Cas enzyme-based electrochemical luminescence nanoprobe, sensor, detection method and detection kit

Abstract

The present invention relates to a Cas enzyme-based ECL nanoprobe using Pdots, a sensor, a detection method, and a detection kit. The preparation method of the ECL nano-probe comprises the following steps: a Pdots nano-particle synthesis step of polymerizing a conjugated polymer and a copolymer molecule to synthesize Pdots nano-particles, and a Pdots nano-particle modification step of modifying the obtained Pdots nano-particles by using an oligonucleotide chain modified by a quenching agent molecule. According to the ECL detection method disclosed by the invention, a double enzyme digestion system of Cas enzyme and excision enzyme can be used, and the ECL sensor disclosed by the invention can be a disposable detection chip. According to the present invention, the amplification-free, high-sensitivity, high-accuracy, high-specificity and high-flexibility ECL detection can be achieved.

Description

Technical Field

[0001] The present invention relates to an electrochemiluminescence (ECL) nanoprobe based on Cas enzyme using semiconductor polymer quantum dots (Pdots), a sensor, a detection method and a detection kit. Background Art

[0002] Electrochemiluminescence (ECL) is an electrochemically induced energy relaxation process. Since the excitation mode is separated from the signal detection, it has the characteristics of electrochemical controllability and low background. Due to the relatively simple temporal and spatial control of ECL technology, ECL imaging using a charge-coupled device (CCD) camera for signal acquisition has developed rapidly and has been used for the analysis of a variety of target molecules. It has also been extended to the screening of genomic toxins, immunoassays, fingerprint analysis, cell detection, and the study of reaction mechanisms. The ECL detection method retains the advantages of the chemiluminescence method, such as high sensitivity, wide linear range, convenient observation, and simple instrumentation; at the same time, it also has advantages that the chemiluminescence method cannot match, such as good reproducibility, stable reagents, easy control, and some reagents can be reused.

[0003] Current electrochemiluminescence imaging mostly uses inorganic complex luminescence systems such as ruthenium and iridium complexes, but the insolubility of these complexes in aqueous solutions and the cytotoxicity caused by the heavy metals they contain limit their application in biological analysis.

[0004] Semiconducting Polymer Quantum Dots (Pdots) are a new type of organic fluorescent material. Due to their non-toxicity, large light absorption cross-section, high fluorescence quantum efficiency, photostability and biocompatibility, they have been widely used in biological detection, cell biology and clinical medicine.

[0005] In the prior art, there have been many attempts to make semiconductor polymer quantum dots into fluorescent probes for electrochemiluminescence detection (see non-patent literature 1, 2). Electrochemiluminescence sensors prepared in the form of semiconductor polymer quantum dots have advantages over traditional electrochemiluminescence platforms, such as higher luminescence brightness, better photostability, and better biocompatibility.

[0006] However, as a signal detection platform, the electrochemiluminescence platform itself does not have the recognition function of the target molecule to be detected, and usually needs to be used with a special probe to realize the recognition and analysis of the specific target molecule, so its recognition specificity / accuracy for the object to be detected depends on the special probe it is matched with. The selection of such probes and the nucleic acid sequences that can be targeted are usually limited due to the requirements of specificity, that is, the accuracy / specificity of the detection is limited, which is not convenient for detecting any nucleic acid sequence. For example, in non-patent literature 2, Pdots are connected to DNA or RNA oligonucleotide probes to thereby realize the recognition and analysis of trace nucleic acid molecules.

[0007] Cas enzyme is a nucleic acid recognition and cleavage enzyme protein with a checkpoint mechanism, which enables it to have single-base resolution accuracy. The biggest advantage of nucleic acid detection based on Cas enzyme is that it utilizes the unique checkpoint mechanism of Cas enzyme, so that the detection of nucleic acids can achieve single-base resolution sensitivity. In addition, by designing and synthesizing the guide RNA (gRNA) of Cas enzyme, any nucleic acid sequence can be detected at low cost.

[0008] For example, a Cas12a-based electrochemiluminescent biosensor was designed in non-patent document 3 and used for the detection of human papillomavirus (HPV-16) DNA.

[0009] Although such technology has ultra-high nucleic acid recognition accuracy, it is not sensitive enough to identify trace amounts of nucleic acid, so nucleic acid pre-amplification is usually required to improve the detection limit. Although the use of pre-amplification improves the sensitivity of detection, it also reduces the accuracy of detection, because the amplification of a large number of nucleic acid fragments may cause aerosol contamination and produce false positive results. In addition, the pre-amplification step also makes the detection step complicated and prolongs the detection time.

[0010] Prior art literature

[0011] Non-patent literature

[0012] Non-patent literature 1: Ningning Wang, Yaqiang Feng, et al.; ElectrochemiluMinescent Imaging for Multi-immunoassay Sensitized by Dual DN AAmplification of Polymer Dot Signal; Anal.Chem.2018,90,7708-7714.

[0014] Non-patent document 2: Guangming Li et al.; Ratiometric fluorescent detect ion ofmiRNA-21via pH-regulated adsorption of DNA on polymer do ts and exonucleaseIII-assisted amplification, Analytica Chimica Acta 1

[0015] 232(2022)340450.

[0016] Non-patent literature 3: Peng-Fei Liu et al.; Cas12a-based electrochemical biosensor for target amplification-free DNA detection; Biosensors and Bioelectronics 176 (2021) 112954. Summary of the invention

[0017] Problems to be solved by the invention

[0018] As mentioned above, the Cas enzyme has the advantage of being able to target any nucleic acid fragment, but its own sensitivity is limited, and it needs to be combined with other technologies to improve sensitivity (such as the commonly used pre-amplification), but this will cause false positives, and it is difficult to quantify the original nucleic acid. Electrochemiluminescence methods, such as electrochemiluminescence detection methods based on semiconductor polymer quantum dots (Pdots), have the characteristics of signal amplification, good accuracy, and quantification, and do not require pre-amplification, which can avoid false positives caused by aerosols. However, it is highly dependent on the oligonucleotide probes used in conjunction, and the accuracy / specificity of the detection is limited, which is not convenient for detecting any nucleic acid sequence.

[0019] The present invention is accomplished in view of the above circumstances, and an object of the present invention is to provide an electrochemiluminescence detection method with high sensitivity, high accuracy, high specificity and high flexibility.

[0020] The inventors of the present invention have conducted detailed and in-depth repeated studies on the problems existing in the above-mentioned prior art. As a result, it was found that by applying the Cas enzyme system to the electrochemiluminescence method based on semiconductor polymer quantum dots (Pdots), an electrochemiluminescence detection method based on Cas enzyme was constructed, which can make up for 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, high specificity and high flexibility.

[0021] Specifically, the target nucleic acid is identified and the Cas enzyme activity is activated by a complex of Cas protein and crRNA (Cas protein-crRNA), and the Pdots particles are modified with an oligonucleotide chain (Q-DNA) modified with a quencher molecule to prepare a nanoprobe, and the Q-DNA on the nanoprobe is sheared by the shearing activity of the Cas protein-crRNA activated by the target nucleic acid, thereby realizing the switch control of the Pdots ECL signal. In addition, by adopting the Cas enzyme and EXO III dual enzyme catalytic system, the sensitivity of the Pdots ECL signal switch can be further improved.

[0022] Methods for solving problems

[0023] The present invention provides the following technical solutions.

[0024] <1> A method for preparing an electrochemiluminescent nanoprobe, comprising the following steps:

[0025] The Pdots nanoparticle synthesis process comprises polymerizing the conjugated polymer and the copolymer molecules to synthesize the Pdots nanoparticles, and

[0026] In the Pdots nanoparticle modification step, the obtained Pdots nanoparticles are modified using an oligonucleotide chain modified with a quencher molecule.

[0027] <2> The preparation method according to <1>, wherein

[0028] The conjugated polymer is PDHF, PFO, PPE, MEH-PPV, PFPV, PFBT, CN-PPV, PF-DBT5, or PBOC;

[0029]

[0030] <3> The preparation method according to <1> or <2>, wherein

[0031] The copolymer molecule is a polystyrene-polyacrylic acid block copolymer (PS-PAA), a polystyrene-maleic anhydride copolymer (PSMA) or a poly(isobutylene-alt-maleic anhydride) (PIMA),

[0032]

[0033]

[0034] <4> The production method according to any one of <1> to <3>, wherein

[0035] The conjugated polymer and the copolymer molecules are synthesized into Pdots particles through a nano-coprecipitation method.

[0036] <5> The production method according to any one of <1> to <4>, wherein

[0037] The quencher molecule is a black hole quencher, a dark quencher, or an amine-reactive quencher.

[0038] <6> The production method according to any one of <1> to <5>, wherein

[0039] The oligonucleotide chain is a single-stranded DNA.

[0040] <7> The preparation method according to <6>, wherein

[0041] The single-stranded DNA is a single-stranded DNA with a hairpin structure.

[0042] <8> The production method according to any one of <1> to <5>, wherein

[0043] The oligonucleotide chain is double-stranded DNA.

[0044] <9> An electrochemiluminescent nanoprobe prepared by the method described in any one of <1> to <8>.

[0045] <10> An electrochemiluminescent sensor obtained by dropping the electrochemiluminescent nanoprobe described in <9> onto a working electrode.

[0046] <11> The electrochemiluminescence sensor according to <10>, wherein the working electrode is one selected from a glassy carbon electrode, an indium tin oxide electrode, and a screen-printed electrode.

[0047] <12> An electrochemiluminescence detection method based on Cas enzyme, comprising the following steps:

[0048] Enzymatic reaction step: adding the sample to be tested to the Cas enzyme catalytic system, wherein the Cas enzyme catalytic system comprises a guide nucleic acid capable of binding to the target nucleic acid;

[0049] Pdots chip manufacturing process: fixing the electrochemiluminescent nanoprobe described in <9> onto the working electrode; and

[0050] Sample detection step: adding the reaction solution obtained in the enzymatic reaction step to the working electrode, collecting the electrochemical luminescence signal and analyzing it.

[0051] <13> The electrochemiluminescence detection method according to <12>, wherein:

[0052] The Cas enzyme comprises at least one Cas protein selected from Cas12 (VA type), Cas13 (VI type) and Cas14 (VF type).

[0053] <14> The electrochemiluminescence detection method according to <13>, wherein:

[0054] The Cas enzyme comprises at least one Cas protein selected from Cas12a, Cas13a, Cas13b, Cas14a, Cas14b and Cas14c.

[0055] <15> The electrochemiluminescence detection method according to any one of <12> to <14>, wherein

[0056] The oligonucleotide chain in the electrochemiluminescent nanoprobe is single-stranded DNA.

[0057] <16> The electrochemiluminescence detection method according to <15>, wherein

[0058] The single-stranded DNA is a single-stranded DNA with a hairpin structure.

[0059] <17> The electrochemiluminescence detection method according to any one of <12> to <14>, wherein

[0060] The oligonucleotide chain in the electrochemiluminescent nanoprobe is a double-stranded DNA, and EXO III enzyme is further used in the enzymatic reaction process.

[0061] <18> A kit for electrochemiluminescence detection based on Cas enzyme, comprising:

[0062] An enzymatic reaction solution: comprising a Cas enzyme catalytic system, wherein the Cas enzyme catalytic system comprises a guide nucleic acid capable of binding to a target nucleic acid; and

[0063] Pdots disposable detection chip: the electrochemiluminescent nanoprobe described in <9> is fixed on the working electrode of the screen-printed three-electrode sheet.

[0064] <19> The electrochemiluminescence detection kit according to <18>, wherein

[0065] The oligonucleotide chain in the electrochemiluminescent nanoprobe is double-stranded DNA, and the enzymatic reaction solution further contains EXO III enzyme.

[0066] Effects of the Invention

[0067] The present invention provides an electrochemiluminescence detection method that does not require amplification and has high sensitivity, high accuracy, high specificity and high flexibility. Specifically:

[0068] (1) By combining the Pdot nanoprobe and the Cas enzyme system's cleavage activity, the detection sensitivity of the target nucleic acid can be improved without the need for pre-amplification;

[0069] (2) The single-base resolution specificity of the Cas enzyme can improve the accuracy of ECL signal detection and avoid false positive signals caused by misidentification of target nucleic acids;

[0070] (3) Through customized Cas enzyme guide RNA (crRNA), it is possible to achieve targeting of any nucleic acid sequence and highly sensitive signal output based on ECL;

[0071] (4) When the quenching group is connected to a rigid dsDNA structure, the dual enzyme catalytic system of Cas12 and EXO III can be used to expand the application range of Cas12 enzyme cleavage and further improve the sensitivity of ECL detection.

[0072] (5) The ECL nanoprobe of the present invention can also be made into a disposable detection chip, which is beneficial for POCT (point-of-care testing) and can achieve convenient and rapid nucleic acid detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1 Schematic diagram showing the structures of linear single-stranded DNA modified Pdots nanoprobes (ssDNA-Pdots), hairpin single-stranded DNA modified Pdots nanoprobes (hDNA-Pdots) and double-stranded DNA modified Pdots nanoprobes (dsDNA-Pdots).

[0074] Figure 2 Schematic diagram of target nucleic acid detection based on the ECL switching principle of hDNA-Pdots nanoprobes.

[0075] Figure 3 Schematic diagram of target nucleic acid detection based on the dual-enzyme catalyzed ECL switching principle of dsDNA-Pdots nanoprobes.

[0076] Figure 4 2 is a diagram showing the TEM characterization results of the PFBT Pdots prepared in Example 1.

[0077] Figure 5 : is a graph showing the UV and DLS characterization results of the DNA-Pdots prepared in Example 1. Wherein, Figure 5 (a) is the UV characterization result, Figure 5 (b) is the DLS characterization result.

[0078] Figure 6 : is a graph showing the comparison of ECL intensities of different ECL sensing electrodes prepared in Example 2 before and after the enzyme is activated. Figure 6 (a) is a graph showing the comparison of ECL intensity of ssDNA-Pdots modified electrode before and after Cas enzyme activation. Figure 6 (b) is a graph showing the comparison of ECL intensity of hDNA-Pdots modified electrode before and after Cas enzyme activation. Figure 6 (c) is a graph showing the comparison of ECL intensity of the dsDNA-Pdots modified electrode before and after the dual enzyme is activated.

[0079] Figure 7 3 is a graph showing the detection results of HPV16 DNA using the hDNA-Pdots sensing electrode prepared in Example 2. DETAILED DESCRIPTION

[0080] The following is a detailed description of the specific embodiments of the present invention. It should be noted that the following description of the embodiments is only for the purpose of illustrating the inventive concept of the present invention, and is not intended to limit the present invention.

[0081] <Electrochemiluminescent nanoprobe and preparation method thereof>

[0082] One embodiment of the present invention relates to a method for preparing an electrochemiluminescent nanoprobe (hereinafter sometimes also referred to as an "ECL nanoprobe"), which comprises the following steps:

[0083] The Pdots nanoparticle synthesis process comprises polymerizing the conjugated polymer and the copolymer molecules to synthesize the Pdots nanoparticles, and

[0084] In the Pdots nanoparticle modification step, the obtained Pdots nanoparticles are modified using an oligonucleotide chain modified with a quencher molecule.

[0085] (Semiconductor Polymer Quantum Dots)

[0086] Semiconductor polymer quantum dots (hereinafter sometimes referred to as “Pdots”) are nanoparticles mainly composed of π-conjugated polymers, with small particle size (usually 20-30 nm in diameter) and high brightness.

[0087] Conjugated polymers can be divided into polyacetylene, polythiophene, polyaniline, polypyrrole, polyparaphenylene, polyfluorene, polyphenylene vinylene, etc. according to the structure of the main chain. The main chain structure of the most commonly used conjugated polymers is shown in the following structural formula.

[0088]

[0089] 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, for example, it can be a compound selected from PDHF, PFO, PPE, MEH-PPV, PFPV, PFBT, CN-PPV, PF-DBT5, and PBOC;

[0090]

[0091]

[0092] It should be noted that the conjugated polymers listed above are merely examples, and those skilled in the art will appreciate that derivatives of the above compounds formed by substitution with one or more substituents (e.g., alkyl, hydroxyl, halogen, etc.), as well as their spatial isomers, etc., can also be used in the present invention.

[0093] The method for producing the conjugated polymer used in the present invention is not particularly limited, and the conjugated polymer may be synthesized by chemical reaction through synthesis routes such as Suzuki coupling reaction, Stiller coupling reaction, and cross coupling reaction, or may be purchased through commercial channels, without particular limitation.

[0094] (Synthesis of Pdots Nanoparticles)

[0095] In the present invention, Pdots nanoparticles are synthesized by polymerizing conjugated polymer and copolymer molecules.

[0096] The nanoparticles in the present invention refer to particles with at least one dimension in nanometer size (0.1-100 nm) in three-dimensional space. Specifically, the nanoparticles can be various geometric forms such as nanospheres, nanotubes, nanorods, or nano-onions. By forming the semiconductor polymer quantum dots into nanoforms, the ECL response signal can be effectively amplified, and the detection limit, detection range, selectivity and stability of the ECL sensor can be improved. In the present invention, as described in the examples below, materials in the form of nanospheres were successfully prepared, but the nanoparticles that can be used in the present invention are not limited to nanospheres, and other nanoforms such as nanorods can also be formed according to the different compound raw materials and synthesis routes used.

[0097] In the present invention, the conjugated polymer and the copolymer molecule can be synthesized by a method for preparing an organic material nanostructure known in the prior art, such as a nano-coprecipitation method, a microemulsion method, and a self-assembly method (Liu Ronghua, Preparation and Application of Novel Fluorescent Conjugated Polymer Nanoparticles, PhD Dissertation, University of Science and Technology Beijing, 2018), without particular limitation. Among them, from the perspective of biocompatibility, microemulsion method and nano-coprecipitation method are more preferably used.

[0098] In the microemulsion method, for example, by using polyethylene glycol (PEG), polyethylene glycol-phosphatidylethanolamine (PEG-PE) and other polyethylene glycol molecules, biomacromolecules such as phospholipids and other non-toxic, non-immunogenic hydrophilic polymers to coat the luminescent molecules, luminescent molecular nanoparticles with good biocompatibility and high stability can be obtained.

[0099] The nano-coprecipitation method is to dissolve the luminescent molecule (conjugated polymer) in a good solvent, and then inject a large amount of poor solvent under ultrasonic conditions. The solubility of the luminescent molecule drops sharply and agglomerates, thereby precipitating out in the form of small particles. Then, the residual organic solvent is removed to obtain luminescent molecule nanoparticles. The nano-coprecipitation method is preferred because it is simple to operate and the size of the prepared nanoparticles is small and uniform.

[0100] In the nano-coprecipitation method, amphiphilic polymers can also be used to functionalize the surface of luminescent molecular nanoparticles. During the preparation process, the luminescent molecules and amphiphilic polymers will self-assemble. Since one end of the amphiphilic polymer has a hydrophobic functional group and the other end has a hydrophilic functional group, it is possible to obtain luminescent molecular nanoparticles coated with amphiphilic polymers, and the functional groups of the amphiphilic polymers (such as amino and carboxyl groups) will be exposed on the outer surface of the nanoparticles, thereby obtaining surface functionalized luminescent nanoparticles. Functional groups of nucleic acids, polypeptides, sugars, or proteins can react with the functional groups of the above-mentioned amphiphilic polymers exposed on the outer surface of the nanoparticles through condensation reactions or bioorthogonal click reactions to obtain luminescent molecular nanoparticles with recognition functions.

[0101] In the present invention, the above-mentioned amphiphilic polymer is also referred to as a copolymer molecule. As the copolymer molecule, copolymer molecules commonly used in the art can be used, such as polystyrene-polyacrylic acid block copolymer (PS-PAA), polystyrene maleic anhydride copolymer (PSMA) or poly (isobutylene-alt-maleic anhydride) (PIMA), etc., without particular limitation.

[0102]

[0103] The diameter of the nanoparticles obtained by the nano-coprecipitation method can be as small as 1nm to 2nm.

[0104] In addition, it should be noted that the surface functionalization modification of Pdots nanoparticles is not limited to the above-mentioned modification method of amphiphilic polymers, but can also adopt other modification methods such as direct functionalization method (i.e., directly modifying functional groups such as alkoxy chains, amino / carboxyl groups, or biological molecules with targeting functions to the luminescent molecules through covalent coupling methods), physical coating (i.e., coating nanoparticles with phospholipids or silica, etc.).

[0105] By functionalizing the surface of luminescent molecular nanoparticles, each nanoparticle surface can have a large number of functional groups, which can then bind to a large number of biological molecules such as oligonucleotides, thereby amplifying the electrochemiluminescence signal and improving its detection sensitivity.

[0106] (Modification of Pdots nanoparticles)

[0107] In the present invention, the modification of Pdots nanoparticles refers to the oligonucleotide modification of Pdots nanoparticles. Specifically, the surface functionalized Pdots nanoparticles are modified with oligonucleotide chains modified with quencher molecules to obtain oligonucleotide-modified Pdots nanoparticles (hereinafter sometimes also referred to as "ECL nanoprobes").

[0108] The connection method between oligonucleotides and Pdots nanoparticles is not limited to covalent reaction (such as cross-linking through NH2-COOH), and can also be through other connection methods such as electrostatic interaction and affinity adsorption.

[0109] The oligonucleotide chain used for modification can be a double-stranded oligonucleotide chain (such as dsDNA, etc.) or a single-stranded oligonucleotide chain (such as RNA, ssDNA), etc., and can be appropriately selected according to the type of Cas enzyme used.

[0110] From the perspective of bringing the quencher molecules close to Pdots and enhancing the amplification of the ECL signal, the above-mentioned oligonucleotide chain is preferably a double-stranded oligonucleotide chain, or a single-stranded oligonucleotide chain with a secondary structure that can bring the quencher molecules close to Pdots, such as a single-stranded oligonucleotide chain with a hairpin structure.

[0111] In addition, since the associated cleavage activity of the Cas enzyme is non-specific, the nucleotide sequence constituting the oligonucleotide chain is not particularly limited, and any sequence is acceptable. The length of the oligonucleotide chain is also not particularly limited, as long as the quencher molecule can be connected to the Pdots and resonance energy transfer can be generated between the quencher molecule and the Pdot particles, and can be designed with reference to various oligonucleotide chains commonly used in the prior art for connection.

[0112] When the oligonucleotide chain is a straight single-stranded oligonucleotide chain, from the perspective of the sensitivity of Cas enzyme cleavage, the number of nucleotides should not be too many, preferably 3 to 20, more preferably 4 to 11, and further preferably 5, 6, 7, 8, 9, 10, 11 or 12.

[0113] When the oligonucleotide chain is a single-stranded oligonucleotide chain with a hairpin structure, the number of nucleotides thereof may be, for example, 20 or more, wherein the number of nucleotides constituting the circular fragment of the hairpin is not particularly limited, and a circular shape may be formed. From the perspective of the sensitivity of Cas enzyme shearing, the number of nucleotides constituting the stem fragment of the hairpin should not be too many, preferably 4 to 20, more preferably 4 to 10, and further preferably 4, 5, 6, 7 or 8.

[0114] When the oligonucleotide chain is a double-stranded oligonucleotide chain, the double-stranded oligonucleotide chain preferably includes a chain directly connected to Pdots and a chain indirectly connected to Pdots by hybridization. The chain directly connected to Pdots preferably includes a single-stranded terminal portion that can be cut by the Cas enzyme, and a double-stranded portion that can be cut by a DNA exonuclease formed with the hybrid chain. From the perspective of the sensitivity of Cas enzyme and DNA exonuclease shearing, the number of nucleotides at the end portion of the single strand of the double-stranded oligonucleotide chain is preferably 3 to 20, more preferably 4 to 10, and further preferably 4, 5 or 6, and the double-stranded portion of the double-stranded oligonucleotide chain is calculated as a single chain, and the number of nucleotides is preferably 15 to 24, more preferably 16 to 20, and further preferably 17, 18 or 19.

[0115] The oligonucleotide chain of the present invention is modified with a quencher molecule, and the dynamic quenching of the quencher molecule occurs through fluorescence resonance energy transfer (FRET) or collision quenching. Since the light absorption of the quencher molecule overlaps with the fluorescence emission of the Pdots nanoparticles, the resonance energy can be transferred from the excited nanoparticles to the quencher molecule, thereby quenching the emission of the conjugated polymer molecule.

[0116] The quencher molecule may be a quencher molecule with resonance energy transfer properties commonly used in the art, without particular limitation, for example, a black hole quencher may be used. T M Reagents, i.e. BHQ series quenchers), dark quenchers (e.g. DABC YL series quenchers, etc.), BlackBerry TM Quenchers (i.e., BBQ series quenchers), Blueberry quenchers (i.e., BLU series quenchers), or amine reactive quenchers (i.e., QSY series quenchers), etc. Among them, BHQ quencher is the most commonly used. These quenchers can be obtained through commercial channels (e.g., LGC, Biosearch Technologies TM etc.) to obtain.

[0117] The method for connecting the quencher molecule to the oligonucleotide is not particularly limited, and the method commonly used in the art can be used. In addition, the quencher molecule can be connected to the tail end of the oligonucleotide or inserted into the oligonucleotide sequence.

[0118] In the present invention, the oligonucleotide modified with the quencher molecule acts as a signal switch of the present invention together with the Cas enzyme described below. Specifically, when the oligonucleotide is not cut by the activated Cas enzyme, the quencher molecule is connected to the surface of the Pdots nanoparticles through the oligonucleotide, exerts a quenching effect on the Pdots, and the sensing electrode shows a low ECL intensity (ECL OFF), and when the oligonucleotide is cut by the activated Cas enzyme, the quencher molecule is detached from the surface of the Pdots nanoparticles, so that the sensing electrode shows a strong ECL signal (ECL ON).

[0119] <Electrochemiluminescence sensor>

[0120] The electrochemiluminescence sensor (hereinafter sometimes referred to as an ECL sensor) of the present invention can be obtained by dropping the above-mentioned ECL nanoprobe onto a working electrode.

[0121] The working electrode used in the present invention is not particularly limited, and a common electrode used in ECL detection can be used, such as a glassy carbon electrode (GCE), an indium tin oxide (ITO) electrode, or a screen printed electrode (SPE). Preferably, an indium tin oxide electrode is used, and more preferably, a three-electrode system is used, which is configured with, for example, a platinum wire counter electrode, an Ag / AgCl reference electrode, and an indium tin oxide (ITO) working electrode.

[0122] <Electrochemiluminescence detection method>

[0123] One embodiment of the present invention relates to an electrochemiluminescence detection method based on Cas enzyme (hereinafter sometimes also referred to as "ECL detection method"), which comprises the following steps:

[0124] Enzymatic reaction step: adding the sample to be tested to the Cas enzyme catalytic system, wherein the Cas enzyme catalytic system comprises a guide nucleic acid capable of binding to the target nucleic acid;

[0125] Pdots chip manufacturing process: fixing the electrochemiluminescent nanoprobes onto the working electrode; and

[0126] Sample detection step: adding the reaction solution obtained in the enzymatic reaction step to the working electrode, collecting the electrochemical luminescence signal and analyzing it.

[0127] In addition, the ECL detection method of the present invention may not include the above-mentioned Pdots chip production step and directly use the above-mentioned ECL sensor (also referred to as ECL chip).

[0128] The ECL detection method of the present invention can detect the target molecule in the sample to be tested. The sample to be tested is not particularly limited, for example, it can be urine, blood, serum, cerebrospinal fluid or saliva. The target molecule is not particularly limited, for example, it can be nucleic acid, protein, small chemical molecule, etc.

[0129] In the present invention, enzyme catalysis or other specific chemical reactions are used as target recognition and signal conversion units. When the target molecule is added, the above enzymatic or chemical reaction can change the signal switch state of the ECL nanoprobe, thereby achieving qualitative or quantitative detection of the target signal.

[0130] As the enzyme used in the enzyme catalyzed reaction, for example, the Cas enzyme with collateral cleavage activity in the CRISPR / Cas enzyme can be used. This activity is a non-specific nuclease cleavage activity. When targeted to the target nucleic acid molecule, the Cas protein will be activated and obtain the activity of non-specific cleavage of any nucleic acid molecule.

[0131] The Cas enzyme, for example, includes at least one Cas protein selected from 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. Wherein, Cas12 targets dsDNA (double-stranded DNA, double-stranded DNA), Cas13 targets ssRNA (single-stranded RNA, single-stranded RNA), and Cas14 targets ssDNA. In addition, Cas12a also has non-specific shearing activity (also referred to as trans-shearing activity) of ssDNA (single-stranded DNA, single-stranded DNA), so it is also possible to target ssDNA.

[0132] In addition, the Cas12 enzyme is more preferred because it targets DNA and is more commercially mature, and can be used in other non-nucleic acid detection fields (such as proteins, small biological molecules, etc.) in addition to DNA.

[0133] In the present invention, by designing and synthesizing the guide RNA (gRNA) of the Cas enzyme, any nucleic acid sequence can be targeted. The gRNA consists of two parts, one is CRISPR RNA (crRNA), which is a nucleotide sequence of 17 to 20 bases in length that is complementary to the target nucleic acid; and a tracr RNA, which can serve 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, and the activated Cas enzyme changes the signal switch state of the ECL nanoprobe by cleaving the oligonucleotide connected to the ECL nanoprobe with the associated cleavage activity.

[0134] Next, the principle of the signal switch and the ECL detection method of the present invention are described in detail.

[0135] The principle of the signal switch of the present invention is as follows:

[0136] In the present invention, oligonucleotide chain-modified Pdots particles are used as ECL nanoprobes. Since the oligonucleotide chain (ssDNA, hDNA and dsDNA used in the embodiment) is end-modified with a quencher molecule, Pdots is in an ECL OFF state. When the target molecule does not exist and the Cas enzyme is not activated by the target molecule, the oligonucleotide chain modified by the quencher molecule will not be separated from the Pdots surface, and the sensing electrode shows a low ECL intensity (ECL OFF); on the contrary, when the target molecule exists and the Cas enzyme is activated, because the Cas enzymes such as Cas12 enzymes have associated shearing activity, they can shear the oligonucleotide chain connected to Pdots, causing the quencher molecule to be separated from the Pdots surface, recovering the ECL of Pdots, so that the sensing electrode produces a strong ECL signal (ECL ON).

[0137] Figure 1 Schematic diagram showing the structures of linear single-stranded DNA modified Pdots nanoprobes (ssDNA-Pdots), hairpin single-stranded DNA modified Pdots nanoprobes (hDNA-Pdots) and double-stranded DNA modified Pdots nanoprobes (dsDNA-Pdots). Figure 1 As shown, the end of the oligonucleotide (DNA) chain is connected to a quencher molecule. In the hDNA-Pdots and dsDNA-Pdots structures, since the quencher molecules are closer to the Pdots, they have better signal quenching effects and stronger signal amplification, and are therefore expected to have better detection sensitivity.

[0138] When hDNA-Pdots are used as reporter probes, only Cas enzymes are used in the enzymatic reaction, e.g. Figure 2 As shown, hDNA itself is a single-stranded DNA fragment. When the target molecule exists and the Cas enzyme is activated, the circular single-stranded fragment of the hairpin is first cut by the Cas enzyme due to the associated cleavage activity of the Cas enzyme. After the hairpin single-stranded fragment is cut, the double-stranded part formed by hydrogen bonding will dehybridize due to the weakened binding force, thereby causing BHQ to detach from the Pdots surface, restoring the ECL of the Pdots, and generating a strong ECL signal (ECL ON) at the sensing electrode.

[0139] When dsDNA-Pdots nanoprobes are used as reporter probes, a dual enzyme catalytic system of Cas enzyme and DNA exonuclease III is required in the enzymatic reaction, such as Figure 3As shown, when the target molecule exists and the Ca s enzyme is activated, it will first cut the short single-stranded fragments exposed at the outer end of the dsDNA, and then activate the exonuclease III (sometimes also referred to as "EXO III") to cut the remaining double-stranded fragments, so that the quencher molecules are separated from the surface of Pdots, the ECL of Pdots is restored, and the sensing electrode generates a strong ECL signal (ECL ON).

[0140] The above-mentioned explanation of DNA-Pdots nanoprobe is only an example, and it is clear to those skilled in the art that the oligonucleotides that can be used in the present invention are not limited to DNA, but can also be RNA, etc.; Cas enzyme is also not limited to Cas12a shown in the figure, and other types of Cas12 enzymes or Cas13, Cas14 enzymes can also be used. In addition, the above-mentioned enzyme for shearing double-stranded DNA is also not limited to EXO III, and other commonly used shearing enzymes in the art can also be used.

[0141] The specific steps of the ECL detection method of the present invention are as follows:

[0142] Enzymatic reaction step: adding the sample to be tested into a Cas enzyme system containing Cas protein and crRNA to obtain a sample reaction solution. The crRNA can specifically recognize the target nucleic acid in the sample to be tested.

[0143] Pdots chip production process: the ECL nanoprobe of the present invention is added dropwise onto the working electrode to obtain the Pdots chip of the present invention (i.e., ECL sensor, Pdots ECL chip). Of course, the ECL chip can also directly use the ECL sensor of the present invention without being prepared when in use. The ECL sensor is preferably a disposable detection chip (sometimes also referred to as "Pdots disposable detection chip" in the present invention), which can be obtained by fixing the ECL nanoprobe of the present invention on the screen-printed electrode.

[0144] Sample detection process: drip the sample reaction solution onto the Pdots chip, incubate for a period of time at a certain temperature, collect the electrochemical luminescence signal and analyze it. The ECL detection of the present invention can be achieved through the signal switch.

[0145] <Electrochemiluminescence detection kit>

[0146] One embodiment of the present invention relates to a Cas enzyme-based electrochemiluminescence detection kit (hereinafter sometimes also referred to as an "ECL detection kit"), comprising:

[0147] An enzymatic reaction solution: comprising a Cas enzyme catalytic system, wherein the Cas enzyme catalytic system comprises a guide nucleic acid capable of binding to a target nucleic acid; and

[0148] Pdots disposable detection chip: The above-mentioned electrochemiluminescent nanoprobe is fixed on the working electrode of the screen-printed three-electrode sheet.

[0149] As mentioned above, when the oligonucleotide chain in the ECL nanoprobe of the present invention is a double-stranded DNA, the enzymatic reaction solution may further comprise EXO III enzyme.

[0150] In addition, the ECL detection kit of the present invention may further include other reagents and instructions required for ECL detection.

[0151] Above, the electrochemiluminescent nanoprobe of the present invention and its preparation method and application are described based on the embodiments, but the present invention is not limited to these. As long as it does not depart from the gist of the present invention, the embodiments are implemented in a manner that can be obtained by various modifications that can be thought of by those skilled in the art, or other methods constructed by combining some of the constituent elements in the embodiments are also included in the scope of the present invention.

[0152] Example

[0153] The following examples are given to specifically illustrate the Pdots nanoparticles, ECL nanoprobes, ECL detection methods, etc. of the present invention, but it should be noted that the present invention is not limited to these examples.

[0154] The reagents, experimental methods and equipment used in the examples are as follows:

[0155] (1) Reagents:

[0156] LbaCas12a was purchased from New England Biolabs (Ipswich, MA, UK).

[0157] 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 purchased from Sigma-Aldrich (Shanghai, China).

[0158] 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 Co., Ltd. (Beijing, China).

[0159] Disposable screen-printed electrodes were purchased from Nanjing Jingjie Biotechnology Co., Ltd.

[0160] All oligonucleotides listed in Table 1 were purchased from Sangon Biotechnology Co., Ltd. (Shanghai, China).

[0161] In addition, phosphate buffered saline (PBS, 0.1 M, pH 7.4) was prepared by mixing stock solutions of KH2PO4 and Na2HPO4.

[0162] The oligonucleotide sequences used in the examples are as follows:

[0163] Table 1

[0164]

[0165] (2) Experimental methods and equipment

[0166] TEM images were obtained using a TECNAI G2 F20 transmission electron microscope (FEI, USA).

[0167] Zeta potential was recorded using a 90Plus DynaPro NanoStar (Brookhaven InstruMent Corporation, USA).

[0168] The UV absorption spectra were obtained using a UV-3600 spectrophotometer (Shimadzu, Japan).

[0169] The cyclic voltammetry experiments were performed on a CHI 630D electrochemical workstation (CHI Instrument Co., Ltd., China).

[0170] The ECL experiments were performed on a homemade reaction cell of an MPI-EII ECL analyzer (Xi’an Ruimai Analytical Instrument Co., Ltd., China) using a three-electrode system.

[0171] Example 1: Preparation of ECL nanomaterials

[0172] 1. Preparation of PFBT Pdots:

[0173] The preparation process can be found in the article Anal.Chem.2018,90,7708-7714 published by the inventor.

[0174] First, 1 mg PFBT and PSMA were dissolved in 1 mL tetrahydrofuran (THF) to prepare 1 mg / mL PFBT and PSMA respectively; then, 500 μL PFBT (1 mg / mL) and 100 μL PSMA (1 mg / mL) were added to 9.4 mL THF, mixed, and ultrasonically degassed for 20 minutes to obtain a precursor solution; then, 2 mL of the precursor solution was quickly injected into 8 mL of ultrapure water under ultrasonic conditions for 5 times, and ultrasonication was continued for 3 minutes; then, excess ultrapure water and tetrahydrofuran were removed by rotary distillation; finally, PFBT-Pd ots was obtained by filtering using a 0.22 μm pore size polyethersulfone syringe.

[0175] The morphology of the prepared PFBT Pdots was characterized by transmission electron microscopy (TEM). Figure 4 .like Figure 4 As shown, the Pdots particles are well-regularly spherical and uniform in size, with an average particle size of 24±2 nm.

[0176] 2. Preparation of ECL nanoprobes

[0177] 1) Preparation of nucleic acid-modified PFBT Pdots probes:

[0178] a. Preparation of ssDNA-Pdots

[0179] Take 600 μL PFBT Pdots, 12 μL 5% PEG and 12 μL 1M HEPES and shake to mix, then adjust the pH to 7.3 with 0.1 M NaOH, add 12 μL 10 mg / mL EDC and 3 μL 10 mg / mL NHS, and shake to mix for 30 min, then add 10 μL 100 μM ssDNA, stir at 200 rpm for 48 h, and use a 30 kd ultrafiltration tube to ultrafilter to remove free ssDNA (10000 rpm, 5 min / time, repeated 8 times) to obtain ssDNA-Pdots.

[0180] b. Preparation of hDNA-Pdots:

[0181] Take 600 μL PFBT Pdots, 12 μL 5% PEG and 12 μL 1M HEPES and shake to mix, then adjust the pH to 7.3 with 0.1 M NaOH, add 12 μL 10 mg / mL EDC and 3 μL 10 mg / mL NHS, and shake to mix for 10 min, then add 10 μL 100 μM hairpin nDNA, stir at 200 rpm for 48 h, and use 30 kd ultrafiltration tube to remove free hairpin DNA (10000 rpm, 5 min / time, repeated 8 times) to obtain hDNA-Pdots.

[0182] c. Preparation of dsDNA-Pdots:

[0183] Take 600 μL PFBT Pdots, 12 μL 5% PEG and 12 μL 1M HEPES and shake to mix, then adjust the pH to 7.3 with 0.1 M NaOH, add 12 μL 10 mg / mL EDC and 3 μL 10 mg / mL NHS, and shake to mix for 10 min, then add 60 μL 15.5 μM dsDNA, stir at 200 rpm for 48 h, and use a 30 kd ultrafiltration tube to ultrafilter to remove free dsDNA (10000 rpm, 5 min / time, repeated 8 times) to obtain dsDNA-Pdots.

[0184] The above-mentioned ssDNA, hairpinDNA and dsDNA are shown in Table 1, and are all pre-connected with the quencher molecule BHQ2.

[0185] d. Characterization of DNA-Pdots:

[0186] The synthesized ssDNA-Pdots, dsDNA-Pdots and hDNA-Pdots were characterized by UV and DLS. Figure 5 .in, Figure 5 (a) is the UV characterization result, Figure 5 (b) is the DLS characterization result.

[0187] from Figure 5 (a) It can be seen that compared with Pdots, ssDNA-Pdots (not shown), dsDNA-Pdots and hDNA-Pdots all have the characteristic peak of DNA at 260nm. Figure 5 (b) It can be seen that the absorption and particle size of ssDNA-Pdots (not shown), dsDNA-Pdots and hDNA-Pdots are increased compared to Pdots. These results show that the synthesis of ssDNA-Pdots, dsDNA-Pdots and hDNA-Pdots was successful.

[0188] Example 2: Detection of signal switch based on Cas12a conversion catalysis and HPV DNA

[0189] 1) Principle of signal switch:

[0190] a. Basic principles

[0191] Since the ends of the oligonucleotide chains (the above-mentioned ssDNA, dsDNA and hDNA) are modified with BHQ, the Pdots are in the ECLOFF state. When the target molecule is not present and the Cas enzyme is not activated by the target molecule, the BHQ-modified oligonucleotide chain will not detach from the Pdots surface, and the sensing electrode shows a low ECL intensity (ECL OFF); conversely, when the target molecule is present and the Cas enzyme is activated, the oligonucleotide chain is cleaved due to its associated cleavage activity, causing BHQ to detach from the Pdots surface, restoring the ECL of the Pdots, and causing the sensing electrode to generate a strong ECL signal (ECL ON).

[0192] b. ECL switch based on ssDNA-Pdots and hDNA-Pdots nanoprobes:

[0193] When ssDNA-Pdots and hDNA-Pdots are used as reporter probes, both ssDNA and hDNA are single-stranded DNA fragments. As described in a above, when the target molecule is present and the Cas enzyme is activated, the single-stranded DNA will be cut due to the associated cleavage activity, thereby causing the quencher molecule to detach from the Pdots surface.

[0194] In hDNA fragments, such as Figure 2 As shown, the circular single-stranded fragment of the hairpin is first cut by the Cas enzyme, and the double-stranded part formed by hydrogen bonding will dehybridize after the single-strand is cut due to the weakened binding force, thereby causing BHQ to detach from the Pdots surface, restoring the ECL of the Pdots, and the sensing electrode generates a strong ECL signal (ECL ON).

[0195] C. Dual-enzyme catalyzed ECL switch based on dsDNA-Pdots nanoprobes

[0196] When dsDNA-Pdots nanoprobes are used as reporter probes, when the target molecule is present and the Cas12a enzyme is activated, the short single-stranded fragments exposed at the outer end of the dsDNA will be cut first, and then exonuclease III (EXO III) will be activated to cut the remaining double-stranded fragments, thereby causing BHQ to detach from the Pdots surface, restoring the ECL of the Pdots, and the sensing electrode will generate a strong ECL signal (ECLON).

[0197] 2) Verification of signal switch:

[0198] 1. Preparation of ECL sensing electrodes:

[0199] 2 μl of ssDNA-Pdots, hDNA-Pdots, and dsDNA-Pdots were dropped onto the surface of the screen-printed electrode, dried and fixed, to prepare sensing electrodes of ssDNA-Pdots, hDNA-Pdots, and dsDNA-Pdots.

[0200] 2. Detection:

[0201] a. ECL detection based on ssDNA-Pdots or hDNA-Pdots nanoprobes

[0202] First, a sample reaction solution containing 50 nM Cas12a protein and 0.25 μM crRNA was prepared, and 1 μL HPV16 DNA was added to 19 μL sample reaction solution and mixed well to prepare a Cas12a detection solution.

[0203] Then, 4 μL of Cas12a detection solution was added dropwise to the surfaces of ssDNA-Pdots and hDNA-Pdots sensing electrodes, respectively, incubated at 37 °C for 1 h, rinsed with 1xPBS, and dried naturally;

[0204] Finally, the ECL response was recorded in an ECL detection electrolyte (0.1 M pH 7.4 PBS containing 0.1 M TPrA and 0.1 M KNO3). ECL was detected using cyclic voltammetry, scanning from 0 to +1.50 V at a scan rate of 100 mV / s.

[0205] b. Dual-enzyme catalyzed ECL detection based on dsDNA-Pdots nanoprobes

[0206] First, Cas12a detection solution (10-fold diluted Buffer II containing 50 nM Cas12a, 0.25 μM crRNA, 50 nM HPV16 DNA) was prepared as above.

[0207] Then, 4 μl of Cas12a detection solution was added dropwise to the surface of the dsDNA-Pdots sensor electrode, incubated at 37 °C for 1 h, rinsed with 1xPBS, and dried naturally;

[0208] Next, 3 μl of EXOⅢ solution (10-fold diluted Buffer I, which contains 100 U / ml EXOⅢ) was added dropwise to the surface of the sensing electrode, incubated at 37°C for 30 min, and then rinsed with 1xPBS;

[0209] Finally, the ECL response was recorded in an ECL detection electrolyte (0.1 M pH 7.4 PBS containing 0.025 M TPrA and 0.1 M KNO3). The specific ECL detection conditions are as follows:

[0210] Detection method: Cyclic voltammetry

[0211] Maximum potential: +1.5V

[0212] Minimum potential: 0V

[0213] Scan speed: 100mV / s

[0214] PMT: High voltage 500V

[0215] The ECL test results are shown in Figure 6 .in, Figure 6 (a) is a graph showing the comparison of ECL intensity of ssDNA-Pdots modified electrode before and after Cas enzyme activation. Figure 6 (b) is a graph showing the comparison of ECL intensity of hDNA-Pdots modified electrode before and after Cas enzyme activation. Figure 6 (c) is a graph showing the comparison of ECL intensity of the dsDNA-Pdots modified electrode before and after the dual enzyme is activated.

[0216] from Figure 6 As shown in (a), (b) and (c), when the sensing electrodes modified with the above-mentioned different Pdots nanoprobes are used, the ECL intensity decreases, and the ECL intensity is restored after the enzyme is activated by the target nucleic acid. This result shows the feasibility of the direct ECL switch system based on Pdots nanoprobes of the present invention.

[0217] In addition, from Figure 6 (b) It can be seen that compared with the Pdots modified electrode, the ECL intensity of the hDNA-Pdots modified electrode dropped to ~20%, indicating that the sensing interface was in the ECL OFF state; when 1 μM target molecule was added, the Cas12a enzyme was activated, an ECLON response was generated, and the ECL intensity of the modified electrode recovered to ~80%. That is, the hDNA-Pdots modified electrode showed better detection sensitivity than the ssDNA-Pdots modified electrode. This result is considered to be due to the fact that in the ssDNA with a hairpin structure, the quencher molecule is closer to the Pdots, so it has a better signal quenching effect and a stronger signal amplification.

[0218] from Figure 6(c) It can be seen that compared with the Pdots modified electrode, the ECL intensity of the dsDNA-Pdots modified electrode dropped to ~30%, indicating that the sensing interface is in the ECL OFF state; when 1 μM target molecule is added, the Cas12a enzyme is activated, the Cas12a-EXO III dual enzyme shearing reaction is started, and an ECL ON response is generated, and the ECL intensity of the modified electrode is restored to ~65%. This result not only shows the feasibility of the dual enzyme catalytic ECL switch system based on dsD NA-Pdots nanoprobe, but also shows that it has good detection sensitivity. Since the dsDNA structure is similar to the hDNA structure, the quencher molecule is closer to Pdots than in the straight-chain ssDNA, so it also has good detection sensitivity. In addition, since the dual enzyme digestion system of Cas enzyme and exonuclease is used in this detection, thanks to the high activity of the dual enzyme digestion system, the time required to obtain the ECL signal is shorter (less than 30 minutes).

[0219] c. Detection performance experiment:

[0220] Using the hDNA-Pdots sensing electrode prepared as above, different concentrations of HPV16 DNA were detected. Figure 7 As shown, there is still a response when the HPV16 DNA concentration reaches 10 pM, indicating that the prepared ECL nanoprobe and ECL sensor of the present invention can be actually used for the detection of target nucleic acid.

[0221] Industrial Applicability

[0222] As can be seen from the above embodiments, the ECL nanoprobes prepared in the present invention, especially the hDNA-Pdots nanoprobes and dsDNA-Pdots nanoprobes, can switch the ECL signal from off to on by combining with the Cas enzyme system, thereby providing an electrochemiluminescence detection method that does not require amplification, has high sensitivity, high accuracy and specificity, and high flexibility. In addition, the ECL nanoprobes of the present invention can also be made into disposable detection chips, such as disposable plastic chips or paper-based chips, which are beneficial to POCT and can achieve convenient and fast nucleic acid detection.

Claims

1. A method for preparing an electrochemiluminescent nanoprobe, comprising the following steps: The Pdots nanoparticle synthesis process comprises polymerizing the conjugated polymer and the copolymer molecules to synthesize the Pdots nanoparticles, and In the Pdots nanoparticle modification step, the obtained Pdots nanoparticles are modified using an oligonucleotide chain modified with a quencher molecule.

2. The preparation method according to claim 1, wherein The conjugated polymer is PDHF, PFO, PPE, MEH-PPV, PFPV, PFBT, CN-PPV, PF-DBT5, or PBOC; 3. The preparation method according to claim 1 or 2, wherein The copolymer molecule is a polystyrene-polyacrylic acid block copolymer (PS-PAA), a polystyrene maleic anhydride copolymer (PSMA) or a poly(isobutylene-alt-maleic anhydride) (PIMA), 4. The preparation method according to any one of claims 1 to 3, wherein The conjugated polymer and the copolymer molecules are synthesized into Pdots particles through a nano-coprecipitation method.

5. The preparation method according to any one of claims 1 to 4, wherein The quencher molecule is a black hole quencher, a dark quencher, or an amine-reactive quencher.

6. The preparation method according to any one of claims 1 to 5, wherein The oligonucleotide chain is a single-stranded DNA.

7. The preparation method according to claim 6, wherein: The single-stranded DNA is a single-stranded DNA with a hairpin structure.

8. The preparation method according to any one of claims 1 to 5, wherein The oligonucleotide chain is double-stranded DNA. 9 . An electrochemiluminescent nanoprobe, which is prepared by the preparation method according to any one of claims 1 to 8.

10. An electrochemiluminescent sensor, which is obtained by dropping the electrochemiluminescent nanoprobe according to claim 9 onto a working electrode.

11. The electrochemiluminescence sensor according to claim 10, wherein: The working electrode is one selected from a glassy carbon electrode, an indium tin oxide electrode, and a screen-printed electrode.

12. An electrochemiluminescence detection method based on Cas enzyme, comprising the following steps: Enzymatic reaction step: adding the sample to be tested to the Cas enzyme catalytic system, wherein the Cas enzyme catalytic system comprises a guide nucleic acid capable of binding to the target nucleic acid; Pdots chip manufacturing process: fixing the electrochemiluminescent nanoprobe according to claim 9 to the working electrode; and Sample detection step: adding the reaction solution obtained in the enzymatic reaction step to the working electrode, collecting the electrochemical luminescence signal and analyzing it.

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

14. The electrochemiluminescence detection method according to claim 13, wherein: The Cas enzyme comprises at least one Cas protein selected from Cas12a, Cas13a, Cas13b, Cas14a, Cas14b and Cas14c.

15. The electrochemiluminescence detection method according to any one of claims 12 to 14, wherein: The oligonucleotide chain in the electrochemiluminescent nanoprobe is single-stranded DNA.

16. The electrochemiluminescence detection method according to claim 15, wherein: The single-stranded DNA is a single-stranded DNA with a hairpin structure.

17. The electrochemiluminescence detection method according to any one of claims 12 to 14, wherein: The oligonucleotide chain in the electrochemiluminescent nanoprobe is a double-stranded DNA, and EXO III enzyme is further used in the enzymatic reaction process.

18. A kit for electrochemiluminescence detection based on Cas enzyme, comprising: An enzymatic reaction solution: comprising a Cas enzyme catalytic system, wherein the Cas enzyme catalytic system comprises a guide nucleic acid capable of binding to a target nucleic acid; and Pdots disposable detection chip: the electrochemiluminescent nanoprobe according to claim 9 is fixed on the working electrode of the screen-printed three-electrode sheet.

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

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