups on the electrochemiluminescent part. It is also understandable that the specific range of y is not easy to determine. y>0 indicates that there are oxygen-containing or F-containing functional groups on the electrochemiluminescent part.

[0080] In the chemical formula, M' is a luminescent metal element, i.e., a luminophore as an electrochemiluminescent probe, which can occupy the M atomic vacancies on the MXenes structure. At the same time, since the MXenes structure has a large specific surface area, it can effectively prevent the aggregation of luminescent metals, and thus the free radicals are not easily self-quenched, thereby enhancing the electrochemiluminescence intensity. M' can be selected from one or more of Pt, Ru, Au, Ag, Rh, and Pd according to actual conditions.

[0081] In the general chemical formula, a represents the amount of substitution of the luminescent metal element for the M atom vacancies in the MXenes structure. It is understandable that the substitution amount is not easy to measure, but the substitution amount of the luminescent metal element for the M atom vacancies will not exceed the concentration of the M atom vacancies, that is, 0<a≤x, indicating that the luminescent metal element can completely or partially replace the M atom vacancies in the MXenes structure.

[0082] In the general chemical formula, R represents an organic ligand group bound to M'. Since it is difficult to obtain a single atomic metal from a luminescent metal element due to a strong metallic bond, a luminescent metal precursor is required to obtain a single atomic metal, thereby further occupying the M atomic vacancies on the MXenes structure. The luminescent metal precursor is generally a luminescent metal organic compound in which a luminescent metal is bound to an organic ligand. During the deposition of the luminescent metal precursor on the MXenes structure, the luminescent metal precursor will break and some organic ligands will fall off. Therefore, some organic ligands are also bound to the luminescent metal elements on the MXenes structure. It can be understood that the specific type of organic ligand group is related to the luminescent metal precursor and has no effect on the structure of the electrochemical luminescent part. z represents the content of the organic ligand group brought by M' on the electrochemical luminescent part, which is also difficult to quantify and is related to the process in the preparation process. z>0 indicates that there is an organic ligand group bound to M' on the electrochemical luminescent part. In this application, unless otherwise specified, the luminescent metal precursor may refer to the luminescent metal precursor molecule itself, or to a molecule from which some organic ligand groups fall off during atomic layer deposition. The structure of the luminescent metal precursor changes with the temperature of atomic layer deposition.

[0083] In some embodiments of the present application, the transition metal element is selected from one or more of Sc, Ti, Zr, V, Nb, Cr, Mo, and Hf.

[0084] In some of the above embodiments, specific types of transition metal elements in the MXenes structure are specifically listed. The transition metal elements and the X element together constitute the skeleton of the MXenes structure. The above types of transition metals all have good electrical conductivity, which can ensure that the electrochemical luminescence part also has good electrical conductivity.

[0085] In some embodiments of the present application, the electrochemical luminescent portion is composed of the chemical formula Pt a -R z -Ti3-x C 2 T y express,

[0086] Wherein, R is an organic ligand group that binds to Pt, and z>0;

[0087] T is an oxygen-containing or F-containing functional group bound to the surface of the MXenes structure, and y>0.

[0088] In some of the above embodiments, the elements in the chemical formula of the electrochemiluminescent portion are further defined, and a MXenes structure with Ti vacancies is formed by Ti and C, thereby obtaining an electrochemiluminescent portion with good conductivity; Pt, as a luminescent metal element, has high catalytic activity, which can ensure that the electrochemiluminescent portion has a strong electrochemiluminescence intensity.

[0089] When preparing the electrochemical luminescent part, a chemical formula of M can be provided first. n+1 AX n The MAX phase material is etched to remove the A element in the MAX phase material, and the MA bond is damaged to obtain a MXenes structure containing atomic vacancies, and then an atomic layer deposition method is used to embed a luminescent metal precursor in the atomic vacancies. In some embodiments, the luminescent metal precursor includes (trimethyl) methylcyclopentadienyl platinum (strem chemicals, MeCpPtMe 3 ), bis(ethylcyclopentadienyl)ruthenium. The chemical formula of MAX phase material is Ti 3 AlC 2 .

[0090] In a second aspect, the present application provides an electrochemiluminescent sensor, comprising:

[0091] A glassy carbon electrode loaded with an electrochemiluminescent probe according to any embodiment of the first aspect.

[0092] In the present application, the electrochemiluminescent probe according to any embodiment of the first aspect can be loaded on the surface of the glassy carbon electrode and used as a working electrode to obtain an electrochemiluminescent sensor. It can be understood that the electrochemiluminescent sensor has the beneficial effects of the first aspect described above.

[0093] In some embodiments, the electrochemiluminescent probe according to any embodiment of the first aspect is ultrasonically dispersed in water to obtain a suspension with a concentration of 1 to 5 mg / mL, 1 to 10 μL of the suspension is dropped onto the surface of a pre-polished glassy carbon electrode, and the electrode is incubated and dried to obtain a glassy carbon electrode carrying the electrochemiluminescent probe.

[0094] In a third aspect, the present application provides the use of the electrochemiluminescent sensor according to the second aspect in detecting biomarkers carried by exosomes, wherein the biomarkers are polynucleotides or proteins.

[0095] Nucleic acid aptamers can bind specifically and selectively to the biomarker to be tested. When the nucleic acid aptamer binds specifically to the target biomarker, the configuration of the nucleic acid aptamer itself will change accordingly, thereby detaching from the electrochemical luminescence part. The ECL signal emitted by the electrochemical luminescence part is restored, so that the ECL signal can be detected to detect whether the biological sample contains the target biomarker, and the concentration of the target biomarker in the biological sample can also be judged based on the intensity of the ECL signal.

[0096] Optionally, the biomarkers may be CD9 protein, CD63 protein, or CD81 protein carried by exosomes.

[0097] Hereinafter, the embodiments of the present application will be described. The embodiments described below are exemplary and are only used to explain the present application, and should not be construed as limiting the present application. If no specific techniques or conditions are indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. If the manufacturer is not indicated in the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0098] Example 1

[0099] (1) Tiv-Ti with atomic vacancies 3-x C 2 T y Preparation of MXenes structures.

[0100] In a polytetrafluoroethylene container, 1.2 g LiF was dissolved in 10 mL 9 mol / L hydrochloric acid, and then 0.5 g MAX phase material Ti was added under stirring. 3 AlC 2 Powder (particle size <38 μm), react at 35 ° C for 24 h to obtain clay-like Tiv-Ti 3-x C 2 T y MXenes suspension; clay-like Tiv-Ti 3-x C 2 T y The MXenes suspension was centrifuged and the precipitate was collected and washed with water until the pH was 6-7. During this process, the Ti-Al bond was damaged and some Ti atoms were peeled off. 3-x C 2 T y MXenes structures are negatively charged and hydrophilic due to their surface functional groups (e.g., -O, -OH, and -F).

[0101] The washed precipitate was redispersed to obtain a MXenes suspension, which was then ultrasonically treated for 2 h under argon flow protection and then centrifuged at 3500 rpm for 1 h to obtain Tiv-Ti 3-x C 2 T y MXenes homogenized supernatant and directly stored the homogenized supernatant at 4 °C for later use.

[0102] (2) Electrochemical luminescence part Pt-Cp-Ti 3-x C 2 T y Preparation of MXenes:

[0103] 2 mg / mL Tiv-Ti 3-x C 2 T y MXenes were dispersed in 200 mL of water and ultrasonicated for half an hour. 2 mL was dripped onto a quartz glass sheet and placed in the deposition chamber of an atomic layer deposition device (Savannah 100, Cambridge Nanotechnology Inc., USA) after drying. The temperature was set to 65 °C and nitrogen (purity: 99.9995%) was continuously blown into the atomic layer deposition chamber.

[0104] Then, nitrogen gas is used as a carrier to blow the vaporized luminescent metal precursor MeCpPtMe into the atomic layer deposition chamber in a pulsed mode. 3 The pulse cycle includes: 1s blowing stage, 5s holding stage, 20s pumping stage, the pulse cycle cycle number is 5 times, the nitrogen flow rate is 50sccm, and the total amount of MeCpPtMe3 blown is 10mg;

[0105] Ozone was continuously blown into the atomic layer deposition chamber for 30 min, and the oxygen flow rate was 0.5 L / min to obtain the electrochemical luminescence part Pt-Cp-Ti 3-x C 2 T y MXenes.

[0106] (3) Preparation of glassy carbon electrode carrying electrochemiluminescence probe.

[0107] The prepared electrochemical luminescent part Pt-Cp-Ti 3-x C 2 T y MXenes were ultrasonically dispersed in water to obtain a suspension with a concentration of 1 mg / mL. 3 μL of the suspension was dropped onto the surface of a pre-polished glassy carbon electrode, and the glassy carbon electrode carrying the electrochemical luminescence part was obtained after incubation and drying.

[0108] Please refer to Table 1. Design and synthesize nucleic acid aptamers according to the biomarkers to be detected, connect the light absorbing part MB molecule to the nucleic acid aptamer, and connect the connected nucleic acid aptamer and light absorbing part to the electrochemical luminescence part.

[0109]

[0110] The ECL intensity test uses a three-electrode system, where the working electrode is a glassy carbon electrode carrying an electrochemiluminescent probe, the reference electrode is Ag / AgCl (saturated KCl), and the counter electrode is a platinum wire placed in a working solution of potassium ferrocyanide solution.

[0111] Embodiment 2:

[0112] Using tripropylamine (TPrA) as a coreaction reagent, the electrochemiluminescence of the glassy carbon electrode carrying the electrochemiluminescent portion and the glassy carbon electrode carrying the chemiluminescent probe prepared in Example 1 were tested.

[0113] See also Figure 1 , where curve a is the ECL signal of the glassy carbon electrode carrying the electrochemiluminescent part, and curve b is the ECL signal of the glassy carbon electrode carrying the chemiluminescent probe. From curve a and curve b, it can be seen that the quenching efficiency of the glassy carbon electrode carrying the chemiluminescent probe on the ECL signal reaches 99.8%.

[0114] See also Figure 2 , which are the absorption spectra of MB molecules in the absorption part and Pt-Cp-Ti molecules in the electrochemical luminescence part 3-x C 2 T y Luminescence spectrum of MXenes. The absorption part MB has obvious absorption peaks at about 610nm and 666nm. 3-x C 2 T y MXenes show a broad ECL signal emission peak at 625 nm, the absorption spectrum of MB in the absorption part and the electrochemical luminescence spectrum of Pt-CpTiv-Ti in the electrochemical luminescence part. 3-x C 2 T y The emission spectra of MXenes have significant overlap. Figure 2 It can be seen that Pt-Cp-Ti 3-x C 2 T y The ECL signal emitted by MXenes can be absorbed by MB molecules, thereby quenching the electrochemiluminescence of the electrochemiluminescence part.

[0115] Example 3: Isolation and counting of exosomes

[0116] Human breast cancer cell line (MCF-7 cells), human breast ductal carcinoma cell line (BT474 cells) and HeLa cell line were cultured in a culture medium containing 10% exosome-free fetal bovine serum (FBS) and 1% penicillin / streptomycin, and then the culture medium was placed in an incubator with 5% carbon dioxide and 37°C. After 48 hours of cell culture, when the cell concentration reached 70%, the culture supernatant containing exosomes was collected.

[0117] Exosomes were isolated from the collected culture supernatant at 4°C. 300 mL of the culture supernatant was centrifuged at 500 g for 10 min, then at 2000 g for 20 min, filtered through a 0.8 μm pore size membrane, centrifuged at 10,000 g for 90 min, then centrifuged at 3,000 g for 15 min, then filtered through a 0.2 μm pore size membrane, and ultracentrifuged at 100,000 g for 90 min to obtain exosome samples.

[0118] Nanoparticle tracking analysis (NTA) was used to count the exosome samples to determine the recovery rate, separation purity, and particle size distribution of the exosomes. The exosome samples were diluted to a particle concentration of 2×10 8 and 1×10 9 paticles / mL and stored at -20℃ until used.

[0119] The isolated exosome samples were observed. The exosomes isolated from BT474 showed a round or oval vesicle structure with a coating membrane and a double lipid layer vesicle structure. The NTA analysis results showed that the size of the vesicles isolated from BT474 was mainly distributed in the range of 30-200nm, with an average vesicle diameter of 107±3nm. The exosomes isolated from MCF-7 showed a round vesicle structure, and the size of the isolated vesicles was mostly in the range of 30-300nm, with an average vesicle size of 117±3nm. The exosomes isolated from HeLa cells showed a round vesicle structure. The size of the isolated vesicles was mostly in the range of 30-300nm, with an average vesicle size of 123±3nm.

[0120] Example 4

[0121] The glassy carbon electrode carrying the chemiluminescent probe prepared in Example 1 was immersed in the exosome solution separated from MCF-7 in Example 3, and the exosome concentration was 5.0×10 2 particlesμL –1 . See Figure 2 , where curve c is MB-CD63 aptamer / Pt-Cp-Ti 3-x C 2 T yThe electrochemiluminescence intensity of MXenes modified electrode; Curve d is when the exosome concentration increases to 5.0×10 5 particlesμL –1 When MB-CD63 aptamer / Pt-Cp-Ti 3-x C 2 T y Electrochemical luminescence intensity of MXenes modified electrode. From curves c and d, we can see that MB-CD63 aptamer / Pt-Cp-Ti 3-x C 2 T y After the nucleic acid aptamers on the surface of MXenes bind to the CD63 protein in the exosomes, the MB-CD63 aptamer is transferred from the Pt-Cp-Ti 3-x C 2 T y MXenes surface desorption, thus Pt-Cp-Ti 3-x C 2 T y The electrochemiluminescence of the MXenes-modified electrode gradually recovered.

[0122] Example 5

[0123] A control nucleic acid chain with a random sequence was designed, and the base sequence was: 5′-AGCTTAGCGTACGATTCGACTGACTATCGATG-3′, and the MB molecule was connected to the 3′ end of the control nucleic acid chain.

[0124] MB-control nucleic acid chain / Pt-Cp-Ti 3-x C 2 T y MXenes glassy carbon electrode, MB-CD9 aptamer / Pt-Cp-Ti prepared in Example 1 3-x C 2 T y MXenes glassy carbon electrodes and Pt-Cp-Ti 3-x C 2 T y The MXenes glassy carbon electrodes were immersed in the exosome solution separated from MCF-7 in Example 3, and the exosome concentration was 5.0×10 5 particlesμL –1 TPrA was used as a co-reactant to detect the ECL signal intensity.

[0125] See also Figure 3 , where curve a is the load Pt-Cp-Ti 3-x C 2 T yElectrochemical luminescence intensity of MXenes glassy carbon electrode, curve b is MB-control nucleic acid chain / Pt-Cp-Ti 3-x C 2 T y Electrochemical luminescence intensity of MXenes glassy carbon electrode; curve c is MB-CD9 aptamer / Pt-Cp-Ti 3-x C 2 T y Electrochemical luminescence intensity of MXenes modified electrode. From curves a and c, we can see that MB-CD9 aptamer / Pt-Cp-Ti 3-x C 2 T y After the nucleic acid aptamers on the surface of MXenes bind to the CD9 protein in the exosomes, the MB-CD9 aptamer is transferred from the Pt-Cp-Ti 3-x C 2 T y MXenes surface desorption, thus Pt-Cp-Ti 3-x C 2 T y The electrochemiluminescence of MXenes modified electrodes gradually recovered, and the MB-CD63 aptamer was 3-x C 2 T y The quenching efficiency of MXenes is 98.7%. 3-x C 2 T y The nucleic acid aptamers on the surface of MXenes cannot bind to the CD9 protein in the exosomes, making it impossible to restore the electrochemiluminescence.

[0126] Example 6

[0127] MB-CD63 aptamers with concentrations of 10 μM, 30 μM, 50 μM, and 100 μM were respectively 3-x C 2 T y MXenes glassy carbon electrode connection. Pt-Cp-Ti 3-x C 2 T y MXenes is Pt-Cp-Ti with a concentration of 1 mg / ml 3-x C 2 T y 3 μl of MXenes was dropped onto the surface of the glassy carbon electrode, and the electrode was incubated at 37 °C to dry.

[0128] See also Figure 4 , is a graph showing the ECL quenching degree of MB-CD63 aptamers at different concentrations. Figure 4 As shown, the degree of ECL quenching gradually increased with the increase of MB-CD63 aptamer concentration and reached more than 97% at 50 mM.

[0129] The exosome samples of the same concentration were mixed with MB-CD63\Pt-Cp-Ti 3-x C 2 T y The MXenes glassy carbon electrode was incubated for 10 min, 30 min, 60 min, 90 min and 120 min.

[0130] See also Figure 5 , is the ECL signal intensity line graph of exosome samples with the same concentration and different incubation times. Figure 5 As shown in the figure, the recovery degree of ECL luminescence intensity gradually increased with the increase of incubation time of exosome samples, reaching the maximum value at 60 min of incubation.

[0131] Exosome samples with different concentrations were respectively treated with MB-CD63\Pt-Cp-Ti 3-x C 2 T y The MXenes glassy carbon electrode was incubated for 60 min and the ECL luminescence intensity was detected.

[0132] like Figure 6 and Figure 7 As shown in Figure 2, the ECL signal intensity gradually increased with the increase of exosome concentration. 5 particlesμL –1 In the range of 1.5, the ECL signal intensity is linearly related to the logarithm of the exosome sample concentration (log(Cexo), and the linear equation is I(ECL signal intensity) = 377.5log[Cexo(exoμL –1 )] + 38.5, the correlation coefficient is 0.987 (n = 3). The minimum detection limit is calculated according to log (LOD) = (3θ-b) / k, where θ is the background standard deviation, b is 38.5, and k is 377.5), and the minimum detection limit is 35particlesμL –1 (S / N=3). It is proved that the electrochemiluminescence sensor provided has good sensitivity and can be used to detect low-concentration exosome samples.

[0133] Example 7

[0134] MB-CD63\Pt-Cp-Ti 3-x C 2 T yMXenes electrochemical sensors were used to detect exosome samples extracted from MCF-7 cells, BT474 cells and HeLa cells, respectively.

[0135] See also Figure 8 , which is a bar graph of the ECL signal intensity of exosome samples extracted from different cell lines. As can be seen from the figure, the ECL signal produced by exosomes from MCF-7 cells is the strongest, followed by the ECL signal of exosomes from BT474 cells, and the ECL signal of exosomes from HeLa cells is the weakest. This shows that exosomes produced by breast cancer cell lines usually show a higher level of CD63 protein expression, while the expression level of CD63 protein in exosomes produced by HeLa cells is relatively low, which is consistent with the expression of CD63 protein in cancers such as breast cancer. Based on MB-CD63\Pt-Cp-Ti 3-x C 2 T y MXenes electrochemical sensors can be used for analysis of exosomes and their surface protein expression.

[0136] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit it. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. An electrochemiluminescent probe, characterized in that: The electrochemiluminescent probe comprises an electrochemiluminescent part, a nucleic acid aptamer and a light absorbing part connected in sequence, the luminescence spectrum of the electrochemiluminescent part at least partially overlaps with the light absorbing spectrum of the light absorbing part, and the nucleic acid aptamer is used to specifically bind to the biomarker to be detected; When the nucleic acid aptamer specifically binds to the biomarker, the nucleic acid aptamer is separated from the electrochemical luminescence part.

2. The electrochemiluminescent probe according to claim 1, characterized in that The nucleic acid aptamer is a single-stranded polynucleic acid, and the nucleic acid aptamer includes one or more of deoxyribonucleic acid, ribonucleic acid, and nucleic acid analogs.

3. The electrochemiluminescent probe according to claim 1, characterized in that The biomarker is one of nucleic acid and protein.

4. The electrochemiluminescent probe according to claim 1, characterized in that The nucleic acid aptamer meets any of the following conditions: (1) The nucleic acid aptamer is a CD63 nucleic acid aptamer, and the sequence of the CD63 nucleic acid aptamer is shown in SEQ ID NO.1; (2) The nucleic acid aptamer is a CD9 nucleic acid aptamer, and the sequence of the CD9 nucleic acid aptamer is shown in SEQ ID NO.2; (3) The nucleic acid aptamer is a CD81 nucleic acid aptamer, and the sequence of the CD81 nucleic acid aptamer is shown in SEQ ID NO.

3.

5. The electrochemiluminescent probe according to claim 1, characterized in that The light absorbing part includes one or more of methylene blue, ascorbic acid, diphenyl ether, ferrocene, trivalent iron ions, and divalent copper ions.

6. The electrochemiluminescent probe according to claim 1, characterized in that The electrochemical luminescence part is a single-atom electrochemical luminescence part, which includes a MXenes structure with atomic vacancies and a luminescent metal element nucleated at the atomic vacancies, and the nucleic acid aptamer is connected to the MXenes structure.

7. The electrochemiluminescent probe according to claim 6, characterized in that The electrochemical luminescent part is represented by the chemical formula M' a -R z -M n+1-x X n T y express, Wherein, M is a transition metal element, X is selected from carbon or nitrogen, n is 1 or 2, x>0, and M and X form a MXenes structure containing M atomic vacancies; M' is one or more luminescent metal elements selected from Pt, Ru, Au, Ag, Rh, and Pd, and M' occupies the vacancy of the M atom, 0<a≤x; R is an organic ligand group that binds to M', z>0; T is an oxygen-containing or F-containing functional group bound to the surface of the MXenes structure, and y>0.

8. The electrochemiluminescent probe according to claim 7, characterized in that The transition metal element is selected from one or more of Sc, Ti, Zr, V, Nb, Cr, Mo, and Hf.

9. The electrochemiluminescent probe according to claim 7, characterized in that The electrochemical luminescent part has a chemical formula of Pt a -R z -Ti 3-x C2T y express, Wherein, R is an organic ligand group that binds to Pt, and z>0; T is an oxygen-containing or F-containing functional group bound to the surface of the MXenes structure, and y>0.

10. An electrochemiluminescence sensor, characterized in that: include: The electrochemiluminescent probe according to any one of claims 1 to 9; The glassy carbon electrode is loaded with the electrochemiluminescent probe.

11. Use of the electrochemiluminescence sensor according to claim 10 in detecting biomarkers carried by exosomes, wherein the biomarkers are polynucleotides or proteins.

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