Method for producing electrochemiluminescent nanoprobe, electrochemiluminescent nanoprobe, electrochemiluminescent sensor, method for detecting electrochemiluminescence, and kit for detecting electrochemiluminescence

Abstract

To provide an electrochemiluminescent nanoprobe that exhibits high sensitivity, high accuracy and specificity while offering high flexibility without the need for amplification.SOLUTION: A method for producing an electrochemiluminescent nanoprobe according to an embodiment includes: a Pdots nanoparticle synthesis step for polymerizing conjugated polymers and copolymer molecules to synthesize Pdots nanoparticles; and a Pdots nanoparticle modification step for using oligonucleotide strands with quencher molecule modifications to modify the obtained Pdots nanoparticles.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] SUMMARY OF THE PRESENT EMBODIMENTS The present invention relates to a method for producing an electrochemiluminescent nanoprobe, an electrochemiluminescent nanoprobe, an electrochemiluminescent sensor, an electrochemiluminescent detection method, and a kit for electrochemiluminescence detection. [Background technology]

[0002] Electrochemiluminescence (ECL) is an electrochemical energy relaxation process, and the excitation mode and signal detection are separated, so it has the characteristics of electrochemical controllability and low background. Due to the relatively simple time and space control of electrochemiluminescence technology, electrochemiluminescence imaging, which uses a charge-coupled device (CCD) camera to collect signals, has been rapidly developed and is used to analyze various target molecules, and is widely applied in gene toxin screening, immunoassay, fingerprint analysis, cell detection, and reaction mechanism research. The electrochemiluminescence detection method retains the advantages of the chemiluminescence method, such as high sensitivity, wide linear range, convenient observation, and simple equipment, and also has the incomparable advantages of the chemiluminescence method, such as high reproducibility, stable reagents, easy control, and some reagents can be used repeatedly.

[0003] Conventional electrochemiluminescence imaging often employs inorganic complex luminescence systems, such as ruthenium and iridium complexes, but the insolubility of these complexes in aqueous solutions and the cytotoxicity issues due to the heavy metals contained therein limit its application to biological analysis.

[0004] Semiconducting polymer quantum dots (Pdots) are novel organic fluorescent materials that are non-toxic and have the characteristics of large light absorption cross section, high fluorescence quantum efficiency, photostability and biocompatibility, and are therefore widely used in biodetection, cell biology and clinical medicine.

[0005] In the prior art, various attempts have been made to use semiconducting polymer quantum dots as fluorescent probes for electrochemiluminescence detection (see Non-Patent Documents 1 and 2). Compared with conventional electrochemiluminescence platforms, electrochemiluminescence sensors fabricated in the form of semiconducting polymer quantum dots have advantages such as higher luminescence brightness, better photostability, and better biocompatibility.

[0006] However, the electrochemiluminescence platform is a signal detection platform, and does not have a function of identifying the target molecule to be measured by itself. It is generally necessary to add a special probe to identify and analyze a specific target molecule. Therefore, the identification specificity / accuracy of the object to be measured depends on the special probe added thereto. The selection of such probes and targetable nucleic acid sequences are generally restricted by specific requirements, that is, the detection accuracy / specificity is limited, and it is difficult to detect any nucleic acid sequence. For example, in Non-Patent Document 2, Pdots were linked to DNA or RNA oligonucleotide probes to identify and analyze trace amounts of nucleic acid molecules.

[0007] Cas enzymes are nucleic acid recognition and cleavage enzyme proteins with a checkpoint mechanism, and therefore have single-base resolution accuracy. The greatest advantage of nucleic acid detection based on Cas enzymes is that by utilizing the checkpoint mechanism specific to Cas enzymes, detection of nucleic acids can reach single-base resolution sensitivity. In addition, by designing and synthesizing guide RNA (gRNA) for Cas enzymes, any nucleic acid sequence can be detected at low cost.

[0008] For example, Non-Patent Document 3 designs an electrochemiluminescence biosensor based on Cas12a and uses it to detect human papillomavirus (HPV-16) DNA.

[0009] Although such a technique has high accuracy in nucleic acid recognition, the sensitivity of identifying trace amounts of nucleic acid is insufficient. Therefore, it is generally necessary to perform pre-amplification of nucleic acid to improve the detection limit. The use of pre-amplification improves the detection sensitivity but reduces the detection accuracy. This is because the amplification of a large amount of nucleic acid fragments may cause aerosol contamination, resulting in false positive results. In addition, the pre-amplification step also complicates the detection step and extends the detection time. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Ningning Wang, Yaqiang Feng,et al.,”ElectrochemiluMinescent Imaging for Multi-immunoassay Sensitized by Dual DNA Amplification of Polymer Dot Signal”, Anal. Chem., 2018, 90, 7708-7714 [Non-Patent Document 2] Guangming Li et al., “Ratiometric fluorescent detection of miRNA-21 via pH-regulated adsorption of DNA on polymer dots and exonuclease III-assisted amplification”, Analytica Chimica Acta, 1232(2022)340450 [Non-Patent Document 3] Peng-Fei Liu et al., “Cas12a-based electrochemiluminescence biosensor for target amplification-free DNA detection”, Biosensors and Bioelectronics, 176, (2021), 112954 Summary of the Invention [Problem to be solved by the invention]

[0011] The problem to be solved by the present invention is to provide a method for producing an electrochemiluminescent nanoprobe that does not require amplification, has high sensitivity, high accuracy, specificity, and is highly flexible, an electrochemiluminescent nanoprobe, an electrochemiluminescent sensor, an electrochemiluminescent detection method, and a kit for electrochemiluminescence detection. [Means for solving the problem]

[0012] The manufacturing method of the electrochemiluminescent nanoprobe of the embodiment includes a Pdots nanoparticle synthesis step of polymerizing a conjugated polymer and a copolymer molecule to synthesize Pdots nanoparticles, and a Pdots nanoparticle modification step of modifying the obtained Pdots nanoparticles with an oligonucleotide chain having a quencher molecule modification. [Brief description of the drawings]

[0013] [Figure 1] FIG. 1 is a schematic diagram showing an example of the structure of a nanoprobe according to an embodiment. [Diagram 2] FIG. 2 is a schematic diagram of target nucleic acid detection illustrating the ECL switch principle based on hDNA-Pdots nanoprobes. [Diagram 3] Figure 3 is a schematic diagram of target nucleic acid detection illustrating the dual enzyme-catalyzed ECL switch principle based on dsDNA-Pdots nanoprobes. [Figure 4] FIG. 4 shows the results of TEM analysis of the PFBT Pdots produced in Example 1. [Figure 5A] FIG. 5A shows the results of ultraviolet analysis of the DNA-Pdots produced in Example 1. [Figure 5B] FIG. 5B shows the results of DLS analysis of the DNA-Pdots produced in Example 1. [Figure 6A] FIG. 6A shows the comparison results of ECL intensity before and after enzyme activation of different ECL sensor electrodes prepared in Example 2. [Figure 6B] FIG. 6B shows the comparison results of ECL intensity before and after enzyme activation of different ECL sensor electrodes prepared in Example 2. [Figure 6C] FIG. 6C shows the comparison results of ECL intensity before and after enzyme activation of different ECL sensor electrodes prepared in Example 2. [Figure 7] FIG. 7 shows the results of detection of HPV 16 DNA using the hDNA-Pdots sensor electrode produced in Example 2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] As mentioned above, Cas enzymes have the advantage of being able to target any nucleic acid fragment, but their sensitivity is limited and they need to be combined with other techniques (such as the commonly used preamplification) to improve their sensitivity. However, as mentioned above, they can cause false positives and are difficult to quantify the original nucleic acid. Electrochemiluminescence methods, such as electrochemiluminescence detection methods based on semiconductor polymer quantum dots (Pdots), have the advantages of being able to amplify signals, being highly accurate, and being able to be quantified, and they do not require preamplification, which can avoid false positives caused by aerosols. However, they are highly dependent on the oligonucleotide probes that are formulated and used, and the detection accuracy / specificity is limited, making it difficult to detect any nucleic acid sequence.

[0015] The present invention has been made in view of the above circumstances, and an object of the present invention is to provide an electrochemiluminescence detection method which has high sensitivity, high accuracy, specificity and high flexibility.

[0016] As a result of intensive research into the problems existing in the above-mentioned conventional techniques, the inventors of the present invention have found that by applying a Cas enzyme system to an electrochemiluminescence method based on semiconductor polymer quantum dots (Pdots), an electrochemiluminescence detection method based on Cas enzymes can be constructed, which can complement the shortcomings of the Pdots-based ECL and Cas12-based methods, thereby providing an electrochemiluminescence detection method that does not require amplification, has high sensitivity, high accuracy, specificity, and is highly flexible.

[0017] Specifically, the Cas protein-crRNA complex (Cas protein-crRNA) identifies the target nucleic acid and activates the Cas enzyme activity, and a Pdots nanoparticle is modified with an oligonucleotide chain (Q-DNA) modified with a quencher molecule to produce a nanoprobe, and the cleavage activity of the Cas protein-crRNA activated by the target is used to cleave the Q-DNA on the nanoprobe, thereby realizing control over the Pdots ECL signal switch. In addition, the sensitivity of the Pdots ECL signal switch can be further improved by adopting a dual enzyme catalytic system of Cas enzyme and exonuclease III (hereinafter sometimes referred to as "EXO III").

[0018] According to the present invention, there is provided an electrochemiluminescence detection method that does not require amplification, has high sensitivity, high accuracy, specificity, and is highly flexible. (1) By combining Pdots nanoprobes with the joint cleavage activity of Cas enzymes, the detection sensitivity for target nucleic acids can be improved without the need for pre-amplification. (2) The single-base specificity of Cas enzymes can improve the accuracy of ECL signal detection and avoid false positive signals due to misrecognition of target nucleic acids. (3) Customized guide RNA (crRNA) for Cas enzyme can target any nucleic acid sequence and output a highly sensitive signal based on ECL. (4) When the quenching group is linked via a dsDNA rigid structure, it is hoped that the dual enzyme catalytic system of Cas12 and EXO III can be employed to broaden the cleavage scope of the Cas12 enzyme and further improve the sensitivity of ECL detection. (5) The ECL nanoprobe of the present invention can be fabricated into a disposable detection chip, which is useful for point-of-care testing (POCT) and can realize convenient and rapid nucleic acid detection.

[0019] Hereinafter, specific embodiments of the present invention will be described in detail. Note that the following description of the embodiments is only for the purpose of explaining the inventive concept of the present invention, and is not intended to limit the present invention.

[0020] <Electrochemiluminescent nanoprobe and its manufacturing method> One embodiment of the present invention relates to a method for producing an electrochemiluminescent nanoprobe (hereinafter sometimes referred to as an "ECL nanoprobe"), comprising a Pdots nanoparticle synthesis step of synthesizing Pdots nanoparticles by polymerizing conjugated polymer and copolymer molecules, and a Pdots nanoparticle modification step of modifying the obtained Pdots nanoparticles with an oligonucleotide chain having a quencher molecule modification.

[0021] (Semiconductor polymer quantum dots) Semiconducting polymer quantum dots (hereafter sometimes referred to as "Pdots") are small (typically 20-30 nm in diameter) and highly luminous nanoparticles composed primarily of π-conjugated polymers.

[0022] Conjugated polymers are classified into polyacetylene, polythiophene, polyaniline, polypyrrole, polyparaphenylene, polyfluorene, polyphenylenevinylene, etc. depending on the structure of the main chain, and the main chain structure of the currently commonly used conjugated polymer is as shown in the structural formula (1) below.

[0023] [ka]

[0024] The specific conjugated polymer is not particularly limited, and for example, the conjugated polymer described in Angew. Chem. Int. Ed. 2013, 52, 3086-3109 can be used, and may be, for example, a compound selected from PDHF, PFO, PPE, MEH-PPV, PFPV, PFBT, CN-PPV, PF-DBT5, and PBOC shown in the following chemical formula (2).

[0025] [ka]

[0026] It should be noted that the conjugated polymers listed above are merely examples, and as will be apparent to those skilled in the art, derivatives formed by substituting the above compounds with one or more substituents (e.g., alkyl groups, hydroxyl groups, halogens, etc.), and spatial isomers thereof, etc., can be similarly used in the present invention.

[0027] The method for producing the conjugated polymer used in the present invention is not particularly limited, and can be synthesized by chemical reaction via synthetic routes such as Suzuki coupling reaction, Scherrer coupling reaction and cross-coupling reaction, or can be purchased through commercial routes, and is not particularly limited.

[0028] (Synthesis of Pdots nanoparticles) In the present invention, Pdots nanoparticles are synthesized by polymerizing conjugated polymer and copolymer molecules.

[0029] The nanoparticles in the present invention refer to particles in which at least one dimension in a three-dimensional space is nano-sized (0.1 to 100 nm). Specifically, the nanoparticles may be in various geometric forms, such as nanoballs, nanotubes, nanorods, or nano-onions. By forming the semiconductor polymer quantum dots in a nano form, the ECL response signal can be effectively amplified, and the detection limit, detection range, selectivity, stability, etc. of the ECL sensor can be improved. In the present invention, as in the examples described below, a material in the form of nanoballs was successfully produced, but the nanoparticles usable in the present invention are not limited to nanoballs, and other nano forms such as nanorods can also be formed depending on the compound raw material and synthesis route adopted.

[0030] In the present invention, the conjugated polymer and the copolymer molecule can be synthesized by a method for preparing organic material nanostructures known in the prior art, such as, but not limited to, nano-coprecipitation method, micro-emulsion method and self-assembly method (Liu Ronghua, Preparation and Application of Novel Fluorescent Conjugated Polymer Nanoparticles, Beijing University of Science and Technology Doctoral Dissertation, 2018). Here, from the viewpoint of biocompatibility, it is more preferable to use the micro-emulsion method and nano-coprecipitation method.

[0031] In the microemulsion method, highly biocompatible and stable luminescent molecule nanoparticles can be obtained by coating luminescent molecules with non-toxic, non-immunogenic hydrophilic polymers, such as polyethylene glycol (PEG), polyethylene glycol-phosphatidylethanolamine (PEG-PE), and other polyethylene glycol molecules, and biopolymers, such as phospholipids.

[0032] In the nano-coprecipitation method, luminescent molecules (conjugated polymers) are dissolved in a good solvent, and a large amount of poor solvent is injected under ultrasonic conditions, causing the solubility of the luminescent molecules to decrease rapidly and aggregate, thereby precipitating in the form of small particles, and then the remaining organic solvent is removed to obtain luminescent molecule nanoparticles. The nano-coprecipitation method is preferred because it is easy to operate and the nanoparticles produced are small and uniform in size.

[0033] In the nano-coprecipitation method, the luminescent molecular nanoparticles can be surface-functionalized using amphiphilic polymers. In the manufacturing process, the luminescent molecules and the amphiphilic polymers self-assemble, and one end of the amphiphilic polymer has a hydrophobic functional group and the other end has a hydrophilic functional group, so that the luminescent molecular nanoparticles coated with the amphiphilic polymer can be obtained, and the functional groups (e.g., amino and carboxy groups) of the amphiphilic polymer are exposed on the outer surface of the nanoparticles, thereby obtaining surface-functionalized luminescent nanoparticles. Functional groups such as nucleic acids, polypeptides, sugars, or proteins can react with the functional groups exposed on the outer surface of the nanoparticles of the amphiphilic polymers through condensation reaction or bioorthogonal click reaction, to obtain luminescent molecular nanoparticles with recognition function.

[0034] In the present invention, the amphiphilic polymer is also called a copolymer molecule. As the copolymer molecule, copolymer molecules commonly used in this field, such as polystyrene-polyacrylic acid block copolymer (PS-PAA), polystyrene maleic anhydride copolymer (PSMA) or poly(isobutylene-alt-maleic anhydride) (PIMA), etc., as shown in the following chemical formula (3), can be used, but are not limited thereto.

[0035] [ka]

[0036] The diameter of the nanoparticles obtained by the nanocoprecipitation method can reach a minimum of 1 nm to 2 nm.

[0037] The surface functionalization of Pdots nanoparticles is not limited to the above-mentioned modification method using amphiphilic polymers, and other modification methods such as direct functionalization (i.e., directly modifying functional groups such as alkoxy chains, amino / carboxy groups, or biomolecules having targeting functions to luminescent molecules by covalent bonding) and physical coating (i.e., coating nanoparticles with phospholipids or silicon dioxide, etc.) can also be used.

[0038] By surface functionalizing luminescent molecular nanoparticles, it is possible to have a large number of functional groups on the surface of each nanoparticle, and further to bind a large number of biological molecules such as oligonucleotides, thereby amplifying the electrochemiluminescence signal and improving its detection sensitivity.

[0039] (Modification of Pdots nanoparticles) In the present invention, modification of Pdots nanoparticles refers to oligonucleotide modification of Pdots nanoparticles, specifically, the above-mentioned surface-functionalized Pdots nanoparticles are modified with an oligonucleotide chain modified with a quencher molecule to obtain oligonucleotide-modified Pdots nanoparticles (hereinafter sometimes referred to as "ECL nanoprobes").

[0040] The method of binding between oligonucleotides and Pdots nanoparticles is not limited to covalent bond reaction (eg, cross-linking with NH2-COOH), but may be other binding methods such as electrostatic action or affinity adsorption.

[0041] The oligonucleotide chain used for modification may be a double-stranded oligonucleotide chain (e.g., dsDNA, etc.) or a single-stranded oligonucleotide chain (e.g., RNA, ssDNA), etc., and specifically, can be appropriately selected depending on the type of Cas enzyme used.

[0042] From the viewpoint of bringing the quencher molecule into close proximity to the Pdots and enhancing the amplification of the ECL signal, the oligonucleotide chain is preferably a double-stranded oligonucleotide chain or a single-stranded oligonucleotide chain having a secondary structure that can bring the quencher molecule into close proximity to the Pdots, for example, a single-stranded oligonucleotide chain having a hairpin structure.

[0043] In addition, since the solidarity cleavage activity of the Cas enzyme is non-specific, the nucleotide sequence constituting the oligonucleotide chain is not particularly limited and may be any sequence. The length of the oligonucleotide chain is also not particularly limited, and it is sufficient that the quencher molecule can be bound to the Pdots and that resonance energy transfer can occur between the quencher molecule and the Pdots nanoparticle, and it can be designed with reference to various oligonucleotide chains for linking that are commonly used in conventional technologies.

[0044] When the oligonucleotide chain is a linear single-stranded oligonucleotide chain, from the viewpoint of sensitivity of cleavage by Cas enzyme, the number of nucleotides should not be too large, and is preferably 3 to 20, more preferably 4 to 11, and even more preferably 5, 6, 7, 8, 9, 10, 11, or 12.

[0045] When the oligonucleotide strand is a single-stranded oligonucleotide strand having a hairpin structure, the number of nucleotides may be, for example, 20 or more, and the number of nucleotides in the circular fragment constituting the hairpin is not particularly limited as long as it forms a circle. From the viewpoint of the sensitivity of cleavage by Cas enzyme, the number of nucleotides in the stem fragment constituting the hairpin should not be too large, and is preferably 4 to 20, more preferably 4 to 10, and even more preferably 4, 5, 6, 7, or 8.

[0046] When the oligonucleotide chain is a double-stranded oligonucleotide chain, the double-stranded oligonucleotide chain preferably includes a chain directly bound to Pdots and a chain indirectly bound to Pdots by hybridization. The chain directly bound to Pdots preferably includes a single-stranded terminal portion cleavable by Cas enzyme, a hybrid chain, and a double-stranded portion cleavable by DNA exonuclease constituting the double-stranded portion. From the viewpoint of sensitivity of cleavage by Cas enzyme and DNA exonuclease, the number of nucleotides in the single-stranded terminal portion in the double-stranded oligonucleotide chain is preferably 3 to 20, more preferably 4 to 10, more preferably 4, 5 or 6, and the double-stranded portion in the double-stranded oligonucleotide chain preferably has a number of nucleotides as a single strand, more preferably 15 to 24, more preferably 16 to 20, more preferably 17, 18 or 19.

[0047] The oligonucleotide chain of the present invention is modified with a quencher molecule. The dynamic quenching of the quencher molecule occurs by fluorescence resonance energy transfer (FRET) or collisional quenching. Since the light absorption of the quencher molecule overlaps with the fluorescence emission of the Pdots nanoparticle, the resonance energy can be transferred from the excited nanoparticle to the quencher molecule, and the emission of the conjugated polymer molecule can be quenched.

[0048] The quencher molecule may be a quencher molecule having a resonance energy transfer characteristic commonly used in the art, and is not particularly limited. For example, a black hole quencher may be used. TM Reagents, i.e. BHQ-based quenchers, dark quenchers (e.g. DABCYL series quenchers, etc.), BlackBerry TMQuenchers (i.e., BBQ series quenchers), Blueberry quenchers (i.e., BLU series quenchers), or amine-reactive quenchers (QSY series quenchers) can be used. Among them, BHQ quenchers are often used. All of these quenchers are commercially available (e.g., LGC, BiochTechnologies, Inc., 2006). TM ) etc.) can be obtained.

[0049] The method for linking the quencher molecule to the oligonucleotide is not particularly limited, and any method commonly used in the art can be used. The quencher molecule may be linked to the end of the oligonucleotide or may be inserted into the oligonucleotide sequence.

[0050] In the present invention, the oligonucleotide modified with the quencher molecule functions as a signal switch of the present invention together with the Cas enzyme described below. Specifically, when the oligonucleotide is not cleaved by the activated Cas enzyme, the quencher molecule is linked to the surface of the Pdots nanoparticle via the oligonucleotide, exerts a quenching effect on the Pdots, and the sensor electrode displays a low ECL intensity (ECL OFF). On the other hand, when the oligonucleotide is cleaved by the activated Cas enzyme, the quencher molecule is released from the surface of the Pdots nanoparticle, and the sensor electrode displays a strong ECL signal (ECL ON).

[0051] <Electrochemiluminescence sensor> The electrochemiluminescence sensor (hereinafter, sometimes referred to as ECL sensor) of the present invention can be obtained by dropping the above-mentioned ECL nanoprobe onto a working electrode.

[0052] The working electrode used in the present invention is not particularly limited, and may be one of electrodes commonly used in ECL detection, such as a glass carbon electrode (GCE), an indium tin oxide (ITO) electrode, or a screen-printed electrode (SPE). An indium tin oxide electrode is preferably used, and a three-electrode system is more preferably used. The electrode system includes, for example, a platinum wire counter electrode, an Ag / AgCl reference electrode, and an indium tin oxide (ITO) working electrode.

[0053] <Electrochemiluminescence detection method> One embodiment of the present invention relates to an electrochemiluminescence detection method based on a Cas enzyme (hereinafter, sometimes referred to as an "ECL detection method"), which includes an enzyme reaction step in which a sample to be measured is added to a Cas enzyme catalyst system containing a guide nucleic acid capable of binding to a target nucleic acid, a Pdots chip preparation step in which the above-mentioned electrochemiluminescent nanoprobe is fixed to a working electrode, and a sample detection step in which the reaction solution obtained in the enzyme reaction step is added to the working electrode, and an electrochemiluminescence signal is collected and analyzed.

[0054] Furthermore, the ECL detection method of the present invention may directly use the above-mentioned ECL sensor (also called ECL chip) without including the above-mentioned Pdots chip preparation step.

[0055] The ECL detection method of the present invention can detect a target molecule in a sample to be measured. The sample to be measured is not particularly limited, and may be, for example, urine, blood, serum, cerebrospinal fluid, saliva, etc. The target molecule is not particularly limited, and may be, for example, a nucleic acid, a protein, a small chemical molecule, etc.

[0056] In the present invention, as a target recognition and signal conversion unit in an enzyme-catalyzed reaction or other specific chemical reaction, when a target molecule is dropped, the signal switch state of the ECL nanoprobe can be changed by the enzyme reaction or chemical reaction, thereby realizing qualitative or quantitative detection of the target signal.

[0057] As an enzyme used in an enzymatic catalytic reaction, for example, a Cas enzyme having collateral cleavage activity among CRISPR / Cas enzymes can be used, which belongs to the cleavage activity of non-specific nucleases. When targeted to a target nucleic acid molecule, the Cas enzyme is activated and acquires the non-specific activity of cleaving any nucleic acid molecule.

[0058] The Cas enzyme may include at least one Cas protein selected from, for example, Cas12 (VA type), Cas13 (VI type) and Cas14 (VF type), for example, at least one Cas protein selected from Cas12a, Cas12b, Cas13a, Cas13b, Cas14a, Cas14b and Cas14c. Here, Cas12 targets dsDNA (double-stranded DNA), Cas13 targets ssRNA (single-stranded RNA), and Cas14 targets ssDNA. In addition, Cas12a further has non-specific cleavage activity (also called trans-cleavage activity) against ssDNA, and therefore can further target ssDNA.

[0059] Additionally, Cas12 enzyme is more preferred because it targets DNA, is commercially mature, and can be used in other non-nucleic acid detection areas in addition to DNA (e.g., proteins, small biological molecules, etc.).

[0060] In the present invention, any nucleic acid sequence can be targeted by designing and synthesizing a guide RNA (gRNA) for the Cas enzyme. The gRNA is composed of two parts, one is CRISPR RNA (crRNA), which is a 17-20 base long nucleotide sequence that complementarily pairs with the target nucleic acid; and the other is tracr RNA, which acts as a scaffold to help the Cas enzyme fold. The guide RNA activates the Cas enzyme by binding to the oligonucleotide of the target nucleic acid. The activated Cas enzyme cleaves the oligonucleotide linked to the ECL nanoprobe by its solidarity cleavage activity, changing the signal switch state of the ECL nanoprobe.

[0061] The principle of the signal switch and the ECL detection method of the present invention will be specifically described below. The principle of the signal switch of the present invention is as follows.

[0062] In the present invention, Pdots nanoparticles modified with oligonucleotide chains are used as ECL nanoprobes, and the ends of the oligonucleotide chains (for example, ssDNA, hDNA, and dsDNA used in the examples) are modified with quencher molecules, so that the Pdots are in the ECL OFF state. When the target molecule is not present and the Cas enzyme is not activated by the target molecule, the oligonucleotide chains modified with the quencher molecule do not leave the Pdots surface, and the sensor electrode displays low ECL intensity (ECL OFF). Conversely, when the target molecule is present and the Cas enzyme is activated, the Cas enzyme, such as Cas12 enzyme, has a solidarity cleavage activity, so that the oligonucleotide chains linked to the Pdots are cleaved, which causes the quencher molecule to leave the Pdots surface, restoring the ECL of the Pdots, and the sensor electrode generates a strong ECL signal (ECL ON).

[0063] Fig. 1 is a schematic diagram showing an example of the structure of a nanoprobe according to an embodiment. Fig. 1 shows schematic diagrams illustrating the structures of a Pdots nanoprobe modified with a linear single-stranded DNA (ssDNA-Pdots), a Pdots nanoprobe modified with a hairpin-structured single-stranded DNA (hDNA-Pdots), and a Pdots nanoprobe modified with a double-stranded DNA (dsDNA-Pdots).

[0064] As shown in Figure 1, a quencher molecule is linked to the end of an oligonucleotide (DNA) strand. In the structures of hDNA-Pdots and dsDNA-Pdots, the quencher molecule is close to the Pdots, and therefore it is expected to have a better signal quenching effect and stronger signal amplification, resulting in better detection sensitivity.

[0065] When using hDNA-Pdots as a reporter probe, only Cas enzyme is required for the enzyme reaction. As shown in Figure 2, hDNA itself is a single-stranded DNA fragment. When the target molecule is present and the Cas enzyme is activated, the circular single-stranded fragment of the hairpin is first cut by the Cas enzyme due to the solidarity cleavage activity of the Cas enzyme. After the single-stranded fragment of the hairpin is cut, the double-stranded part bound by hydrogen bonds weakens the binding force and the hybridization is eliminated, so that BHQ detaches from the Pdots surface, restoring the ECL of the Pdots, and the sensor electrode generates a strong ECL signal (ECL ON). Figure 2 is a schematic diagram of target nucleic acid detection based on the hDNA-Pdots nanoprobe, showing the ECL switch principle.

[0066] When using dsDNA-Pdots nanoprobes as reporter probes, it is necessary to use a dual enzyme catalytic system of Cas enzyme and DNA exonuclease III for the enzymatic reaction. As shown in Figure 3, when the target molecule is present and the Cas enzyme is activated, it first cuts the short single-stranded fragment exposed at the outer end of the dsDNA, and then the exonuclease III is activated and cuts the remaining double-stranded fragment, which causes the quencher molecule to detach from the Pdots surface, restoring the ECL of the Pdots, and the sensor electrode generates a strong ECL signal (ECL ON). Figure 3 is a schematic diagram of target nucleic acid detection based on the dual enzyme catalytic ECL switch principle based on dsDNA-Pdots nanoprobes.

[0067] The above description of the DNA-Pdots nanoprobe is merely illustrative, and as will be understood by those skilled in the art, the oligonucleotides that can be used in the present invention are not limited to DNA, but may be RNA, etc.; the Cas enzyme is not limited to Cas12a shown in the drawings, but other types of Cas12 enzymes, Cas13, and Cas14 enzymes can be used as well. In addition, the enzyme for cleaving the double-stranded DNA is not limited to EXO III, but other cleavage enzymes available in the field can also be used.

[0068] Specific steps of the ECL detection method of the present invention are, for example, as follows.

[0069] Enzyme reaction step: the sample to be measured is added to a Cas enzyme system containing Cas protein and crRNA to obtain a sample reaction solution, where the crRNA can specifically identify the target nucleic acid in the sample to be measured.

[0070] Pdots chip preparation step: Drop the ECL nanoprobe of the present invention onto the working electrode to obtain the Pdots chip of the present invention (i.e., ECL sensor, Pdots ECL chip). Of course, the ECL sensor of the present invention can be used directly without manufacturing the ECL chip at the time of use. The ECL sensor is preferably a disposable detection chip (sometimes referred to as "Pdots disposable detection chip" in the present invention). It can be obtained by immobilizing the ECL nanoprobe of the present invention on a screen-printed electrode.

[0071] Sample detection step: the above sample reaction solution is dropped onto the Pdots chip, incubated at a certain temperature for a certain time, and then the electrochemiluminescence signal is collected and analyzed. The ECL detection of the present invention can be realized by the above signal switch.

[0072] <Electrochemiluminescence detection kit> One embodiment of the present invention relates to a Cas enzyme-based electrochemiluminescence detection kit (hereinafter, sometimes referred to as an "ECL detection kit") that includes an enzyme reaction solution containing a Cas enzyme catalytic system including a guide nucleic acid capable of binding to a target nucleic acid, and a Pdots disposable detection chip in which the above-mentioned electrochemiluminescent nanoprobe is fixed to a screen-printed electrode.

[0073] As described above, when the oligonucleotide strand in the ECL nanoprobe of the present invention is double-stranded DNA, the enzyme reaction solution may further contain EXO III enzyme.

[0074] Furthermore, the ECL detection kit of the present invention may further include other reagents necessary for ECL detection, an instruction manual, and the like.

[0075] Although the electrochemiluminescent nanoprobe and its manufacturing method and applications of the present invention have been described above based on the embodiments, the present invention is not limited thereto. As long as it does not deviate from the spirit of the present invention, the scope of the present invention also includes forms obtained by applying various modifications to the embodiments that a person skilled in the art can conceive, and other forms constructed by combining some of the components in the embodiments.

[0076] (Example) The Pdots nanoparticles, ECL nanoprobes, ECL detection methods, etc. of the present invention will be specifically described below with reference to examples, although the present invention is not limited to these examples.

[0077] The reagents, experimental methods and devices used in the examples are as follows. (1) Reagents EnGen® Lba Cas12a was purchased from New England Biolabs (Ipswich, MA, UK). Polystyrene maleic anhydride (PSMA, average Mw: 1700), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), polyethylene glycol (PEG, average Mw: 3350), and 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride (EDC) were all purchased from Sigma-Aldrich Co., Ltd. (Shanghai, China). Poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(1,4-benzo-{2,1′,3}-thiazole)] (PFBT) and tripropylamine (TPrA, ] 98%) were purchased from J&K Chemical Ltd. (Beijing, China). Disposable screen-printed electrodes were purchased from Nanjing Jingjie Biotechnology Co., Ltd (Nanjing, China). All oligonucleotides listed in Table 1 were purchased from Sangon Bioengineering Co. Ltd. (Shanghai, China). Also, phosphate buffer solution (PBS, 0.1 M, pH 7.4) is prepared by mixing stock solutions of KH2PO4 and Na2HPO4.

[0078] The oligonucleotide sequences used in the examples are as follows:

[0079] [Table 1]

[0080] (2) Experimental method and equipment Transmission electron microscopy (TEM) was obtained with a TECNAIG2F 20 transmission electron microscope (FEI, USA).

[0081] Zeta potentials are recorded using a 90 Plus DynaPro NanoStar (Brookhaven Instrument Corporation, USA).

[0082] Ultraviolet absorption spectra were obtained with a UV-3600 spectrophotometer (Shimadzu, Japan).

[0083] Cyclic voltammetry experiments are performed on a CHI 630D electrochemical workstation (CHI instruments Inc., China).

[0084] ECL experiments were carried out in a home-made reaction chamber of an MPI-EII ECL analyzer (Xi'an Remex, China), which employs a three-electrode system.

[0085] Example 1: Preparation of ECL nanomaterials 1. Preparation of PFBT Pdots The manufacturing process is described in the inventor's publication Anal.Chem. 2018, 90, 7708-7714.

[0086] First, 1 mg of PFBT and PSMA were dissolved in 1 mL of tetrahydrofuran (THF) to prepare 1 mg / mL of PFBT and PSMA. Next, 500 μL of PFBT (1 mg / mL) and 100 μL of PSMA (1 mg / mL) were taken and added to 9.4 mL of THF, mixed, and degassed by ultrasonic for 20 minutes to obtain a precursor solution. In addition, 2 mL of the precursor solution was rapidly injected into 8 mL of ultrapure water under ultrasonic conditions for 5 times, and after ultrasonic treatment for 3 minutes, excess ultrapure water and tetrahydrofuran were removed by rotary distillation. Finally, the mixture was filtered using a polyethersulfone syringe with a pore size of 0.22 μm to produce PFBT-Pdots.

[0087] The morphology of the produced PFBT Pdots was analyzed by transmission electron microscopy (TEM), and the results are shown in Figure 4. Figure 4 shows the TEM analysis results of the PFBT Pdots produced in Example 1. As shown in Figure 4, the Pdots nanoparticles have a good regular spherical shape and are uniform in size, with an average particle size of 24 ± 2 nm.

[0088] 2. Fabrication of ECL Nanoprobes 1) Preparation of nucleic acid-modified PFBT Pdots probes a. Preparation of ssDNA-Pdots 600μL of PFBT Pdots, 12μL of 5% PEG and 12μL of 1M HEPES were taken and mixed uniformly by shaking, then the pH was adjusted to 7.3 with 0.1M NaOH. 12μL of 10mg / mL EDC and 3μL of 10mg / mL NHS were added and mixed uniformly by shaking for 30min. Next, 10μL of 100μM ssDNA was added and stirred at 200rpm for 48h, after which the free ssDNA was removed by ultrafiltration in a 30kd ultrafiltration tube (10000rpm, 5min / time, repeated 8 times) to obtain ssDNA-Pdots.

[0089] b. Preparation of hDNA-Pdots 600μL of PFBT Pdots, 12μL of 5% PEG and 12μL of 1M HEPES were taken and mixed uniformly by shaking, then the pH was adjusted to 7.3 with 0.1M NaOH. 12μL of 10mg / mL EDC and 3μL of 10mg / mL NHS were added and mixed uniformly by shaking for 10min. Next, 10μL of 100μM hairpin DNA was added and stirred at 200 rpm for 48h, after which the free hairpin DNA was removed by ultrafiltration in a 30kd ultrafiltration tube (10000 rpm, 5min / time, repeated 8 times) to obtain hDNA-Pdots.

[0090] c. Preparation of dsDNA-Pdots 600μL of PFBT Pdots, 12μL of 5% PEG and 12μL of 1M HEPES were taken and mixed uniformly by shaking, then the pH was adjusted to 7.3 with 0.1M NaOH. 12μL of 10mg / mL EDC and 3μL of 10mg / mL NHS were added and mixed uniformly by shaking for 10min. Next, 60μL of 15.5μM dsDNA was added and stirred at 200rpm for 48h, after which the free dsDNA was removed by ultrafiltration using a 30kd ultrafiltration tube (10000rpm, 5min / time, repeated 8 times) to obtain dsDNA-Pdots.

[0091] As shown in Table 1, the ssDNA, hairpin DNA, and dsDNA were all previously linked to the quencher molecule BHQ2.

[0092] d. Characterization of DNA-Pdots The synthesized ssDNA-Pdots, dsDNA-Pdots and hDNA-Pdots were subjected to ultraviolet and DLS analysis, and the results are shown in Figures 5A and 5B. Figure 5A shows the results of ultraviolet analysis of the DNA-Pdots produced in Example 1. Figure 5B shows the results of DLS analysis of the DNA-Pdots produced in Example 1.

[0093] As can be seen from Figure 5A, ssDNA-Pdots (not shown), dsDNA-Pdots and hDNA-Pdots all have a DNA characteristic peak at 260 nm compared to Pdots. Also, as can be seen from Figure 5B, the absorption and particle size of ssDNA-Pdots (not shown), dsDNA-Pdots and hDNA-Pdots all increased compared to Pdots. These results indicate that the synthesis of ssDNA-Pdots, dsDNA-Pdots and hDNA-Pdots was successful.

[0094] Example 2: Validation of the signal switch based on Cas12a conversion catalyst and detection of HPV DNA 1) Principle of signal switch a. Basic principle Since the ends of the oligonucleotide chains (ssDNA, dsDNA, and hDNA as mentioned above) are modified with BHQ, the Pdots are in the ECL OFF state. When the target molecule is not present and the Cas enzyme is not activated by the target molecule, the oligonucleotide chain modified with BHQ does not leave the Pdots surface, and the sensor electrode displays a low ECL intensity (ECL OFF). Conversely, when the target molecule is present and the Cas enzyme is activated, the oligonucleotide chain is cleaved by its solidarity cleavage activity, which causes BHQ to leave the Pdots surface, restoring the ECL of the Pdots, and the sensor electrode generates a strong ECL signal (ECL ON).

[0095] b. ECL switch based on ssDNA-Pdots and hDNA-Pdots nanoprobes When ssDNA-Pdots and hDNA-Pdots are used as reporter probes, both ssDNA and hDNA are single-stranded DNA fragments, and as shown in (a) above, when a target molecule is present and the Cas enzyme is activated, the single-stranded DNA is cleaved by the solidarity cleavage activity, causing the quencher molecule to detach from the Pdots surface.

[0096] In the case of the hDNA fragment, as shown in Figure 2, the circular single-stranded fragment of the hairpin is first cut by the Cas enzyme, and after the single strand of the double-stranded portion connected by hydrogen bonds is cut, the binding strength weakens and hybridization is eliminated, so that BHQ detaches from the Pdots surface, restoring the ECL of the Pdots, and the sensor electrode generates a strong ECL signal (ECL ON).

[0097] c. Dual enzyme-catalyzed ECL switch based on dsDNA-Pdots nanoprobes When the dsDNA-Pdots nanoprobe is used as a reporter probe, in the presence of a target molecule, the Cas12a enzyme is activated; it first cleaves a short single-stranded fragment exposed at the outer end of the dsDNA, and then exonuclease III (EXO III) is activated to cleave the remaining double-stranded fragment, causing BHQ to detach from the Pdots surface, restoring the ECL of the Pdots, and the sensor electrode generates a strong ECL signal (ECL ON).

[0098] 2) Verification of signal switches 1. Fabrication of ECL Sensor Electrodes 2 μl of each of ssDNA-Pdots, hDNA-Pdots, and dsDNA-Pdots are dropped onto the surface of the screen-printed electrode, and then dried and fixed to produce sensor electrodes of ssDNA-Pdots, hDNA-Pdots, and dsDNA-Pdots.

[0099] 2. Detection a. ECL detection based on ssDNA-Pdots or hDNA-Pdots nanoprobes First, a sample reaction solution containing 50 nM Cas12a protein and 0.25 μM crRNA was prepared. 1 μL of HPV16 DNA was added to 19 μL of the sample reaction solution and mixed uniformly to prepare a Cas12a detection solution.

[0100] Next, take 4 μL of Cas12a detection solution and drop it onto the surface of the ssDNA-Pdots and hDNA-Pdots sensor electrodes, respectively, incubate at 37 °C for 1 h, wash with 1x PBS, and air-dry.

[0101] Finally, the ECL response was recorded in the ECL detection electrolyte (0.1 M pH 7.4 PBS containing 0.1 M TPrA and 0.1 M KNO3), where the ECL detection was performed using cyclic voltammetry, scanning from 0 to +1.50 V at a scan rate of 100 mV / s.

[0102] b. Dual enzyme-catalyzed ECL detection based on dsDNA-Pdots nanoprobes First, a Cas12a detection solution (10-fold diluted Buffer II containing 50 nM Cas12a, 0.25 μM crRNA, and 50 nM HPV16 DNA) was prepared as described above.

[0103] Then, take 4 μl of Cas12a detection solution and drop it onto the surface of the dsDNA-Pdots sensor electrode, incubate it at 37 °C for 1 h, wash it with 1x PBS, and air dry it.

[0104] Next, 3 μl of EXO III solution (10-fold diluted Buffer I containing 100 U / ml EXO III) is dropped onto the surface of the sensor electrode, incubated at 37° C. for 30 minutes, and washed with 1×PBS.

[0105] Finally, the ECL response was recorded in the ECL detection electrolyte (0.1M pH 7.4 PBS containing 0.025M TPrA and 0.1M KNO3). The specific ECL detection conditions are as follows: Detection method: Cyclic voltammetry Maximum voltage: +1.5V Lowest potential: 0V Scan speed: 100 mV / s PMT: High voltage 500V

[0106] The ECL detection results are shown in Figures 6A to 6C. Figures 6A to 6C are diagrams showing the comparison results of ECL intensity before and after enzyme activation of different ECL sensor electrodes prepared in Example 2. Here, Figure 6A is a diagram showing the comparison results of ECL intensity before and after Cas enzyme activation of an ssDNA-Pdots modified electrode, Figure 6B is a diagram showing the comparison results of ECL intensity before and after Cas enzyme activation of an hDNA-Pdots modified electrode, and Figure 6C is a diagram showing the comparison results of ECL intensity before and after dual enzyme activation of a dsDNA-Pdots modified electrode.

[0107] As can be seen from Figures 6A, 6B and 6C, when the sensor electrodes modified with the above different Pdots nanoprobes are used, the ECL intensities are all decreased, and after the enzyme is activated by the target nucleic acid, the ECL intensities are all restored. This result demonstrates the feasibility of the direct ECL switch system based on the Pdots nanoprobes of the present invention.

[0108] In addition, as can be seen from FIG. 6B, the ECL intensity of the hDNA-Pdots modified electrode was reduced to 20% compared with the Pdots modified electrode, indicating that the sensor interface was in the ECL OFF state. When 1 μM of target molecule was added, the Cas12a enzyme was activated, generating an ECL ON response, and the ECL intensity of the modified electrode was restored to 80%. That is, the hDNA-Pdots modified electrode showed better detection sensitivity than the ssDNA-Pdots modified electrode. This result is believed to be due to the fact that in the hairpin structure of ssDNA, the quencher molecule is close to the Pdots, so it has a better signal quenching effect and stronger signal amplification.

[0109] As can be seen from FIG. 6C, the ECL intensity of the dsDNA-Pdots modified electrode was reduced by 30% compared with the Pdots modified electrode, indicating that the sensor interface was in the ECL OFF state. When 1 μM of target molecule was added, the Cas12a enzyme was activated, triggering the cleavage reaction by Cas12a-EXO III dual enzymes, generating an ECL ON response, and the ECL intensity of the modified electrode was restored to 65%. This result not only demonstrated the feasibility of the dual enzyme catalysis ECL switch system based on the dsDNA-Pdots nanoprobe, but also demonstrated its good detection sensitivity. Since the dsDNA structure is similar to the hDNA structure, the quencher molecule is closer to Pdots than linear ssDNA, and therefore it is believed to have similarly good detection sensitivity. In addition, the dual enzyme cleavage system of Cas enzyme and exonuclease was adopted for the detection, which contributed to the high activity of the dual enzyme cleavage system, and the time required to obtain the ECL signal was shorter (less than 30 minutes).

[0110] c. Detection performance experiment Using the hDNA-Pdots sensor electrode prepared as above, different concentrations of HPV 16 DNA were detected. Figure 7 shows the results of detecting HPV 16 DNA using the hDNA-Pdots sensor electrode prepared in Example 2. As shown in Figure 7, there was still a response when the HPV 16 DNA concentration reached 10 pM, demonstrating that the ECL nanoprobe and ECL sensor prepared in the present invention can actually be used to detect target nucleic acid.

[0111] Industrial Applicability As can be seen from the above examples, the ECL nanoprobes prepared by the present invention, particularly the hDNA-Pdots nanoprobes and dsDNA-Pdots nanoprobes, can be combined with the Cas enzyme system to switch the ECL signal from off to on, thereby providing an electrochemiluminescence detection method with high sensitivity, high accuracy, specificity and high flexibility without the need for amplification. In addition, the ECL nanoprobes of the present invention can be further manufactured into disposable detection chips, such as disposable plastic chips or paper-based chips, which are useful for POCT and can realize convenient and rapid nucleic acid detection.

Claims

1. a Pdots nanoparticle synthesis step of polymerizing conjugated polymer and copolymer molecules to synthesize Pdots nanoparticles; a Pdots nanoparticle modification step of modifying the obtained Pdots nanoparticles with an oligonucleotide chain having a quencher molecule modification; The method for producing an electrochemiluminescent nanoprobe comprises:

2. The conjugated polymer is one of PDHF, PFO, PPE, MEH-PPV, PFPV, PFBT, CN-PPV, PF-DBT5, and PBOC, as shown in the following chemical formula (1): The method of claim 1 . [Formula 1]

3. The copolymer molecule is one of polystyrene-polyacrylic acid block copolymer (PS-PAA), polystyrene maleic anhydride copolymer (PSMA), and poly(isobutylene-alt-maleic anhydride) (PIMA), as shown in the following chemical formula (2): The method of claim 1 . [Case 2]

4. The conjugated polymer and the copolymer molecules are subjected to nano-coprecipitation to synthesize the Pdots nanoparticles; The method of claim 1 .

5. the quencher molecule is one of a black hole quencher, a dark quencher, and an amine-reactive quencher; The method of claim 1 .

6. The oligonucleotide strand is a single-stranded DNA. The method of claim 1 .

7. The single-stranded DNA is a hairpin single-stranded DNA. The method according to claim 6.

8. The oligonucleotide strand is a double-stranded DNA. The method of claim 1 .

9. A Pdots nanoparticle in which a conjugated polymer and a copolymer molecule are polymerized, and the Pdots nanoparticle is modified with an oligonucleotide chain having a quencher molecule modification; Electrochemiluminescent nanoprobes.

10. A working electrode on which an electrochemiluminescence nanoprobe is dropped, the Pdots nanoparticle being a Pdots nanoparticle modified with an oligonucleotide chain having a quencher molecule modification, the Pdots nanoparticle being a polymerized conjugated polymer and copolymer molecule; Electrochemiluminescence sensor.

11. the working electrode is one of a glassy carbon electrode, an indium tin oxide electrode, and a screen printed electrode; The electrochemiluminescence sensor according to claim 10.

12. An enzyme reaction step in which the sample to be measured is added to a Cas enzyme catalytic system containing a guide nucleic acid capable of binding to a target nucleic acid; a sample detection step of adding the reaction solution obtained in the enzyme reaction step to a working electrode onto which an electrochemiluminescent nanoprobe, which is a Pdots nanoparticle in which a conjugated polymer and a copolymer molecule are polymerized and which is modified with an oligonucleotide chain having a quencher molecule modification, is dropped, and collecting and analyzing an electrochemiluminescent signal; Electrochemiluminescence detection method comprising:

13. The Cas enzyme includes at least one Cas protein selected from Cas12 (type VA), Cas13 (type VI), and Cas14 (type VF); The electrochemiluminescence detection method according to claim 12.

14. The Cas enzyme comprises at least one Cas protein selected from the group consisting of Cas12a, Cas13a, Cas13b, Cas14a, Cas14b, and Cas14c; The electrochemiluminescence detection method according to claim 13.

15. The oligonucleotide strand in the electrochemiluminescent nanoprobe is a single-stranded DNA. The electrochemiluminescence detection method according to claim 12.

16. The single-stranded DNA is a hairpin single-stranded DNA. The electrochemiluminescence detection method according to claim 15.

17. the oligonucleotide strand in the electrochemiluminescent nanoprobe is a double-stranded DNA, and the enzyme reaction step further uses an EXO III enzyme; The electrochemiluminescence detection method according to claim 12.

18. An enzyme reaction solution containing a Cas enzyme catalytic system including a guide nucleic acid capable of binding to a target nucleic acid; A detection chip having an electrochemiluminescence nanoprobe fixed to a working electrode, the electrochemiluminescence nanoprobe being a Pdots nanoparticle in which a conjugated polymer and a copolymer molecule are polymerized, and the Pdots nanoparticle is modified with an oligonucleotide chain having a quencher molecule modification; A kit for electrochemiluminescence detection comprising:

19. The oligonucleotide strand in the electrochemiluminescent nanoprobe is a double-stranded DNA, and the enzyme reaction solution further contains an EXO III enzyme. The electrochemiluminescence detection kit according to claim 18.

20. The detection chip is a disposable Pdots detection chip in which a screen-printed three-electrode sheet is used as the working electrode. The electrochemiluminescence detection kit according to claim 18.

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