Electrochemical luminescence nanoprobe based on heat exciton organic luminescent molecules, sensor, detection method and detection kit

Abstract

The present invention relates to an electrochemical luminescence (ECL) nanoprobe based on a heat exciton organic light-emitting molecule, a sensor using the electrochemical luminescence nanoprobe, a detection method, and a detection kit. The preparation method of the electrochemical luminescence nano probe comprises the following steps: a heat exciton nano particle synthesis step: polymerizing heat exciton organic luminescence molecules and copolymer molecules to synthesize heat exciton nano particles; and a heat exciton nano-particle modification process: modifying the obtained heat exciton nano-particles by using an oligonucleotide chain modified by quenching agent molecules to obtain the modified heat exciton nano-particles. According to the present invention, the overall efficiency of the electrochemical luminescence can be improved so as to achieve the rapid non-amplification high-throughput detection of the target molecule, and the wide detection range and the extremely high sensitivity are provided.

Description

Technical Field

[0001] The present invention relates to an electrochemiluminescence (ECL) nanoprobe based on a thermoexciton organic luminescent molecule, a sensor using the electrochemiluminescence nanoprobe, a detection method and a detection kit. Background Art

[0002] Electroluminescence (EL) refers to the phenomenon that luminescent materials emit light under the action of electric field and are excited by current and voltage. It is a process that directly converts electrical energy into light energy. Organic electroluminescence refers to the phenomenon that thin film devices made of organic luminescent materials emit light under the excitation of electric field. The luminescence principle of organic electroluminescent devices is that, driven by external voltage, electrons and holes injected by electrodes recombine in organic matter to release energy, and transfer energy to the molecules of organic luminescent materials, so that they are excited and transition from the ground state to the excited state. When the excited molecules return to the ground state from the excited state, they undergo radiative transition and produce luminescence. The excited state can be divided into singlet (S) excitons and triplet (T) excitons according to the difference in spin multiplicity. The excited state molecules can be in the lowest energy singlet excited state S1 or triplet excited state T1, or in a higher singlet excited state (S2, S3, S4, ...) or triplet excited state (T2, T3, T4, ...). When a molecule in the S1 excited state returns to the S0 ground state through a radiative transition, "fluorescence emission" is produced. When a molecule in the T1 excited state returns to the S0 ground state through a radiative transition, "phosphorescence emission" is produced (see Fig.19 ).

[0003] Electrochemiluminescence (ECL) technology is a method that uses electrochemical principles to generate excited states on the electrode surface to induce specific luminescence. The excited state in the electrochemiluminescence process is generated by electron transfer reactions between intermediate free radicals formed by electron transfer between electroactive substances and electrodes.

[0004] Electrochemiluminescent systems can be divided into metal complex electrochemiluminescent systems and organic compound electrochemiluminescent systems (hereinafter sometimes referred to as "organic luminescent materials") according to the luminescent reagent, such as polyaromatic hydrocarbons such as organic fluorene, BODIPY derivatives, fluorene-based polymers and various organic nanomaterials. Commonly used electrochemiluminescent metal complexes include tris(bipyridine)ruthenium(II) complex ions (Ru(bpy)3 2+ ), which belongs to the phosphorescent luminescence system. When it is used as an ECL luminescent probe, the exciton utilization rate can reach 100%. Therefore, based on the tris(bipyridine) ruthenium(II) complex ion (Ru(bpy)

[0005] 3 2+) has become the standard method for electrochemiluminescence detection. However, the above-mentioned organic luminescent materials belong to the fluorescent luminescence system, and their exciton utilization rate can only reach 25%.

[0006] In terms of excited state, there are two traditional excited state modes for organic luminescent materials currently used in the field of electrochemical luminescence, namely, the localized excited state (LE) and the charge transfer excited state (CT). The electron-hole distribution of these two excited states and the exciton binding energy characteristics formed by them make the exciton utilization rate of the localized excited state low and the radiation transition rate / quantum yield (Φ PL ) is high, the exciton utilization rate of the charge transfer excited state is high while the radiative transition rate / quantum yield is low. The two cannot simultaneously achieve both high exciton utilization rate and high radiative transition rate / quantum yield, making it impossible for its luminous efficiency and intensity to reach the ideal optimal level, thereby limiting downstream applications, such as the efficiency of electrochemical luminescence.

[0007] In 2012, Ma Yuguang, Yang Bing and others innovatively applied the photophysical phenomenon of reverse intersystem crossing (RISC) to the design of organic electroluminescent materials and successfully developed a series of donor-acceptor fluorescent materials with different light colors. Through the effective high-energy reverse intersystem crossing process, the exciton utilization rate of their electroluminescent devices was greatly improved. n , n≥2) and the singlet state (S m , m≥1) is called the thermoexciton process, the luminescence mechanism based on the thermoexciton process is called the thermoexciton mechanism, and the luminescent material with obvious thermoexciton antisystem crossing is called the thermoexciton material. The thermoexciton material usually has a large T n -T1 energy level difference and small T n -S m The energy level difference of the state. Large T n -T1 energy level difference will inhibit T n Inward conversion to T1, small T n -S m Energy level difference can increase T n To S m The anti-system crossing rate. n To S m Intersystem crossing and T n The internal conversion to T1 competes with each other. In theory, when the intersystem crossing rate is high enough, the internal conversion is completely suppressed, all high-energy triplet excitons will be converted into singlet excitons, and the exciton utilization rate of the hot exciton material can reach 100% (see non-patent literature 1, Fig.19). Therefore, the hot exciton material can increase the generation ratio of singlet S1 excitons by realizing anti-intersystem crossing (hRISC) through an independent "hot exciton" channel. At the same time, the S1 state exciton has both the high exciton utilization rate of the CT state and the high radiative luminescence property of the LE state. Therefore, the hot exciton material has the property of hybrid localized-charge transfer excited state (Hybrid Locally-excitedand Charge-transfer, HLCT), which can have both high exciton utilization rate and high quantum yield. The hybrid localized-charge transfer excited state refers to a state that contains both the properties of the localized excited state (LE) and the characteristics of the charge transfer (CT) excited state, but the localized excited state and the charge transfer excited state are not a simple mixture, but a single excited state formed after hybridization.

[0008] At present, thermoexciton materials are mainly used in the field of semiconductor materials, such as the field of organic light-emitting diodes (OLEDs) (see non-patent document 2), which are new types of light-emitting materials after the first-generation organic light-emitting materials (fluorescent materials), the second-generation organic light-emitting materials (phosphorescent materials), and the third-generation organic light-emitting materials (thermally activated delayed fluorescence, TADF) materials.

[0009] Due to the excellent optical properties of thermoexciton materials, their application in biological detection is expected. Patent document 1 attempts to use thermoexciton materials for fluorescence detection, but there is no successful example of their use in the field of ECL detection.

[0010] Prior art literature

[0011] Patent Literature

[0012] Patent document 1: CN110981821A

[0013] Non-patent literature

[0014] Non-patent literature 1: Xu Yuwei, Research on high-energy antisystem crossing process and optoelectronic properties of hot exciton luminescent materials, PhD dissertation of South China University of Technology, 2020.

[0015] Non-patent literature 2: Xiaojie Chen, Dongyu Ma et al., Hybridized Local and Charge-Transfer Excited-State Fluorophores through the Regulation of theDonor–Acceptor Torsional Angle For Highly Efficient Organic Light-EmittingDiodes, CCS Chem. 2022, 4, 1284–1294. Summary of the invention

[0016] Problems to be solved by the invention

[0017] As is known to all, organic light-emitting diodes (OLEDs) are all-solid-state devices without liquid components. In organic light-emitting diodes, the luminescent molecules are excited by external voltage and transition from the ground state to the excited state. When the excited molecules return to the ground state from the excited state, they undergo radiative transitions to produce light. The detection environment of electrochemical luminescence is liquid, and the luminescent molecules undergo electrochemical reactions on the electrode surface to produce excited states, and then undergo radiative transitions to emit light. Therefore, the luminescence principles and luminescence environments of organic light-emitting diodes and electrochemical luminescence are different. When considering using the thermoexciton materials currently used in the field of organic light-emitting diodes (OLEDs) as luminescent molecules for electrochemical luminescence (ECL) detection, the first thing to consider is feasibility and reliability.

[0018] The inventors of the present application have conducted detailed and in-depth studies on the above-mentioned issues and found that by using nanotechnology to prepare thermoexciton organic luminescent molecules into electrochemiluminescent nanoprobes (hereinafter sometimes also referred to as "ECL nanoprobes") and modifying them with oligonucleotide chains modified with quencher molecules, the overall efficiency of electrochemiluminescence can be improved, thereby achieving rapid non-amplified high-throughput detection of target molecules with a wide detection range and extremely high sensitivity.

[0019] Methods for solving problems

[0020] The present invention provides the following technical solutions.

[0021] <1> A method for preparing an electrochemiluminescent nanoprobe, comprising the following steps:

[0022] a thermoexciton nanoparticle synthesis step, polymerizing the thermoexciton organic light-emitting molecules and the copolymer molecules to synthesize the thermoexciton nanoparticles; and

[0023] The thermoexciton nanoparticle modification step is to modify the obtained thermoexciton nanoparticles using an oligonucleotide chain modified with a quencher molecule to obtain modified thermoexciton nanoparticles.

[0024] <2> The preparation method according to <1>, wherein

[0025] The thermoexciton organic light-emitting molecule comprises an electron donor (D) structure selected from the following group (a) and an electron acceptor (A) structure selected from the following group (b);

[0026]

[0027] <3> The preparation method according to <2>, wherein

[0028] The electron donor (D) is carbazole, and the electron acceptor (A) is benzothiadiazole.

[0029] <4> The preparation method according to <3>, wherein

[0030] The thermal exciton organic light-emitting molecule is the following BCzP-BT:

[0031]

[0032] <5> The production method according to any one of <1> to <4>, wherein

[0033] 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),

[0034]

[0035] <6> The preparation method according to <5>, wherein

[0036] The thermoexciton organic luminescent molecules and the copolymer molecules are synthesized into thermoexciton nanoparticles through a nano coprecipitation method.

[0037] <7> The production method according to any one of <1> to <6>, wherein

[0038] The nanoparticles are nanospheres, nanotubes, nanorods or nanoonions.

[0039] <8> The production method according to any one of <1> to <7>, wherein

[0040] The quencher molecule is a black hole quencher, a dark quencher, or an amine-reactive quencher.

[0041] <9> An electrochemiluminescent nanoprobe prepared by the method described in any one of <1> to <8>.

[0042] <10> An electrochemiluminescent sensor obtained by dropping the electrochemiluminescent nanoprobe described in <9> onto a working electrode.

[0043] <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.

[0044] <12> The electrochemiluminescence sensor according to <11>, wherein

[0045] The working electrode is a gold-indium tin oxide electrode.

[0046] <13> An electrochemiluminescence detection method using the electrochemiluminescence sensor according to any one of <10> to <12>.

[0047] <14> The electrochemiluminescence detection method according to <13>, comprising the following steps:

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

[0049] Sample detection step: adding the sample reaction solution to the electrochemiluminescence sensor, collecting the electrochemiluminescence signal and analyzing it.

[0050] <15> The electrochemiluminescence detection method according to <14>, wherein the concentration of the Cas protein in the Cas enzyme in the sample reaction solution is 40 nM or more.

[0051] <16> The electrochemiluminescence detection method according to <14> or <15>, wherein the amount of crRNA used is greater than that of Cas protein.

[0052] <17> The electrochemiluminescence detection method according to any one of <13> to <16>, wherein the incubation time of the sample reaction solution on the electrochemiluminescence sensor is 30 minutes or more.

[0053] <18> An electrochemiluminescence detection kit comprising the electrochemiluminescence sensor according to any one of <10> to <12> and a detection reagent containing a Cas enzyme.

[0054] Effects of the Invention

[0055] In the present invention, thermoexciton organic light-emitting molecules are applied to the field of electrochemiluminescence for the first time, and nanoparticles are prepared using the thermoexciton organic light-emitting molecules as light-emitting bodies for the first time, thereby providing electrochemiluminescence (ECL) nanoprobes based on thermoexciton organic light-emitting molecules that can achieve rapid non-amplified high-throughput detection of target molecules and have a wide detection range and extremely high sensitivity, sensors using the electrochemiluminescence nanoprobes, detection methods and detection kits. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 It is a schematic diagram showing the preparation method of the electrochemiluminescent nanoprobe of the present invention.

[0057] Figure 2Schematic diagram of the ECL switch based on ssDNA-BB NRs nanoprobe of the present invention.

[0058] Figure 3 Schematic diagram of the preparation and detection of the sensor array based on ssDNA-BB NRs of the present invention.

[0059] Figure 4 A schematic diagram showing the comparison of the luminescence principles of TADF compounds and thermoexciton materials.

[0060] Figure 5 To show the NMR ( 1 H NMR) and mass spectrometry results, wherein Figure 5 (a) shows the NMR of BCzP-BT molecule ( 1 HNMR) analysis results, Figure 5 (b) is a diagram showing the results of mass spectrometry analysis of BCzP-BT molecules.

[0061] Figure 6 : is a diagram showing the analysis results of the excited state mode of the BCzP-BT molecule prepared in Example 1 of the present invention. Among them, Figure 6 (a) shows the normalized UV-visible absorption and FL spectra of BCzP-BT in different polar solvents. Figure 6 (b) shows that CzP, BT and BCzP-BT in BCzP-BT molecule are 10 -5 M FL spectrum in THF solution.

[0062] Figure 7 Graph showing the excited state properties of the BCzP-BT molecule prepared in Example 1 of the present invention.

[0063] in, Figure 7 (a) represents the Stokes shift (v a -v f ) and the change curve of the orientation polarizability (Δf) of the solvent, Figure 7 (b) is a graph showing the transient PL decay curve of BCzP-BT in THF.

[0064] Figure 8 : is a diagram showing the analysis results of the possibility of the thermal exciton process of BCzP-BT prepared in Example 1 of the present invention. Among them, Figure 8 (a) is a diagram showing the energy level distribution of BCzP-BT molecules, Figure 8 (b) is a diagram showing the natural transition orbital simulation results of the BCzP-BT molecule.

[0065] Fig. 9: is a graph showing the characterization results of BB NRs prepared in Example 1 of the present invention. Among them, Fig. 9 (a) is the TEM image, Fig. 9 (b) is the AFM image. Fig. 9 (c), (d), and (e) are graphs showing the UV and fluorescence spectra and fluorescence lifetimes of BB NRs and BCzP-BT, respectively.

[0066] Fig.10 : is a graph showing the analysis results of the ECL properties of BB NRs prepared in Example 1 of the present invention. Fig.10 (a) is a graph showing the cyclic voltammetry (CV) curve and ECL curve of BB NRs / GCE, where curve a represents the result of first performing an anodic scan, and curve b represents the result of first performing a cathodic scan; Fig.10 (b) is a graph showing the annihilation state ECL curve of BB NRs / GCE.

[0067] Fig.11 The ECL analysis results of the BB NRs prepared in Example 1 of the present invention in the presence of a co-reactant are shown in FIG. Fig.11 (a), (b), and (c) respectively represent the anodic ECL behavior and mechanism of BB NRs when tripropylamine (TPrA) is used as a co-reactant. Fig.11 Curve a in (a) and (b) represents TPrA itself, curve b represents the absence of TPrA, and curve c represents the coexistence with TPrA; Fig.11 (d), (e), and (f) respectively represent the cathode ECL behavior and mechanism of BB NRs when K2S2O8 is used as the co-reactant. Fig.11 Curve a in (d) and (e) represents S2O8 2- The curve b indicates no S2O8 2- , Curve c represents the S2O8 2- coexist.

[0068] Fig.12 To represent Ru(bpy)3 2+ is a graph showing the ECL efficiency of BB NRs prepared in Example 1 of the standard present invention, wherein: Fig.12 (a) and (b) represent BB NRs and Ru(bpy)3 2+ Figure 2 shows the anodic current-time curve and ECL spectrum of the modified GCE. Fig.12 (c) and (d) represent BB NRs and Ru(bpy)3 2+ Cathodic current-time curves and ECL spectra of modified GCE.

[0069] Fig.13: is a graph showing the analysis results of the ECL attribution of the BB NRs prepared in Example 1 of the present invention. Fig.13 (a) is a graph showing the anode and cathode ECL spectra and FL spectra of BB NRs. Fig.13 (b) is a graph showing the CV curve of the glassy carbon electrode modified with BB NRs. Fig.13 (c) is a schematic diagram showing the energy band gap between the lowest unoccupied molecular orbital (LUMO) and the highest occupied molecular orbital (HOMO) of the BCzP-BT molecule.

[0070] Fig.14 : is a diagram showing the characterization results of the ssDNA-BB NRs probe prepared in Example 2 of the present invention. Among them, Fig.14 (a) is a graph showing the UV absorption spectrum and FL spectrum of the ssDNA-BB NRs probe. Curves a, b, c, and d in the figure represent the UV absorption spectra of ssDNA-BB NRs, ssDNA, and BB NRs, and the FL spectrum of BB NRs, respectively. Fig.14 (b) is a graph showing the characterization results of Zeta potential, Fig.14 (c) is a graph showing the UV characterization results of the filtrate from each ultrafiltration during the preparation of the ssDNA-BB NRs probe, and the numbers 1 to 9 in the graph represent the filtrate from the 1st to the 9th time, respectively.

[0071] Fig.15 : is a diagram showing the analysis results of the signal switch feasibility of the ssDNA-BB NRs probe prepared in Example 2 of the present invention. Among them, Fig.15 (a) is a graph showing the results of fluorescence characterization of ssDNA-BB NRs and Cas12a system before (curve a) and after (curve b); Fig.15 (b) is a graph showing the normalized ECL imaging intensity characterization results of different ITO electrodes, where curve a represents the BB NRs modified electrode, curve b represents the ssDNA-BB NRs modified electrode, curve c represents the ssDNA-BB NRs + Cas12a system modified electrode, and curve d represents the ssDNA-BB NRs + Cas12a + crRNA modified electrode.

[0072] Fig.16 Graph showing the results of ECL detection under different detection conditions in Example 3 of the present invention. Fig.16 (a) shows the detection results at different Cas12a concentrations, Fig.16 (b) represents the detection results under different usage ratios of Cas12a and crRNA, Fig.16 (c) shows the detection results at different incubation times on the chip.

[0073] Fig.17 : is a diagram showing the ECL imaging results when using ITO electrode and Au / ITO in Example 3 of the present invention. Fig.17 (a), (b) and (c) show the CV, ECL curves and ECL images of BB NR modified ITO and Au / ITO electrodes, respectively.

[0074] Fig.18 This is a diagram showing the detection results of ECL detection of HPV16 DNA target nucleic acid using a sensor array in Example 3 of the present invention. Fig.18 (a) shows the ECL imaging results and linear curve obtained by detecting HPV16 DNA at a concentration of 1fM to 10nM; Fig.18 (b) shows the analysis results of the detection specificity of ECL detection of HPV16 DNA, wherein a represents 1 nM HPV18 DNA, b represents 1 nM HPV31 DNA, c represents 1 nM HPV58 DNA, d represents 100 pM HPV16 DNA, and e represents a mixed solution containing 1 nM HPV18, 31, 58 DNA and 100 pM HPV16 DNA.

[0075] Fig.19 Schematic diagram showing the luminescence principle of hot exciton materials with hybrid localized charge transfer excited state (HLCT) properties. DETAILED DESCRIPTION

[0076] 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.

[0077] <Electrochemiluminescent nanoprobe and preparation method thereof>

[0078] One embodiment of the present invention relates to a method for preparing an electrochemiluminescent nanoprobe, which comprises the following steps:

[0079] a thermoexciton nanoparticle synthesis step, polymerizing the thermoexciton organic light-emitting molecules and the copolymer molecules to synthesize the thermoexciton nanoparticles; and

[0080] The thermoexciton nanoparticle modification step is to modify the obtained thermoexciton nanoparticles using an oligonucleotide chain modified with a quencher molecule to obtain modified thermoexciton nanoparticles.

[0081] (Thermoexciton organic light-emitting molecules)

[0082] The thermoexciton organic light-emitting molecules that can be used in the present invention are not particularly limited, and any thermoexciton organic light-emitting molecules with localized-charge transfer hybrid excited state properties can be used, such as various thermoexciton organic light-emitting molecules described in non-patent documents 1 and 2.

[0083] Thermionic materials are molecules with the property of localized charge transfer hybridized excited states (HLCT). However, it should be noted that molecules with localized charge transfer hybridized excited states are not necessarily thermonic materials. For example, singlet materials and triplet materials have the property of localized charge transfer hybridized excited states, but they are not thermonic materials.

[0084] Thermionic materials generally have a one-dimensional fused ring structure as a building block and have donor and acceptor units containing heteroatoms.

[0085] Examples of the fused ring structure include a benzene ring, a naphthalene ring, and an anthracene ring. These benzene rings, naphthalene rings, and anthracene rings may further have a substituent.

[0086] The specific structure of the donor-acceptor unit may be, for example, a DA structure or a DAD structure, wherein D represents a donor unit (donor) and A represents an acceptor unit (acceptor).

[0087] Examples of the donor-acceptor unit include a donor-acceptor unit composed of a donor (D) selected from the following group (a) and an acceptor (A) selected from the following group (b).

[0088]

[0089] Thermionic materials are a type of luminescent material that breaks through the spin statistics limitations of traditional fluorescent materials. In terms of the type of light emitted, they can include blue-light thermonic materials, green-light thermonic materials, and red-light thermonic materials.

[0090] The donor and acceptor strength of the thermoexciton material is moderate, so it is easier to obtain pure blue and deep blue light emission in the molecular design of DA-type fluorescent materials. Currently, the blue light thermoexciton materials that have been successfully produced include blue light thermoexciton materials containing phenanthroimidazole units, blue light thermoexciton materials containing anthracene units, and other types of blue light thermoexciton materials.

[0091] Examples of the blue light-emitting thermoexciton material containing a phenanthroimidazole unit include the following materials.

[0092] Chemical structure 1: Blue light-emitting thermoexciton material containing phenanthroimidazole units

[0093]

[0094]

[0095] As the blue light-emitting thermoexciton material containing anthracene units, for example, the following materials can be cited. Chemical Structural Formula 2: Blue light-emitting thermoexciton material containing anthracene units

[0096] As other types of blue light-emitting thermoexciton materials, for example, the following materials can be cited. Chemical structure formula 3: Other types of blue light-emitting thermoexciton materials

[0097] As a representative green light thermoexciton material, for example, the following materials can be cited: Chemical structure 4: Green light thermoexciton material

[0098]

[0099] Representative red light thermoexciton materials include the following:

[0100] Chemical structure 5: Red light thermoexciton material

[0101]

[0102] In addition, for DAD-type hot exciton materials, the connection mode of the acceptor (A) unit and the donor (D) unit can be ortho-connected, meta-connected and para-connected. Among them, when the para-connection is performed, the torsion angle between the acceptor (A) unit and the donor (D) unit is reduced due to the ability to reduce steric hindrance, and a more locally excited component can be given, resulting in faster radiation decay, so that a higher external quantum efficiency (EQE) can be obtained than the ortho- and meta-positions, and therefore it is more preferred. For example, in the embodiment of the present invention, 4,7-bis(4-(9H-carbazole-9-yl)phenyl)benzo[c][1,2,5]thiadiazole (p-BCzP-BT) is exemplified. In this compound, the electron donor (D) is carbazole and the electron acceptor (A) is benzothiadiazole.

[0103] It should be noted that any hot exciton material having the property of localized-charge transfer hybrid excited state (HLCT) can be used in the present invention. The hot exciton materials listed above are only examples, and it is clear to those skilled in the art that derivatives of the above compounds formed by substitution with one or more substituents (such as alkyl, hydroxyl, halogen, etc.), and their spatial isomers, etc. can also be used in the present invention.

[0104] The method for preparing the thermoexciton material used in the present invention is not particularly limited, and it can be synthesized by chemical reaction through synthesis routes such as Suzuki coupling reaction, Stiller coupling reaction and cross coupling reaction, or it can be purchased through commercial channels, without particular limitation.

[0105] The synthesis route of Suzuki coupling reaction is adopted in the embodiment of the present invention, but other synthesis routes can also be adopted as needed. Suzuki coupling reaction is a commonly used carbon-carbon reaction, which uses zero-valent palladium as a catalyst to react aromatic or alkenyl boronic acid with halogenated aromatic hydrocarbons such as chlorine, bromine, and iodine. As a palladium catalyst, for example, tetrakis(triphenylphosphine)palladium (Pd(PPh3)4), Pd[P(o-toly)3]3 and Buchwald catalyst can be used. As aromatic or alkenyl boronic acid, and halogenated aromatic hydrocarbon, the corresponding compound raw materials can be used respectively according to the electron donor (D) and electron acceptor (A) in the desired synthesized thermal exciton organic light-emitting molecule, without special limitation.

[0106] (Synthesis of Thermoexciton Nanoparticles)

[0107] In the present invention, thermoexciton nanoparticles are synthesized by polymerizing thermoexciton organic light-emitting molecules and copolymer molecules.

[0108] 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 thermoexciton organic light-emitting molecules 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 nanorods (NR) were successfully prepared, but the nanoparticles that can be used in the present invention are not limited to nanorods, and other nanoforms such as nanospheres can also be formed according to the different compound raw materials and synthesis routes used.

[0109] In the present invention, the thermoexciton organic light-emitting molecules and the copolymer molecules can be synthesized by methods known in the prior art for preparing organic material nanostructures, such as nano-coprecipitation method, microemulsion method and self-assembly method (Liu Ronghua, Preparation and Application of Novel Fluorescent Conjugated Polymer Nanoparticles, Doctoral Dissertation of 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.

[0110] 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.

[0111] The nano-coprecipitation method is to dissolve the luminescent molecule 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.

[0112] 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, proteins or antibodies 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.

[0113] 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.

[0114]

[0115] The diameter of the nanoparticles obtained by the nano-coprecipitation method can be as small as 1nm to 2nm.

[0116] In addition, it should be noted that the surface functionalization modification of thermoexciton 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.).

[0117] 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 oligonucleotides, thereby amplifying the electrochemical signal and improving the sensitivity of ECL detection.

[0118] (Modification of Thermoexciton Nanoparticles)

[0119] In the present invention, the modification of thermoexciton nanoparticles refers to the oligonucleotide modification of thermoexciton nanoparticles. Specifically, the surface-functionalized thermoexciton nanoparticles are modified using oligonucleotide chains modified with quencher molecules to obtain oligonucleotide-modified thermoexciton nanoparticles (hereinafter sometimes also referred to as "ECL nanoprobes").

[0120] The connection method between the oligonucleotide and the thermoexciton nanoparticle is not limited to covalent reaction (for example, through NH2-COOH cross-linking), and other connection methods such as electrostatic interaction and affinity adsorption can also be used.

[0121] 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.

[0122] 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 thermoexciton nanoparticles and resonance energy transfer can be generated between the quencher molecule and the thermoexciton nanoparticles, and can be designed with reference to various oligonucleotide chains commonly used in the prior art for connection.

[0123] From the perspective of the sensitivity of Cas enzyme cleavage, the number of nucleotides in the oligonucleotide chain should not be too many. Calculated on a single chain, it is preferably 3 to 20, more preferably 4 to 11, and further preferably 5, 6, 7, 8, 9, 10, 11 or 12.

[0124] 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 thermoexciton nanoparticle, the resonance energy can be transferred from the excited nanoparticle to the quencher molecule, thereby quenching the emission of the thermoexciton organic light-emitting molecule.

[0125] 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. TM Reagents, i.e. BHQ series quenchers), dark quenchers (e.g. DABCYL series quenchers, etc.), BlackBerry TMQuencher (i.e., BBQ series quencher), Blueberry quencher (i.e., BLU series quencher), or amine reactive quencher (i.e., QSY series quencher), 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.

[0126] 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.

[0127] Figure 1 The figure is a schematic diagram showing the preparation method of the electrochemiluminescent nanoprobe of the present invention. Taking the thermoexciton organic luminescent molecule BCzP-BT molecule prepared in Example 1 of the present invention as an example, the thermoexciton organic luminescent molecule BCzP-BT molecule and the copolymer molecule PSMA are polymerized by nano-coprecipitation method to obtain thermoexciton nanoparticles (BB NRs), and then the oligonucleotide DNA with the quencher molecule BHQ connected to the end is connected to the thermoexciton nanoparticles by the covalent reaction of NH2-COOH, thereby obtaining the electrochemiluminescent nanoprobe (ECL nanoprobe) of the present invention.

[0128] 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 cleaved by the activated Cas enzyme, the quencher molecule is connected to the surface of the thermoexciton nanoparticle through the oligonucleotide, and the thermoexciton organic light-emitting molecule is quenched, and the sensing electrode shows a low ECL intensity (ECL OFF), and when the oligonucleotide is cleaved by the activated Cas enzyme, the quencher molecule is detached from the surface of the thermoexciton nanoparticle, so that the sensing electrode shows a strong ECL signal (ECL ON).

[0129] <Electrochemiluminescence sensor>

[0130] 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.

[0131] 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.

[0132] In addition, as verified in the embodiments of the present invention later, since gold has a good catalytic effect on co-reactants such as tripropylamine, the gold-indium tin oxide (Au-ITO) electrode can further enhance the ECL signal compared to the ITO electrode. Therefore, the Au-ITO electrode is more preferably used in the present invention.

[0133] <Electrochemiluminescence detection method>

[0134] The electrochemiluminescence detection method of the present invention (hereinafter sometimes referred to as the ECL detection method) uses the above-mentioned ECL sensor and may include the following steps:

[0135] Enzymatic reaction step: adding the sample to be tested to the Cas enzyme catalytic system to obtain a sample reaction solution, wherein the Cas enzyme catalytic system comprises a guide nucleic acid capable of binding to the target nucleic acid; and

[0136] Sample detection step: adding the sample reaction solution to the electrochemiluminescence sensor, collecting the electrochemiluminescence signal and analyzing it.

[0137] In addition, in the ECL detection method of the present invention, the ECL sensor of the present invention may not be used directly, but may be prepared when used, that is, the ECL detection method of the present invention may further include a thermoexciton chip preparation step: the ECL nanoprobe of the present invention is dripped onto the working electrode to obtain the thermoexciton chip of the present invention (that is, the ECL sensor).

[0138] 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.

[0139] 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.

[0140] 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 the Cas enzyme is targeted to the target nucleic acid molecule, the Cas enzyme will be activated and obtain the activity of non-specific cleavage of any nucleic acid molecule.

[0141] The Cas enzyme may, for example, include 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.

[0142] 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.

[0143] 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.

[0144] Next, the principle of the signal switch of the present invention and the preparation and detection method of the ECL detection chip are specifically described by taking the thermoexciton organic nanorods (BB NRs) and the sensor array prepared in the embodiment of the present invention as an example.

[0145] Regarding the principle of the signal switch of the present invention, Figure 2The ECL detection chip shown is specifically described as an example. The thermoexciton nanoparticles (BB NRs) of the present invention are connected to oligonucleotides (ssDNA) modified with quencher molecules (BHQ) at the tail end to obtain the ECL nanoprobe (ssDNA-BB NRs nanoprobe) of the present invention as a reporter probe in ECL detection. Since the ssDNA tail end is modified with BHQ, BB NRs is in the ECL OFF state. The ssDNA-BB NRs nanoprobe is directly drop-coated on the surface of the ITO electrode to construct an ECL sensing interface. The reaction solution is added dropwise to the sensing interface. When the Cas enzyme (Cas12a enzyme) is not activated, BHQ is connected to the BB NRs surface through ssDNA, and the sensing electrode shows a low ECL intensity (ECL OFF); on the contrary, when the target molecule is present and the Cas12a enzyme is activated, ssDNA is sheared, so that BHQ is separated from the BB NRs surface, the ECL of BBNRs is restored, and a strong ECL signal (ECL ON) is generated at the sensing interface.

[0146] Regarding the preparation and detection method of the ECL detection chip of the present invention, Figure 3 The ECL detection method shown is specifically described as an example. First, on an indium tin oxide (ITO) electrode or a gold-indium tin oxide (Au / ITO) electrode obtained by deposition of gold, the ECL nanoprobe of the present invention is added dropwise to obtain the ECL electrode (i.e., ECL sensor) of the present invention. The reaction solution containing Cas protein, crRNA, and the target nucleic acid to be tested that may be contained is added dropwise to the ECL electrode, and by the above-mentioned signal switch, when the target molecule does not exist and the Cas enzyme is not activated, the sensing electrode displays a low ECL intensity (ECL OFF); and when the target molecule exists and the Cas12a enzyme is activated, the ECL of BB NRs is restored, and a strong ECL signal (ECL ON) is generated on the sensing interface, so as to realize the electrochemiluminescence detection based on the thermoexciton organic light-emitting molecule of the present invention.

[0147] From the perspective of obtaining better ECL signal intensity, it is preferred to optimize the ECL detection conditions, such as the concentration of Cas protein in the sample reaction solution, the ratio of Cas protein to crRNA, and the reaction time of the sample reaction solution on the chip (e.g., incubation time).

[0148] There is no particular limitation on the concentration of the Cas protein, but from the perspective of obtaining a better ECL signal intensity, it can be, for example, 40 nM or more, preferably 42 nM or more, more preferably 45 nM to 70 nM, and particularly preferably 50 nM to 60 nM.

[0149] There is no particular limitation on the ratio of Cas protein to crRNA. From the perspective of obtaining better ECL signal intensity, it is preferred that the amount of crRNA used is greater than that of Cas protein. For example, the ratio of Cas protein to crRNA can be 1:1.25 to 1:5, preferably 1:1.35 to 1:4, more preferably 1:1.5 to 1:3, and particularly preferably 1:1.5 to 1:2.

[0150] There is no particular limitation on the incubation time of the sample reaction solution on the chip. From the perspective of obtaining a better ECL signal intensity, it can be, for example, 30 minutes or more, preferably 35 to 120 minutes, more preferably 37 to 90 minutes or more, and particularly preferably 40 to 60 minutes.

[0151] As verified in the examples of the present invention, the ECL efficiency of the thermoexciton material of the present invention can reach 56.7%, and the fluorescence quantum yield (Φ PL ) can reach more than 89%, which is higher than the previously reported Pdots based on the thermally activated delayed fluorescence (TADF) principle (the most efficient reported organic ECL nanomaterial) (Anal. Chem. 2022, 94, 15695-15702) and Pdots based on traditional fluorescent materials (Chem. Sci. 2019, 10, 6815; Anal. Chem. 2016, 88, 845).

[0152] Figure 4 Schematic diagram showing the comparison of the luminescence principle of TADF compounds and thermoexciton materials. Figure 4 As shown in Figure 1, the RISC process of TADF compounds occurs between T1 and S1, so they are also called cold exciton materials. The charge transfer (CT) properties of both the lowest singlet (S1) and triplet (T1) excited states of TADF compounds give them low Φ PL At the same time, the slow reverse intersystem hopping (RISC) of TADF compounds from T1 to S1 easily causes the accumulation of long-lived triplet excitons and leads to serious annihilation. In contrast to the cold exciton pathway of TADF compounds, the RISC of hot exciton materials is in the high-energy excited state (hRISC, T m →S n , m≥2, n≥1), where the large T m -T1 energy gap suppresses the m Internal conversion (IC) to T1, narrow T m -S nEnergy splitting promotes hRISC. Therefore, due to the accelerated RISC, hot exciton materials utilize triplet excitons more fully than TADF materials. The hRISC process alleviates the quenching of T1 excitons. In addition, different from the strong CT characteristics of TADF materials, hot exciton materials have HLCT excited state properties, where the S1 state is an LE or LE-dominated HLCT state, while the T m and S n states are CT states, endowing it with high Φ PL and 100% exciton utilization efficiency.

[0153] In addition, taking the detection of HPV16 DNA as an example, the detection ranges and detection limits of the ECL detection method of the present invention and other methods recorded in the prior art were compared, and the comparison results are shown in Table 1.

[0154] Table 1

[0155] method Detection range (nM) Detection limit <![CDATA[Electrochemiluminescence 1 > 1～100 0.6nM <![CDATA[Electrochemiluminescence 2 > 0.1～200 0.03nM <![CDATA[Electrochemiluminescence 3 > 0.001～10 0.48pM <![CDATA[Electrochemiluminescence 4 > 0.00001～0.015 7.6fM <![CDATA[Electrochemistry 5 > 0.5～100 150pM <![CDATA[Electrochemistry 6 > 0.0000001-0.01 18.6aM The present invention 0.000001-10 0.6fM

[0156] Among them, the superscripts 1 to 6 respectively represent the following documents:

[0157] 1: L. Wang et al., Microchem. J. 2022, 181, 107818.

[0158] 2: Y. Nie et al., Biosens. Bioelectron. 2020, 160, 112217.

[0159] 3: P. Liu et al., Biosens. Bioelectron. 2021, 176, 112954.

[0160] 4: Y. He et al., ACS Appl. Mater. Interfaces, 2021, 13, 298.

[0161] 5: S. Jampasa et al., Sensor. Actuat. B-Chem, 2018, 265, 514.

[0162] 6: Y. He et al., Sensor. Actuat. B-Chem, 2020, 320, 128407.

[0163] As can be seen from Table 1, the ECL chip (sensor) made of the hot exciton organic light-emitting molecule of the present invention has a wider detection range and higher sensitivity compared with other reported ECL chips.

[0164] <ECL detection kit>

[0165] The ECL detection kit of the present invention may directly contain the ECL sensor of the present invention, or may contain the ECL nanoprobe of the present invention, which can be added dropwise to the working electrode during detection. In addition to the ECL sensor, other reagents required for ECL detection, such as detection reagents or instructions containing Cas enzymes, may also be included.

[0166] 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.

[0167] Example

[0168] The following examples are given to specifically illustrate the thermoexciton 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.

[0169] The reagents, experimental methods and equipment used in the examples are as follows:

[0170] (1) Reagents:

[0171] Tetrakis(triphenylphosphine)palladium (Pd(Pph3)4) was purchased from BitPharma Technology Co., Ltd. (Shanghai, China).

[0172] 4-(9H-Carbazol-9-yl)phenylboronic acid pinacol ester was purchased from MacLean Biochemical Technology Co., Ltd. (Shanghai, China).

[0173] 4,7-Dibromobenzo[c]-1,2,5-thiadiazole was purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China).

[0174] LbaCas12a (10 μM solution) was purchased from New England Biolabs (Ipswich, MA, UK).

[0175] Polystyrene-maleic anhydride (PSMA, average Mw: 1700), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), polyethylene glycol (PEG, average Mw: 3350), 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride (EDC), and tetrabutylammonium perchlorate were purchased from Sigma-Aldrich (Shanghai, China).

[0176] Tripropylamine (TPrA, >98%) was purchased from J&K Chemical Co., Ltd. (Beijing, China).

[0177] Indium tin oxide (ITO) glass sheets (100 × 100 mm × 1.1 mm) were purchased from Zhuhai Kaiwei Optoelectronics Technology Co., Ltd. (Zhuhai, China).

[0178] Diethylpyrocarbonate (DEPC) water and all oligonucleotides listed in Table 2 were purchased from Sangon Biotechnology Co., Ltd. (Shanghai, China).

[0179] In addition, phosphate buffered saline (PBS, 0.1 M, pH 7.4) was prepared by mixing stock solutions of KH2PO4 and Na2HPO4.

[0180] The oligonucleotide sequences used in the examples are as follows:

[0181] Table 2

[0182]

[0183] (2) Experimental methods and equipment

[0184] TEM images were obtained using a TECNAI G2 F20 transmission electron microscope (FEI, USA).

[0185] Zeta potential was recorded using a 90Plus DynaPro NanoStar (Brookhaven Instrument Corporation, USA).

[0186] 1 H NMR spectra were measured by Bruker AvanceIII 500HD spectrometer (Germany) with deuterated CDCl3 as solvent and tetramethylsilane (TMS) as internal standard.

[0187] The UV-vis absorption spectra were obtained using a UV-3600 spectrophotometer (Shimadzu, Japan).

[0188] The photoluminescence (PL) spectra and fluorescence lifetime were measured using a FLS-980 fluorescence spectrophotometer (Edinburgh, UK).

[0189] AFM was acquired by an atomic force microscope (BRUKER Dimension Icon, Germany) using the ScanAsyst mode.

[0190] 5 nm Cr and 50 nm Au layers were deposited on the ITO glass slide by PVD75 electron beam evaporation system (Kurt J. Lesker, USA).

[0191] The ECL spectra were obtained using a homemade ECL spectrometer built in the laboratory of Professor Zou Guizheng of Shandong University, which consisted of an Acton SP2300i monochromator equipped with a liquid N2-cooled PyLoN 400BR-eXcelon digital CCD detector (Princeton, USA) and a VersaSTAT3 electrochemical analyzer (Princeton, USA).

[0192] The cyclic voltammetry experiments were performed on a CHI 630D electrochemical workstation (CHI Instrument Co., Ltd., China).

[0193] Anodic and cathodic 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.

[0194] ECL imaging was performed on a home-made ECL imaging system equipped with a focusing lens (EF 50 mmf / 1.2L USM, Canon), an electron multiplying charge-coupled device (EMCCD, iXon Ultra, Andor, UK), and a CHI-660D electrochemical workstation with a setup containing Au / ITO electrode as the working electrode, Ag / AgCl wire as the reference electrode, and Pt wire as the counter electrode.

[0195] Example 1: Preparation and characterization of hot exciton organic nanorods (BB NRs)

[0196] a. Synthesis of thermal exciton molecule BCzP-BT:

[0197] First, 4-(9H-carbazole-9-yl)phenylboronic acid pinacol ester (Cz, 1.1817 g, 3.2 mmol), 4,7-dibromobenzo[c]-1,2,5-thiadiazole (BT, 0.4145 g, 1.41 mmol), K2CO3 (2.0040 g, 14.5 mmol) and tetrakis(triphenylphosphine)palladium (Pd(Pph3)4, 0.2311 g, 0.2 mmol) were added to a mixture of 20 mL toluene and 4 mL water. The Shrek tube was stirred at 90°C for 18 hours under an argon atmosphere. The reaction mixture was extracted with dichloromethane and purified by silica gel column chromatography using petroleum ether and dichloromethane as eluents to obtain 0.7512 g of BCzP-BT product (i.e., 4,7-bis(4-(9H-carbazole-9-yl)phenyl)benzo[c][1,2,5]thiadiazole (p-BCzP-BT, hereinafter referred to as "BCzP-BT") with a yield of 86%. By nuclear magnetic resonance ( 1 H NMR) and mass spectrometry were used to verify the synthesis of the BCzP-BT molecule, and the results are shown in Figure 5 .

[0198] Figure 5 (a) is the NMR ( 1 H NMR) analysis results, Figure 5 (b) is a graph showing the results of mass spectrometry analysis of the BCzP-BT molecule prepared in Example 1 of the present invention. Figure 5 (a) and (b) show that the present invention successfully synthesized the BCzP-BT molecule.

[0199] b. Preparation of hot exciton organic nanorods (BB NRs):

[0200] First, a 2.5 mL THF solution containing 50 μg / mL BCzP-BT and 10 μg / mL polystyrene maleic anhydride copolymer (PSMA, Mn=1700) was ultrasonicated at room temperature for 20 min; then the above solution was quickly injected into 10 mL water and ultrasonicated for 30 seconds; the solvent THF was removed by rotary evaporation at 35°C, and then filtered using a polyethersulfone syringe to obtain BB NRs.

[0201] c. Characterization of the excited state mode and properties of BCzP-BT molecules:

[0202] The excited state mode of the BCzP-BT molecule prepared in Example 1 of the present invention was analyzed and the results are shown in Figure 6 middle.

[0203] First, the normalized UV-visible absorption and fluorescence (FL) spectra of BCzP-BT molecules in different polar solvents were investigated and the results are shown in Figure 6 (a). Due to the transfer of intramolecular charge from Cz to BT unit, BCzP-BT exhibits broad absorption at about 400nm. The shape and position of this UV absorption peak do not change much with the increase of solvent polarity. However, with the increase of solvent polarity, the fluorescence spectrum of BCzP-BT shows a red shift of 68nm and the fluorescence peak becomes broader, indicating that BCzP-BT molecules have a solvent effect. Figure 6 (a) It can be seen that the BCzP-BT molecule has a strong charge transfer state (CT) characteristic.

[0204] Secondly, the CzP, BT and BCzP-BT in the BCzP-BT molecule were analyzed. -5 The difference between the FL spectra in M ​​THF solution is shown in Figure 6 (b) From Figure 6 (b) It can be seen that the luminescence of BCzP-BT molecules originates from the charge transfer from Cz to BT, rather than from the monomer itself.

[0205] Furthermore, in order to explore the excited state properties of BCzP-BT, the Stokes shift (v a -v f ) and the orientation polarizability (Δf) of the solvent, and the dipole moment of S1 is calculated by the slope of the curve. The results are shown in Figure 7 (a).

[0206] exist Figure 7 Two cross-sectional fitting lines are observed in (a), the dipole moment of low polar solvent is smaller, and the dipole moment of high polar solvent is larger, which correspond to the local excited state (LE) and the charge transfer excited state (CT), respectively. For medium polar solvent, the excited state exhibits both LE and CT properties.

[0207] In addition, the fluorescence lifetime of BCzP-BT was characterized and the results are shown in Figure 7 (b). Figure 7 (b) is a graph showing the transient PL decay curve of BCzP-BT in THF. Figure 7 (b) It can be seen that BCzP-BT shows a single exponential decay of 6.72 ns without delay. This result further proves that the S1 excited state of BCzP-BT is a hybrid localized-charge transfer excited state (HLCT) rather than a simple mixture of LE and CT states.

[0208] In addition, to verify the possibility of the hot exciton process, the energy levels of the lowest ten singlet and triplet excited states of BCzP-BT were calculated using TD-m062x / 6-311g(d). The results are shown in Figure 8 (a). Figure 8 The results in (a) show that the large energy gap (1.09 eV) between T2 and T1 suppresses the triplet internal conversion, while the narrow energy gap (0.13 eV) between T2 and S1 promotes the reverse intersystem crossing, thereby alleviating the quenching induced by triplet exciton accumulation and enabling the efficient utilization of triplet excitons through the hot exciton channel.

[0209] In addition, the natural transition orbital simulation (NTO) of the BCzP-BT molecule was performed using Multiwfn software, and the simulation results are shown in Figure 8 (b). Figure 8 The results of (b) show that T1 has local excited state (LE) characteristics, S m and T n It has the characteristics of a charge transfer excited state (CT), and S1 has the characteristics of a hybrid localized charge transfer excited state (because the electrons in the S1 state are concentrated in BT and the adjacent benzene rings, while the holes are distributed throughout the skeleton).

[0210] d. Characterization of BB NRs:

[0211] The BB NRs prepared in Example 1 of the present invention were characterized and the results are shown in Fig. 9 .

[0212] First, the morphology of the synthesized BB NRs was characterized by TEM and AFM. The TEM images and AFM images are shown in Fig. 9 (a) and (b). Fig. 9 The results of (a) and (b) show that BB NRs are rod-shaped with a width of about 30 nm and a length of about 200 nm.

[0213] Fig. 9 (c), (d), and (e) are graphs showing the UV and fluorescence spectra and fluorescence lifetimes of BB NRs and BCzP-BT, respectively. Fig. 9 (c) It can be seen that the BB NRs solution is light green under sunlight, and emits bright yellow-green light under 365nm ultraviolet light. Fig. 9 (d) It can be seen that compared with BCzP-BT, the UV absorption of BB NRs shows a slight red shift around 400 nm due to the polar environment of BB NRs in water, which may reduce the transition energy. Fig. 9 (d) It can be seen that BCzP-BT and BB-NR both show similar FL emission at 554 nm; Fig. 9 (e) It can be seen that the FL lifetime of both is about 6.7ns. These results indicate that BB-NR has the same photophysical properties as BCzP-BT.

[0214] ECL properties of e.BB NRs:

[0215] The ECL properties of the BB NRs prepared in Example 1 of the present invention were analyzed.

[0216] First, annihilation ECL experiments were performed to investigate the stability of the free radical intermediates of BB NRs.

[0217] Among them, the specific conditions of the annihilation ECL experiment are as follows:

[0218] Electrode material: BB NRs / GCE

[0219] Electrolyte: 0.1M PBS pH 7.4, nitrogen flow for 20 minutes

[0220] ECL: Cyclic Voltammetry

[0221] Annihilation State ECL: Pulse Step Method

[0222] Maximum potential: +1.5V

[0223] Minimum potential: -2.0V

[0224] Scan speed: 0.1V / s

[0225] PMT: High voltage 800V

[0226] The experimental results are shown in Fig.10 .

[0227] Fig.10 (a) is a graph showing the cyclic voltammetry (CV) curve and ECL curve of BB NRs / GCE. Fig.10 (a) shows that in N2-saturated PBS, the cyclic voltammetry (CV) curve of the BB NRs modified glassy carbon electrode (BB NRs / GCE) exhibits an oxidation peak at +0.95 V (corresponding to the oxidation of Cz) and a reduction peak at -1.45 V (corresponding to the reduction of BT).

[0228] In addition, from Fig.10 (a) It can be seen that the scanning direction also has a great influence on ECL: when the anodic scan is performed first (curve a), only weak ECL emission appears at +1.05 V (from 0 to +1.5 V, and then to -2.0 V), while when the cathodic scan is performed first (curve b), stronger ECL emission appears at both +1.0 V and -2.0 V (from 0 to -2.0 V, and then to +1.5 V). These results indicate that the reduced state of BB NRs is more stable.

[0229] At the same time, the annihilation state ECL of BB NRs was tested using the pulse step method, and the results are shown in Fig.10 (b), where the left graph represents from -2.0 to +1.5 V, and the right graph represents from +1.5 to -2.0 V. Fig.10 The results of (b) show that the transient ECL emission of BB NRs is only observed at +1.50 V, regardless of whether the initial potential is negative or positive, confirming that the reduced state of BB NRs has better stability.

[0230] In addition, the ECL of BB NRs in the presence of co-reactants was further studied and the results are shown in Fig.11 .

[0231] Firstly, the anodic ECL behavior of BB NRs was investigated using tripropylamine (TPrA) as a co-reactant.

[0232] The specific experimental conditions of the anodic ECL experiment are as follows:

[0233] Electrode material: BB NRs / GCE

[0234] Electrolyte: 0.1M PBS pH 7.4 containing 10mM TPrA

[0235] Test method: Cyclic voltammetry

[0236] Maximum potential: +1.5V

[0237] Minimum potential: 0V

[0238] Scan speed: 0.1V / s

[0239] ECL: PMT, high voltage 400V

[0240] In addition, S2O8 2- As a co-reactant, the cathodic ECL behavior of BB NRs was investigated.

[0241] The specific experimental conditions of the cathode ECL experiment are as follows:

[0242] Electrode material: BB NRs / GCE

[0243] Electrolyte: 0.1M PBS pH 7.4 containing 100mM K2S2O8

[0244] Test method: Cyclic voltammetry

[0245] Maximum potential: 0V

[0246] Minimum potential: -2.0V

[0247] Scan speed: 0.1V / s

[0248] ECL: PMT, high voltage 600V

[0249] The analysis results are shown in Fig.11 ,in, Fig.11 (a), (b), and (c) respectively represent the anodic ECL behavior and mechanism of BB NRs when tripropylamine (TPrA) is used as a co-reactant; Fig.11 (d), (e), and (f) respectively represent the cathodic ECL behavior and mechanism of BB NRs when K2S2O8 is used as a co-reactant.

[0250] from Fig.11 (a) It can be seen that the irreversible oxidation peak of tripropylamine (TPrA) itself (curve a) is observed at +0.98V, and the starting potential is +0.57V, where TPrA · Free radicals are in TPrA ·+ (TPrA oxidation product) produced after deprotonation. Fig.11 (a) and (b) show that in the absence of TPrA (curve b), the oxidation of BB NRs starts at around +0.70 V, generating cationic radicals BB NRs ·+ , thus showing obvious oxidation current and weak ECL emission. Fig.11(a) and (b) show that when TPrA coexists (curve c), BB NRs / GCE observes oxidation peaks of TPrA and BB NRs and a strong ECL emission peaking at +1.07 V. This phenomenon is considered to be attributed to TPrA and BB NRs ·+ Charge transfer between the excited singlet and triplet BB NRs ( 1 BB NRs* and 3 Radiative relaxation of BB NRs*) (see Fig.11 (c)).

[0251] from Fig.11 (d) and (e) show that S2O8 2- Itself (curve a) can be reduced to strong oxidizing SO4 at -1.07V ·- , but its ECL emission is low and can be ignored. Fig.11 (d) and (e) show that in the absence of K2S2O8 (curve b), BB NRs / GCE shows an obvious reduction peak at -1.55 V, accompanied by weak ECL emission, indicating that BB NRs can be reduced to BBNRs. ·- .from Fig.11 (d) and (e) show that in the presence of K2S2O8 (curve c), BB NRs / GCE produces a significant reduction current with a peak potential at -1.40 V, accompanied by strong ECL emission. This phenomenon is considered to be attributed to BB NRs ·- and SO4 ·- The reaction between 1 BB NRs* and 3 Radiation of BB NRs* (see Fig.11 (f)).

[0252] In addition, by adding 10 mM TPrA · or 100mM K2S2O8 with 1mM Ru(bpy)3 2+ As a standard, the current-time curve and ECL spectrum of BB NRs were recorded to verify the ECL efficiency of BB NRs. The specific experimental conditions were the same as the above-mentioned anodic ECL experiment and cathode ECL experiment. The results are shown in Fig.12 and Table 3. Fig.12 To represent Ru(bpy)3 2+ is a graph showing the ECL efficiency of BB NRs prepared in Example 1 of the standard present invention, wherein: Fig.12 (a) and (b) represent BB NRs and Ru(bpy)3 2+ Figure 2 shows the anodic current-time curve and ECL spectrum of the modified GCE. Fig.12(c) and (d) represent BB NRs and Ru(bpy)3 2+ Cathodic current-time curves and ECL spectra of modified GCE.

[0253] Table 3 shows the relationship between BB NRs / GCE and Ru(bpy)3 2+ / Relative ECL efficiency of GCE.

[0254] Table 3:

[0255] system <![CDATA[I / 10 5 ]]> Q <![CDATA[Φ ECL ]]> BB NRs-TPrA 41.7 0.00421 56.7％ <![CDATA[Ru(bpy)3 2+ -TPrA]]> 115 0.00659 100％ <![CDATA[BB NRs-K2S2O8]]> 34.7 0.0500 24.6％ <![CDATA[Ru(bpy)3 2+ -K2S2O8]]> 107 0.0344 100％

[0256] Among them, Φ ECL Calculate using the following formula:

[0257] Φ ECL =Φ° ECL (I / Q) / (I° / Q°).

[0258] Wherein, Φ and Φ°, I and I°, Q and Q° represent the ECL efficiency, ECL intensity integral (integrated ECL spectrum vs wavelength), and consumed charge (integrated CV curve vs time) of the sample and standard, respectively.

[0259] from Fig.12 As can be seen from Table 3, the BB NRs of the present invention show better ECL efficiency than traditional fluorescence-based Pdots (Z. Wang et al., Adv. Funct. Mater. 2020, 30, 2000220; Y. Feng et al., Anal. Chem. 2016, 88, 845; Z. Wang et al., J. Phys. Chem. Lett. 2018, 9, 5296) and TADF Pdots (C. Wang et al., Anal. Chem. 2022, 94, 15695) in the presence of equal or even lower concentrations of co-reactants. This is because the thermal exciton mechanism endows BB NRs with high radiative transition rate and effective exciton utilization efficiency.

[0260] Furthermore, the ECL attribution of BB NRs was investigated and the analysis results are shown in Fig.13 .

[0261] First, the anode and cathode ECL spectra and FL spectra of BB NRs were tested, and the test results are shown in Fig.13 (a) The specific test conditions of the anode and cathode ECL spectra are the same as those of the anode and cathode ECL experiments described above.

[0262] Fig.13 (a) is a graph showing the anode and cathode ECL spectra and FL spectra of BB NRs. Fig.13 (a) It can be seen that the emission peaks of the anode and cathode ECL of BBNRs match well with their FL emission peaks, both at 560 nm, indicating that the ECL and FL processes of BB NRs have the same excited states, which are generated through band gap transitions.

[0263] In addition, the CV curves of BB NRs in organic phase were tested and the test results are shown in Fig.13 (b).

[0264] Among them, the specific test conditions of organic phase CV are as follows:

[0265] Electrode material: BB NRs / GCE

[0266] Electrolyte: 0.1 M tetrabutylammonium perchlorate in tetrahydrofuran solution, purged with nitrogen for 20 minutes.

[0267] Test conditions: Cyclic voltammetry

[0268] Maximum potential: +1.5V

[0269] Minimum potential: -2.2V

[0270] Scan speed: 0.1V / s

[0271] Fig.13 (b) is a graph showing the CV curve of the glassy carbon electrode modified with BB NRs. Fig.13 (c) is a schematic diagram showing the energy band gap between the lowest unoccupied molecular orbital (LUMO) and the highest occupied molecular orbital (HOMO) of the BCzP-BT molecule. Fig.13 (b) Fig.13 (c) It can be seen that the oxidation peak of BB NRs corresponds to the oxidation of two carbazoles. However, since carbazole easily undergoes electrochemical reactions to produce dimers, the CV of BB NRs shows an irreversible oxidation wave; its reduction peak is generated by the reduction of benzothiadiazole. Since benzothiadiazole has two electron-withdrawing imine bonds (C=N), its CV curve also shows two continuous reduction peaks.

[0272] Example 2: ECL nanoprobe

[0273] 1) Oligonucleotide modified BB NRs probe:

[0274] a. Preparation of ssDNA-BB NRs:

[0275] First, 12 μL polyethylene glycol (PEG, Mw=3350) and 12 μL 1 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) were added to 600 μL BB NRs in sequence, and then the pH of the solution was adjusted to 7.4 with 0.1 M NaOH; then 40 μL single-stranded DNA modified with black hole quencher 2 (BHQ2) (ssDNA-BHQ2, 100 μM) and 24 μL freshly prepared EDC (10 mg / mL) were added, and after stirring at room temperature in the dark for 24 hours, free ssDNA-BHQ2 was removed by ultrafiltration and centrifugation to obtain ssDNA-BB NRs, which were diluted so that they had the same absorbance as BB NRs at a wavelength of 410 nm and kept in the dark for later use.

[0276] b. Characterization of ssDNA-BB NRs:

[0277] The UV absorption spectrum and Zeta potential of ssDNA-BB NRs were measured and the results are shown in Fig.14 . Fig.14 middle, Fig.14 (a) is a graph showing the UV absorption spectrum and FL spectrum of the ssDNA-BB NRs probe. Fig.14 (b) is a graph showing the characterization results of Zeta potential, Fig.14 (c) is a graph showing the UV characterization results of the filtrate from each ultrafiltration during the preparation of the ssDNA-BB NRs probe.

[0278] from Fig.14 From the curve a in (a), we can see that the UV-vis absorption of ssDNA-BB NRs shows that it has not only the absorption peak of BB NRs at 410nm, but also the absorption peak of ssDNA at 260nm and the absorption peak of BHQ2 at 580nm. Fig.14 (a) It can also be seen that the broad absorption band of BHQ2 overlaps well with the FL emission peak of BB NRs (curve d), indicating that BHQ2 can quench the emission of BB NRs.

[0279] from Fig.14 (b) It can be seen that the Zeta potential characterization of ssDNA-BB NRs shows that ssDNA-BBNRs have a more negative zeta potential than BB NRs. These results indicate that the preparation of ssDNA-BB NRs probes was successful.

[0280] In addition, when ultrafiltration was performed to purify ssDNABB NRs, the filtrate was collected and measured for UV. Fig.14(c) It can be seen that with the increase in the number of ultrafiltration times, the absorption peaks of DNA and BHQ2 in the solution gradually decrease. This result shows that there is almost no free ssDNA-BHQ2 in the prepared ssDNA-BB NRs probe solution.

[0281] 4) ECL switch based on ssDNA-BB NRs nanoprobes:

[0282] a. Signal switch principle:

[0283] The ssDNA-BB NRs nanoprobe is used as the reporter probe for illustration. Figure 2 As shown, since the ssDNA tail is modified with BHQ, the BB NRs is in the ECL OFF state. The ssDNA-BB NRs nanoprobe is directly drop-coated on the surface of the ITO electrode to construct an ECL sensing interface. The reaction solution is dropped on the sensing interface. When the Cas12a enzyme is not activated, BHQ is connected to the surface of the BB NRs through ssDNA, and the sensing electrode shows a low ECL intensity (ECL OFF); conversely, when the target molecule (target nucleic acid) is present and the Cas12a enzyme is activated, the ssDNA is sheared, thereby causing BHQ to detach from the surface of the BB NRs, restoring the ECL of the BB NRs, and generating a strong ECL signal (ECL ON) at the sensing interface.

[0284] b. Verification of signal switch:

[0285] First, the switching response of Cas12a enzyme to ssDNA-BB NRs nanoprobe was characterized by fluorescence.

[0286] The test conditions for the Cas enzyme-fluorescence test are as follows:

[0287] Detection instrument: Fluorescence excitation detection instrument

[0288] Excitation wavelength: 365nm

[0289] Emission wavelength range: 400~700nm

[0290] PMT: 800V

[0291] Before reaction: 50μL ssDNA-BB NRs + 50μL water

[0292] After reaction: 50μL ssDNA-BB NRs+50μL Cas12a system

[0293] Among them, Cas12a system: 2.5μL Cas12a (1μM) + 5μL cr RNA (1μM) + 5μL NE buffer (10X) + 32.5μL DEPC water + 5μL HPV16 dsDNA (500nM)

[0294] The results of the fluorescence test are shown in Fig.15 .

[0295] Fig.15 (a) is a graph showing the results of fluorescence characterization of ssDNA-BB NRs and Cas12a system before (curve a) and after (curve b). Fig.15 (a) shows that after activating the Cas12a enzyme with 1 nM target molecule, the fluorescence of ssDNA BB NRs is greatly restored (curve b);

[0296] Fig.15 (b) is a graph showing the normalized ECL imaging intensity characterization results of different ITO electrodes. Fig.15 (b) shows that compared with the sensing electrode modified with BB NRs (curve a), the sensing interface modified with ssDNA-BB NRs has almost no ECL imaging signal (curve b), indicating that the sensing interface is in the ECL OFF state; when 1nM target molecule is added, the Cas12a enzyme is activated, cutting off the BHQ-ssDNA on the BB NRs, generating an ECL ON response, and the ECL intensity of the sensing electrode is restored to ~75% (curve c). This result shows the feasibility of the ECL switch system based on ssDNA-BB NRs nanoprobes.

[0297] In addition, from Fig.15 (b) It can also be seen that the connection of DNA-BHQ2 to BB NRs leads to 95.6% quenching of the ECL emission of BB NRs (curves a, b).

[0298] Example 3: HPV gene electrochemiluminescence detection method based on CRISPR / Cas12a conversion catalysis.

[0299] ECL imaging detection based on the sensing array constructed by ssDNA-BB NRs:

[0300] a. Testing plan:

[0301] The detection method is based on the "ECL switch of ssDNA-BB NRs nanoprobe" ( Figure 2 ) was constructed for the detection of HPV genes.

[0302] b. Preparation and testing of detection chips: The specific detection process is as follows Figure 3 shown.

[0303] The specific steps are as follows:

[0304] Step 1: The indium tin oxide (ITO) glass was ultrasonically cleaned with toluene, acetone, ethanol and ultrapure water for 5 minutes each, and then a 5-nanometer-thick chromium layer and a 50-nanometer-thick gold layer were plated on the glass by electron beam evaporation sputtering to obtain an Au / ITO electrode;

[0305] Step 2: Paste a porous sticker (2 mm in diameter, 1 mm in depth, 4*7 holes array) on the surface of the Au / ITO electrode;

[0306] Step 3: Add 3 μL of ssDNA-BB NRs to each well, dry at 37 °C for 30 min, and then carefully wash each well with diethyl pyrocarbonate (DEPC) water;

[0307] Step 4: 0.15 μL LbCas12a (1 μM), 0.3 μL crRNA (750 nM), 0.3 μL NE Buffer (10×) and 1.95 μL DEPC water were reacted at 37°C for 10 minutes, and then 0.3 μL of HPV16 dsDNA or samples of different concentrations were added, and the resulting mixture was quickly dripped into a well and incubated at 37°C for 40 minutes;

[0308] Step 5: Each well on the chip was washed three times with DEPC water to remove the released BHQ2, and then dried at 37°C for 5 minutes before use for ECL imaging detection;

[0309] Step 6: Using Au / ITO electrode as working electrode, Ag / AgCL electrode as reference electrode, platinum wire electrode as counter electrode, electron multiplying charge coupled device (EMCCD, iXon Ultra, Andor, UK) as signal readout device, and 0.1 M pH = 7.4 phosphate buffer solution containing 5 mM tri-n-propylamine (TPrA) as electrolyte, ECL imaging detection was performed in a dark box, and cyclic voltammetry scanning was performed between 0 and +1.0 V, with a scanning speed of 0.1 V / s and an exposure time of 20 seconds.

[0310] c. Optimization of ECL detection conditions:

[0311] First, the ECL test of the present invention was carried out under different test conditions, and the test results are shown in Fig.16 middle.

[0312] Specifically, in Fig.16 ECL detection was performed under the conditions of different Cas12a concentrations, the usage ratio of Cas12a to crRNA, and the reaction time on the chip, and ECL imaging was performed in the electrolyte on the chips under different reaction conditions.

[0313] in, Fig.16 (a) shows the detection results at different Cas12a concentrations, Fig.16 (b) represents the detection results under different usage ratios of Cas12a and crRNA, Fig.16 (c) shows the detection results at different incubation times on the chip.

[0314] The condition when the ECL signal reaches the maximum plateau phase is selected as the optimal detection condition. Fig.16 As shown, the optimized conditions for the ECL detection of the present invention are: the concentration of Cas12a is 50nM, the Cas12a:crRNA is 1:1.5, and the incubation time is 40 minutes.

[0315] Next, the ECL images of BB NR-modified ITO and Au / ITO electrodes in 0.1 M PBS, pH 7.4, containing 5 mM TPrA were compared using ImageJ software. The results are shown in Fig.17 .from Fig.17 It can be seen that compared with BB NR-modified ITO, BB NRs-modified Au / ITO showed an increased oxidation current with an oxidation peak shifted to 0.93 V, a 46-fold stronger ECL response, and improved ECL imaging quality. This difference is considered to be due to the good catalytic ability of gold for the oxidation of TPrA.

[0316] d. Detection performance:

[0317] Detection linearity and sensitivity: Under the optimal detection conditions, the constructed sensor array was used to perform ECL imaging detection of HPV16 DNA target molecules, and the results are shown in Fig.18 (a). Fig.18 The top of (a) shows the results of imaging detection of HPV16 DNA with concentrations ranging from 1fM to 10nM. From the results, it can be seen that the intensity of ECL increases with the increase of HPV16 DNA concentration; the analog-to-digital conversion value (A / D value) of ECL intensity is plotted against the logarithm of HPV16 DNA concentration, and it can be seen that there is a good linear relationship in the range of 1fM to 10nM. Based on the 3S / N calculation, the detection limit of this method for HPV16 DNA is 0.6fM.

[0318] Detection selectivity test: In this example, the detection specificity of the target nucleic acid HPV16 DNA in the presence of 10 times higher concentration of interfering DNA was also tested, and the detection results are shown in Fig.18 (b). Fig.18(b) shows the results of ECL detection of 100pM HPV16DNA (d) in the presence of 1nM HPV18DNA, 1nM HPV31DNA, 1nM HPV58DNA alone (a, b, c) or coexistence (e). The results show that only the sample solution containing HPV16 DNA has an obvious ECL signal, indicating that this method has good selectivity for the detection of HPV16 DNA.

[0319] Industrial Applicability

[0320] As can be seen from the above examples, the present invention uses BCzP-BT as a luminescent molecule and successfully prepares for the first time a hot exciton organic nanorod (BB NR) with sensitive annihilation ECL and strong co-reactant ECL luminescence. PL and high exciton utilization efficiency derived from the charge transfer state, thus showing superior Φ ECL Furthermore, by using the ECL nanoprobe of the present invention and recognizing the target nucleic acid through the CRISPR / Cas enzyme system, the signal is switched from off to on, thereby achieving rapid and high-throughput detection of HPV16 DNA with a simplified operation procedure. Therefore, through the present invention, a high-sensitivity ECL detection platform for HPV16 DNA detection can also be constructed. Its detection effect is close to that of the ECL standard using Ru(bpy)3 2+ Therefore, the ECL detection method of the present invention is a feasible and reliable ECL detection method.

Claims

1. A method for preparing an electrochemiluminescent nanoprobe, comprising the following steps: a thermoexciton nanoparticle synthesis step, polymerizing the thermoexciton organic light-emitting molecules and the copolymer molecules to synthesize the thermoexciton nanoparticles; and The thermoexciton nanoparticle modification step is to modify the obtained thermoexciton nanoparticles using an oligonucleotide chain modified with a quencher molecule to obtain modified thermoexciton nanoparticles.

2. The preparation method according to claim 1, wherein The thermoexciton organic light-emitting molecule comprises an electron donor (D) structure selected from the following group (a) and an electron acceptor (A) structure selected from the following group (b); (a) (b) 3. The preparation method according to claim 2, wherein The electron donor (D) is carbazole, and the electron acceptor (A) is benzothiadiazole.

4. The preparation method according to claim 3, wherein The thermal exciton organic light-emitting molecule is the following BCzP-BT:

5. The preparation method according to any one of claims 1 to 4, 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), 6. The preparation method according to claim 5, wherein: The thermoexciton organic luminescent molecules and the copolymer molecules are synthesized into thermoexciton nanoparticles through a nano coprecipitation method.

7. The preparation method according to any one of claims 1 to 6, wherein The nanoparticles are nanospheres, nanotubes, nanorods or nanoonions.

8. The preparation method according to any one of claims 1 to 7, wherein The quencher molecule is a black hole quencher, a dark quencher, or an amine-reactive quencher. 9 . An electrochemiluminescent nanoprobe 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. The electrochemiluminescence sensor according to claim 11, wherein The working electrode is a gold-indium tin oxide electrode.

13. An electrochemiluminescence detection method using the electrochemiluminescence sensor according to any one of claims 10 to 12.

14. The electrochemiluminescence detection method according to claim 13, comprising the following steps: Enzymatic reaction step: adding the sample to be tested to the Cas enzyme catalytic system to obtain a sample reaction solution, wherein the Cas enzyme catalytic system comprises a guide nucleic acid capable of binding to the target nucleic acid; and Sample detection step: adding the sample reaction solution to the electrochemiluminescence sensor, collecting the electrochemiluminescence signal and analyzing it.

15. The electrochemiluminescence detection method according to claim 14, wherein: In the sample reaction solution, the concentration of the Cas protein in the Cas enzyme is greater than 40 nM.

16. The electrochemiluminescence detection method according to claim 14 or 15, wherein: crRNA is used in greater amounts than Cas proteins.

17. The electrochemiluminescence detection method according to any one of claims 13 to 16, wherein: The incubation time of the sample reaction solution on the electrochemiluminescence sensor is more than 30 minutes. 18 . An electrochemiluminescent detection kit comprising the electrochemiluminescent sensor according to claim 10 and a detection reagent containing a Cas enzyme.

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