Patent Application
20240425751
In accordance with 37 CFR § 1.831-1.835 and 37 CFR § 1.77(b) (5), the specification makes reference to a Sequence Listing submitted electronically as a .xml file named “553524US_ST26”. This .xml file was generated on Jun. 10, 2024 and is 10,178 bytes in size. The entire contents of the Sequence Listing are hereby incorporated by reference.
This application is based upon and claims the benefit of priority from Chinese Patent Application No. 202310682568.5, filed on Jun. 9, 2023, the entire contents of all of which are incorporated herein by reference.
Embodiments described herein relate generally to a method for producing an electrochemiluminescence nanoprobe, the electrochemiluminescence nanoprobe, an electrochemiluminescence sensor, an electrochemiluminescence detection method, and a kit for electrochemiluminescence detection.
Electroluminescence (EL) is a phenomenon in which a luminescent material emits light under current and voltage excitation owing to an electric field and also a process of directly converting electrical energy into light energy. Organic electroluminescence is a phenomenon in which a thin-film element produced from an organic luminescent material emits light under electric field excitation. An organic electroluminescent element has a luminescence principle that, when driven by an external voltage, an electron injected from an electrode and a hole combine and release energy in an organic material, and the energy is transferred to a molecule of an organic luminescent substance to excite the molecule, so that a transition from a ground state to an excited state thus occurs, and, when the excited molecule returns from the excited state to the ground state, a radiative transition occurs to generate a luminescence phenomenon. Excited states can be classified into singlet (S) and triplet (T) excitons in accordance with spin multiplicity. An excited molecule may be in a lowest energy singlet excited state S1 or triplet excited state T1 or may be in a higher energy singlet excited state (S2, S3, S4, . . . ) or triplet excited state (T2, T3, T4, . . . ). When a radiative transition causes a molecule in the S1 excited state to return to the S0 ground state, the molecule generates “fluorescence emission”. When a radiative transition causes a molecule in the T1 excited state to return to the S0 ground state, the molecule generates “phosphorescent emission” (see
The electrochemiluminescence (ECL) technology is a method for generating specific luminescence by performing an electrochemical reaction in an electrode surface by the use of an electrochemical principle to generate an excited state. The excited state in the electrochemiluminescence process is generated by an electron transfer reaction between intermediate radicals formed by electron transfer between an electroactive substance and an electrode.
According to luminescent reagents, electrochemiluminescence-based materials can be classified into metal complex electrochemiluminescence-based materials and organic compound electrochemiluminescence-based materials (hereinafter, sometimes referred to as “organic luminescent materials”), for example, aromatic hydrocarbons such as organic fluorene, BODIPY derivatives, fluorene-based polymers, and various types of organic nanomaterials.
Examples of common electrochemiluminescent metal complexes include tris(bipyridine)ruthenium (II) complex ions (Ru(bpy)32+), which belong to phosphorescent emission-based materials. In the case of using the tris(bipyridine)ruthenium (II) complex ions as an ECL probe, exciton utilization can reach 100%, so that an electrochemiluminescence detection method based on tris(bipyridine)ruthenium (II) complex ions (Ru(bpy)32+) is a standard method for electrochemiluminescence detection. In contrast, the above-mentioned organic luminescent materials belong to fluorescent emission-based materials and exciton utilization thereof can achieve only 25%.
As excited states of organic luminescent materials having been currently used in the electrochemiluminescence field, there are mainly two conventional types of excited states, namely, a locally-excited (LE) state and a charge-transfer (CT) excited state. Due to electron-hole distributions in the two types of excited states and binding energy characteristics of resulting excitons, the utilization of excitons in the locally-excited state is low and radiative transition rate/quantum yield (ΦPL) is high, or the utilization of excitons in the charge-transfer excited state is high and radiative transition rate/quantum yield is low. Thus, high utilization of both the excitons in the locally-excited state and the charge-transfer excited state and high radiative transition rate/quantum yield cannot be achieved at the same time, hence, luminescence efficiency and intensity cannot attain ideally optimal levels. This limits downstream applications, for example, the efficiency of electrochemiluminescence.
In 2012, Ma Eoguang and Yang Bing et al. innovatively applied a photophysical phenomenon of reverse intersystem crossing (RISC) to a design for an organic electroluminescent material and successfully developed a series of donor-acceptor fluorescent materials with different light colors. An effective high-level reverse intersystem crossing process greatly increases the exciton utilization of the electroluminescence element. Here, a conversion process between a higher triplet state (Tn, n≥2) and a singlet state (Sm, m≥1) is referred to as a hot exciton process, a luminescence mechanism realized based on the hot exciton process is referred to as a hot exciton mechanism, and a luminescent material with obvious hot exciton reverse intersystem crossing is referred to as a hot exciton material. The hot exciton material generally has a large Tn-T1 energy level and a small Tn-Sm state energy level. The large Tn-T1 energy level can suppress internal conversion from Tn to T1, meanwhile the small Tn-Sm energy level can increase the rate of reverse intersystem crossing from Tn to Sm. The intersystem crossing from Tn to Sm and the internal conversion from Tn to T1 mutually compete, hence, theoretically, the rate of reverse intersystem crossing can be sufficiently high, the internal conversion can be completely suppressed, high-level triplet excitons can be all converted into singlet excitons, and the utilization of excitons in a hot exciton material can reach 100% (see Yuwei Xu, “Study on high-level reverse intersystem crossing process and photoelectric performance of hot exciton luminescent material”, South China University of Technology, 2020, Ph.D. Thesis, and see
Currently, the hot exciton material is mainly used in the field of semiconductor materials, for example, in the field of organic electroluminescent diodes (OLED) (see Xiaojie Chen, Dongyu Ma et al., “Hybridized Local and Charge-Transfer Excited-State Fluorophores through the Regulation of the Donor-Acceptor Torsional Angle For Highly Efficient Organic Light-Emitting Diodes”, CCS Chem. 2022, 4, 1284-1294), and is a novel luminescent material following the first generation organic luminescent material (fluorescent material), the second generation organic luminescent material (phosphorescent material), and the third generation organic luminescent material (thermally activated delayed fluorescence, TADF).
The hot exciton material has excellent optical properties and therefore has been desired to be applied to bio-detection. The specification of Chinese Patent Application Publication No. 110981821 describes an attempt to use the hot exciton material for fluorescence detection, but there has been no successful example of the use of the hot exciton material in the field of ECL detection yet.
A problem to be solved by the present invention is to provide a method for producing an electrochemiluminescence nanoprobe with a wide detection range and high sensitivity, the electrochemiluminescence nanoprobe, an electrochemiluminescence sensor, an electrochemiluminescence detection method, and a kit for electrochemiluminescence detection.
A method for producing an electrochemiluminescent nanoprobe according to an embodiment includes: a hot exciton nanoparticle synthesis step of polymerizing a hot exciton organic luminescent molecule and a copolymer molecule to synthesize hot exciton nanoparticles; and a hot exciton nanoparticle modification step of modifying the obtained hot exciton nanoparticles with an oligonucleotide chain modified with a quencher molecule to obtain modified hot exciton nanoparticles.
As is well known, an organic electroluminescent diode (OLED) is an all-solid element and includes no liquid component. In the organic electroluminescent diode, a luminescent molecule is excited under the action of an external voltage to make a transition from a ground state to an excited state, and, when the resulting excited molecule returns from the excited state to the ground state, radiative transition is caused to generate luminescence. On the other hand, an environment for detection of electrochemiluminescence is liquid, a luminescent molecule undergoes an electrochemical reaction in an electrode surface to generate an excited state and emits light through radiative transition. Therefore, the organic electroluminescent diode and electrochemiluminescence are different in luminescence principle and luminescence environment. In the case of using a hot exciton material currently used in the field of organic electroluminescent diodes (OLED) as a luminescent molecule for electrochemiluminescence (ECL) detection, problems such as feasibility and reliability are first considered.
The inventors have intensively studied the above-mentioned problem, and, as a result, the inventors found that, by producing an electrochemiluminescent nanoprobe (hereinafter, sometimes referred to as “the ECL nanoprobe”) from a hot exciton organic luminescent molecule by nanotechnology and modifying the electrochemiluminescent nanoprobe with an oligonucleotide chain modified with a quencher molecule, the overall efficacy of electrochemiluminescence can be enhanced, so that quick, non-amplified, high-throughput detection of a target molecule with a wide detection range and extremely high sensitivity can be achieved.
In the present invention, a hot exciton organic luminescent molecule is applied to the field of electrochemiluminescence for the first time and nanoparticles are produced using the hot excitonic organic luminescent molecule as a luminescent material for the first time, whereby quick, non-amplified, high-throughput detection for a target molecule can be achieved, and there can be provided an electrochemiluminescence (ECL) nanoprobe based on the hot exciton organic luminescent molecule with a wide detection range and extremely high sensitivity, and a sensor a detection method, and a detection kit that use the electrochemiluminescence nanoprobe.
Hereinafter, a specific embodiment of the present invention will be described in detail. Note that the following description about the embodiment is only to explain an inventive concept of the present invention and is not intended to limit the present invention.
One embodiment of the present invention relates to a method for producing an electrochemiluminescent nanoprobe, the method including: a hot exciton nanoparticle synthesis step of polymerizing a hot exciton organic luminescent molecule and a copolymer molecule to synthesize hot exciton nanoparticles; and a hot exciton nanoparticle modification step of modifying the obtained hot exciton nanoparticles with an oligonucleotide chain modified with a quencher molecule to obtain modified hot exciton nanoparticles.
The hot exciton organic luminescent molecule to be used in the present invention is not limited to a particular one, and any hot exciton organic luminescent molecule having hybrid locally-excited and charge-transfer excited state properties can be used, and examples thereof include various types of hot exciton organic luminescent molecules described in the above-mentioned “Study on high-level reverse intersystem crossing process and photoelectric performance of hot exciton luminescent material” and “Hybridized Local and Charge-Transfer Excited-State Fluorophores through the Regulation of the Donor-Acceptor Torsional Angle For Highly Efficient Organic Light-Emitting Diodes”.
The hot exciton material is a molecule having hybrid locally-excited and charge-transfer (HLCT) excited state properties. Note that the molecule having the hybrid locally-excited and charge-transfer excited state properties is not necessarily the hot exciton material. For example, singlet and triplet materials have the hybrid locally-excited and charge-transfer excited state properties, but do not belong to the hot exciton material.
The hot exciton material commonly has a one-dimensional fused ring structure as a structural unit and includes a donor cell including a heteroatom.
Examples of the fused ring structure include a benzene ring, a naphthalene ring, and an anthracene ring. These benzene, naphthalene, and anthracene rings may have a substituent.
A specific configuration structure of a donor-acceptor unit may be, for example, a D-A or D-A-D structure. D denotes a donor unit (donor) and A denotes an acceptor unit (Acceptor).
Examples of the donor-acceptor unit include units composed of a donor (D) selected from the following group (a) consisting of chemical formulae (1) and an acceptor (A) selected from the following group (b) consisting of chemical formulae (2).
The hot exciton material is a sort of luminescent material breaking through the spin-statistic limit of conventional fluorescent materials, and includes blue-light hot exciton materials, green-light hot exciton materials, and red-light hot exciton materials in terms of the type of light emitted.
Since the donor-acceptor intensity of the hot exciton material is appropriate, pure-blue light emission and deep-blue light emission are more easily obtained in the molecular design of D-A fluorescent materials. Examples of a current successfully produced blue-light hot exciton material include a phenanthrimidazole unit-containing blue-light hot exciton material, an anthracene unit-containing blue-light hot exciton material, and other types of blue-light hot exciton materials.
Examples of the phenanthrimidazole unit-containing blue-light hot exciton material include phenanthrimidazole unit-containing blue-light hot exciton materials expressed by the following chemical formulae (3) and (4).
Examples of the anthracene unit-containing blue-light hot exciton material include anthracene unit-containing blue-light hot exciton materials expressed by the following chemical formulae 5.
Examples of other types of the blue-light hot exciton materials include blue-light hot exciton materials expressed by the following chemical formulae 6.
Typical examples of the green-light hot exciton material include green-light hot exciton materials expressed by the following chemical formulae 7.
Typical examples of the red-light hot exciton material include red-light hot exciton materials expressed by the following chemical formulae 8.
For the D-A-D hot exciton material, the binding of the acceptor (A) unit to the donor (D) unit may be ortho, meta, or para binding. Here, since spatial resistance can be reduced in the para binding, a twist angle between the acceptor (A) unit and the donor (D) unit decreases, a more locally excited component can be provided, and faster radiation-attenuation is achieved. The para binding is preferred because, according to circumstances, the para binding can achieve higher external quantum efficiency (EQE) than the ortho binding and the meta binding. For example, 4,7-bis(4-(9H-carbazol-9-yl)phenyl)benzo[c][1,2,5]thiadiazole (p-BCzP-BT) is illustrated in examples according to the present invention. In the above-mentioned compound, an electron donor (D) is carbazole and an electron acceptor (A) is benzothiadiazole.
Note that any hot exciton material having hybrid locally-excited and charge-transfer excited state (HLCT) properties can be used in the present invention. The hot exciton materials mentioned above are merely examples. As those skilled in the art will appreciate, derivatives formed by substituting the above-mentioned compound with one or more substituents (for example, an alkyl group, a hydroxy group, and halogen), and spatial isomers thereof, and the likes can be used as well in the present invention.
The method for producing the hot exciton material to be used in the present invention is not limited to a particular one. For example, the hot exciton material can be synthesized by a chemical reaction via a synthetic route of a Suzuki coupling reaction, a Scherrer coupling reaction, a cross-coupling reaction, or the like, or alternatively can be purchased through a commercial channel.
Although a synthetic route of the Suzuki coupling reaction is employed in the examples of the present invention, other synthetic routes may be employed as necessary. The Suzuki coupling reaction is a commonly used carbon-carbon bond formation reaction, in which zero-valent palladium is used as a catalyst and aryl or alkenylboronic acid is allowed to react with halogenated aromatic hydrocarbon such as chlorine, bromine, or iodine. Examples of the palladium catalyst that can be used include tetrakis(triphenylphosphine)palladium (Pd(PPh3)4), Pd[P(o-toly)3]3, and a Buchwald catalyst. As aryl or the alkenyl boronic acid and the halogenated aromatic hydrocarbon, their respective compound raw materials can be employed according to an electron donor (D) and an electron acceptor (A) in a hot exciton organic luminescent molecule synthesized as necessary, and are not limited to particular ones.
In the present invention, hot exciton nanoparticles are synthesized by polymerizing a hot exciton organic luminescent molecule and a copolymer molecule.
Nanoparticles in the present invention are particles within the nano-size range (0.1 to 100 nm) in at least one dimension in a three-dimensional space. Specifically, the nanoparticles may be in various geometric forms such as nanoballs, nanotubes, nanorods, and nano-onions. When a hot exciton organic luminescent molecule is formed in a nanoform, an ECL response signal can be effectively amplified and a detection limit, a detection range, selectivity, stability, and the like of an ECL sensor can be enhanced. In the present invention, a material in the form of nanorods (NR) is successfully produced as in the later-described examples, but, the nanoparticles that can be used in the present invention are not limited to nanorods, but can be formed in other nanoforms such as nanoballs can be formed, depending on the compound raw material and the synthetic route to be employed.
In the present invention, the hot exciton organic luminescent molecule and the copolymer molecule can be synthesized by a known method for producing an organic material nanostructure according to the conventional art, such as a nano-coprecipitation method, a microemulsion method, or a self-assembly method (Liu Ronghua, “Production and Application of Novel Fluorescent Conjugated Polymer Nanoparticles”, University of Science and Technology Beijing, 2018, Ph.D. Thesis), and the method is not limited to a particular one. Here, from the viewpoint of biocompatibility, the microemulsion method and the nano-coprecipitation method are more preferably used.
In the microemulsion method, a luminescent molecule is coated with a non-toxic, non-immunogenic, hydrophilic polymer, for example, a polyethylene glycol-based molecule such as polyethylene glycol (PEG) or polyethylene glycol-phosphatidylethanolamine (PEG-PE), or a biopolymer such as phospholipid, whereby luminescent molecular nanoparticles with high biocompatibility and high stability can be obtained.
In the nano-coprecipitation method, luminescent molecules are dissolved in a good solvent, a poor solvent is injected in large amounts under ultrasonic conditions, whereby the solubility of the luminescent molecules promptly decreases and the luminescent molecules aggregate and thereby precipitate in the form of small particles, and then the remaining organic solvent is removed to obtain luminescent molecular nanoparticles. The nano-coprecipitation method is preferably used because the method can be easily operated and the nanoparticles produced thereby are smaller and uniform in size.
In the nano-coprecipitation method, luminescent molecular nanoparticles can be surface-functionalized using an amphiphilic polymer. In a production process, the luminescent molecules and the amphiphilic polymer are self-organized, and the amphiphilic polymer has a hydrophobic functional group at one end thereof and has a hydrophilic functional group at the other end thereof, hence luminescent molecular nanoparticles coated with the amphiphilic polymer can be obtained and the functional groups (for example, an amino group and a carboxyl group) of the amphiphilic polymer are exposed to outer surfaces of the nanoparticles, and thus surface-functionalized luminescent nanoparticles are obtained. A functional group of a nucleic acid, polypeptide, sugar, protein, an antibody, or the like reacts with the functional groups of the amphiphilic polymer that are exposed to the outer surfaces of the nanoparticles by a condensation reaction or a bioorthogonal click reaction to obtain luminescent molecular nanoparticles having an identification function.
In the present invention, the amphiphilic polymer is also referred to as a copolymer molecule. As the copolymer molecule, copolymer molecules commonly used in the relevant field, such as a polystyrene-polyacrylic acid block copolymer (PS-PAA), a polystyrene-maleic anhydride copolymer (PSMA), and a poly(isobutylene-alt-maleic anhydride) (PIMA), which are expressed by the following chemical formulae 9, can be used, and the copolymer molecule is not limited to a particular one.
The diameter of the nanoparticles obtained by the nano-coprecipitation method can be 1 nm to 2 nm at minimum.
Note that the surface functionalization modification of hot exciton nanoparticles is not limited to the above-described modification method using the amphiphilic polymer, and other modification methods such as direct functionalization (in other words, directly modifying a luminescent molecule with an alkoxy chain, an amino/carboxyl group, or a functional group of a biomolecule or the like having a target function by a covalent-bonding method) and physical coating (in other words, coating nanoparticles with a phospholipid, silicon dioxide, or the like) can be employed.
The surface functionalization of luminescent molecular nanoparticles allows a large number of functional groups to be given to the surface of each nanoparticle, and furthermore allows a large number of oligonucleotides to be bonded, and thus an electrochemical signal can be amplified, so that the sensitivity of ECL detection can be enhanced.
In the present invention, the modification of hot exciton nanoparticles means oligonucleotide modification of hot exciton nanoparticles. Specifically, the above-mentioned surface-functionalized hot exciton nanoparticles are modified with an oligonucleotide chain modified with a quencher molecule to obtain oligonucleotide-modified hot exciton nanoparticles (hereinafter, sometimes referred to as “the ECL nanoprobe”).
A method for binding between oligonucleotide and the hot exciton nanoparticles is not limited to a covalent-bonding reaction (for example, cross-linking by NH2—COOH), and may be other binding methods such as electrostatic action and affinity adsorption.
The oligonucleotide chain used for the modification may be a double-stranded oligonucleotide chain (for example, dsDNA) or may be a single-stranded oligonucleotide chain (for example, RNA or ssDNA). Specifically, the oligonucleotide chain can be suitably selected according to the type of a Cas enzyme to be used.
The collateral cleavage activity of the Cas enzyme is nonspecific, hence a nucleotide sequence constituting the oligonucleotide chain is not limited to a particular one and can be any sequence. The length of the oligonucleotide chain is also not particularly limited as long as a quencher molecule can be bonded to hot exciton nanoparticles and resonance energy can be transferred between the quencher molecule and the hot exciton nanoparticles, and can be designed with reference to various types of oligonucleotide chains for linkage that have been commonly used in the prior art.
From the viewpoint of cleavage sensitivity owing to the Cas enzyme, the number of nucleotides in the oligonucleotide chain as a single chain is better not too large and is preferably within a range of 3 to 20, more preferably within a range of 4 to 11, and still more preferably 5, 6, 7, 8, 9, 10, 11, or 12.
The oligonucleotide chain according to the present invention is modified with a quencher molecule. Dynamic quenching of the quencher molecule is caused by fluorescence resonance energy transfer (FRET) or collisional quenching. In the quencher molecule, optical absorption and the fluorescence emission of the hot exciton nanoparticles overlap, hence resonance energy can be transferred from the excited nanoparticles to the quencher molecule, whereby emission by the hot exciton organic luminescent molecule can be quenched.
The quencher molecule can be any quencher molecule having resonance energy transfer characteristics and being commonly used in the relevant field, and is not limited to a particular one. Examples of the quencher molecule that can be used include black hole quenchers (Black Hole Quencher™ reagents, that is, BHQ-based quenchers), dark quenchers (for example, DABCYL series quenchers), BlackBerry™ quenchers (that is, BBQ-series quenchers), Blueberry quenchers (that is, BLU-series quenchers), and amine reactive quenchers (QSY-series quenchers). Of these quenchers, the BHQ quenchers are often used. Any of these quenchers can be commercially obtained (for example, from LGC, BiochTechnologies™).
A method for binding of the quencher molecule to oligonucleotide is not limited to a particular one, and can be any method commonly used in the relevant field. The quencher molecule may be bound to an end of the oligonucleotide or may be inserted into an oligonucleotide sequence.
In the present invention, the oligonucleotide modified with the quencher molecule functions as a signal switch of the present invention together with a Cas enzyme described below. Specifically, when the cleavage of oligonucleotide by an activated Cas enzyme does not occur, the quencher molecule is bound to surfaces of the hot exciton nanoparticles via the oligonucleotide and exerts a quenching effect on the hot exciton organic luminescent molecule and a sensor electrode indicates a low ECL intensity (ECL OFF). In contrast, when the cleavage of oligonucleotide by the activated Cas enzyme occurs, the quencher molecule leaves the surfaces of the hot exciton nanoparticles, whereby the sensor electrode indicates a strong ECL signal (ECL ON).
An electrochemiluminescence sensor of the present invention (hereinafter, sometimes referred to as “the ECL sensor”) can be obtained by dropping the above-described ECL nanoprobe onto a working electrode.
The working electrode to be used in the present invention is not limited to a particular one and can be one of electrodes commonly used for ECL detection, such as a glass carbon electrode (GCE), an indium tin oxide (ITO) electrode, and a screen printed electrode (SPE). An indium tin oxide electrode is preferably used. A three-electrode system is more preferably used. In the electrode system, for example, a platinum wire counter electrode, an Ag/AgCl reference electrode, and an indium tin oxide (ITO) working electrode are arranged.
As the present invention is verified in the later-described examples, gold has a good catalytic effect on a coreactant tripropylamine or the like, hence a gold-indium tin oxide (Au-ITO) electrode can more greatly enhance an ECL signal than an ITO electrode, and therefore, an Au-ITO electrode is more preferably used in the present invention.
An electrochemiluminescence detection method of the present invention (hereinafter, sometimes referred to as “the ECL detection method”) uses the above-described ECL sensor. The ECL detection method of the present invention may include: an enzyme reaction step of adding a sample to be measured to a Cas enzyme-catalyzed system including a guide nucleic acid capable of binding to a target nucleic acid to obtain a sample reaction solution; and a sample detection step of adding the sample reaction solution to the electrochemiluminescent sensor and collecting and analyzing an electrochemiluminescent signal.
In the ECL detection method of the present invention, the ECL sensor of the present invention can be produced at the time of use, instead of using the ECL sensor as it is. In this case, the ECL detection method of the present invention may further include a hot exciton chip preparation step of dropping the ECL nanoprobe of the present invention onto a working electrode and thereby obtaining a hot exciton chip of the present invention (that is, the ECL sensor).
With the ECL detection method of the present invention, a target molecule contained in the sample to be measured can be detected. The sample to be measured is not limited to a particular one, and may be, for example, urine, blood, serum, cerebrospinal fluid, or saliva. The target molecule is not limited to a particular one, and may be, for example, a nucleic acid, protein, or a chemical small molecule.
In the present invention, when an enzyme-catalyzed reaction or another specific chemical reaction is used as a target-identification and signal-conversion unit and the target molecule is dropped, a signal switch state of the ECL nanoprobe can be changed through the enzyme-catalyzed reaction or the chemical reaction, and thus qualitative or quantitative detection for a target signal can be realized.
As an enzyme used for the enzyme-catalyzed reaction, for example, a Cas enzyme having a collateral cleavage activity in a CRISPR/Cas enzyme can be used. The above-mentioned activity belongs to a nonspecific nuclease cleavage activity. When the Cas enzyme is targeted as a target nucleic acid molecule, the Cas enzyme is activated and acquires the nonspecific activity for the cleavage of any nucleic acid molecule.
The Cas enzyme may include at least one Cas protein selected from the group consisting of Cas12 (type VA), Cas13 (type VI), and Cas14 (type VF), and is, for example, at least one Cas protein selected from the group consisting of Cas12a, Cas12b, Cas13a, Cas13b, Cas14a, Cas14b, and Cas14c. Here, Cas12 targets dsDNA (double-stranded DNA), Cas13 targets ssRNA (single-stranded RNA), and Cas14 targets ssDNA. Furthermore, Cas12a has a nonspecific cleavage activity (also called a trans-cleavage activity) for ssDNA (single-stranded DNA), and is therefore capable of targeting ssDNA.
The Cas12 enzyme targets DNA, is commercially mature, and can be used for other detection areas for non-nucleic acids (for example, protein and a small biomolecule) in addition to DNA, and is therefore more preferably used.
In the present invention, any nucleic acid sequence can be targeted by designing and synthesizing a guide RNA (gRNA) for a Cas enzyme. The gRNA is composed of two parts: one is CRISPR RNA (crRNA), that is, a 17 to 20-base length nucleotide sequence complementary paired with the target nucleic acid; and the other one is tracrRNA serving as an anchor to aid in the folding of the Cas enzyme. The guide RNA activates the Cas enzyme by binding to oligonucleotide in the target nucleic acid. The activated Cas enzyme causes the cleavage of the oligonucleotide bound to the ECL nanoprobe to occur by the collateral cleavage activity and thereby changes a signal switch state of the ECL nanoprobe.
Hereinafter, the principle of a signal switch and a method for the production of and the detection by an ECL detection chip according to the present invention will be specifically described using, as examples, hot exciton organic nanorods (BB NRs) and a sensor array produced in the examples of the present invention.
The principle of the signal switch of the present invention will be specifically described using the ECL detection chip illustrated in
The method for the production of and detection by the ECL detection chip will be specifically described using an ECL detection method illustrated in
From the viewpoint of achieving higher ECL signal intensity, ECL detection conditions, such as the concentration of the Cas protein in a sample reaction solution, the usage ratio of the Cas protein to the crRNA, and a reaction time (for example, an incubation time) of the sample reaction solution on the chip, are preferably optimized.
The concentration of the Cas protein is not particularly limited, but may be, for example, 40 nM or higher, preferably 42 nM or higher, more preferably within a range of 45 nM to 70 nM, and particularly preferably within a range of 50 nM to 60 nM from the viewpoint of achieving higher ECL signal intensity.
Although the usage ratio of the Cas protein to the crRNA is not particularly limited, but, from the viewpoint of achieving higher ECL signal intensity, the amount of the crRNA used is preferably larger than the amount of the Cas protein used. For example, the usage ratio of the Cas protein to the crRNA may be within a range of 1:1.25 to 1:5, preferably within a range of 1:1.35 to 1:4, more preferably within a range of 1:1.5 to 1:3, and particularly preferably within a range of 1:1.5 to 1:2.
The incubation time of the sample reaction solution on the chip is not particularly limited, but may be, for example, 30 minutes or longer, preferably within a range of 35 to 120 minutes, more preferably within a range of 37 to 90 minutes or longer, and particularly preferably within a range of 40 to 60 minutes from the viewpoint of achieving higher ECL signal intensity.
As verified by the later-described examples of the present invention, the ECL efficiency of the hot exciton material of the present invention can reach 56.7% and the fluorescence quantum yield (ΦPL) can reach 89% or higher. The hot exciton material of the present invention is higher in ECL efficiency and fluorescence quantum yield than the previously-reported Pdots based on the principle of the thermally activated delayed fluorescence (TADF) (that have the reported highest organic ECL nanomaterial efficiency) (Anal. Chem. 2022, 94, 15695-15702) and Pdots based on a conventional fluorescent material (Chem. Sci. 2019, 10, 6815; Anal. Chem. 2016, 88, 845).
Using the detection of HPV16 DNA as an example, the ECL detection method according to the present invention will be compared with other methods described in the prior art, in terms of detection range and detection limit. Comparison results are illustrated in Table 1.
1L. Wang et al., Microchem. J. 2022, 181, 107818.
2Y. Nie et al., Biosens. Bioelcetron. 2020, 160, 112217.
3P. Liu et al., Biosens. Bioelectron. 2021, 176, 112954.
4Y. He et al., ACS Appl. Mater. Interfaces, 2021, 13, 298.
5S. Jampasa et al., Sensor. Actuat. B-Chem, 2018, 265, 514.
6Y. He et al., Sensor. Actuat. B-Chem, 2020, 320, 128407.
Note that superior
As seen from Table 1, the ECL chip (sensor) produced based on the hot exciton organic luminescent molecule of the present invention has a wider detection range and higher sensitivity than the other reported ECL chips.
An ECL detection kit of the present invention may directly include the above-described ECL sensor of the present invention. The ECL detection kit beneficially includes the above-described ECL nanoprobe of the present invention and drops the ECL nanoprobe onto a working electrode during detection. Besides the ECL sensor, the ECL detection kit may include, for example, other reagents necessary for ECL detection, such as a Cas enzyme-containing detection reagent, and an instruction manual.
The electrochemiluminescent nanoprobe, the method for producing the electrochemiluminescent nanoprobe, and the application of the electrochemiluminescent nanoprobe, and the like according to the present invention have been described above, based on the embodiments, but the present invention is not limited to them. Other aspects obtained by applying various modifications conceivable by those skilled in the art to the embodiments and other aspects constructed by combining some of the constituents in the embodiments are also included in the scope of the present invention as long as the aspects do not deviate from the gist of the present invention.
Hereinafter, the hot exciton nanoparticles, the ECL nanoprobe, the ECL detection method, and the like according to the present invention will be specifically described with reference to examples. Note that the present invention is not limited to these examples.
Reagents, an experimental method, and apparatuses that are used in the examples are as follows.
Oligonucleotide sequences used in the examples are as follows.
TEM is obtained by TECNAIG2F 20 transmission electron microscope (FFI, USA).
Zeta potentials are recorded using 90 Plus DynaPro NanoStar (Brookhaven Instrument Corporation, USA).
1H NM/R spectra are measured using deuterated CDCl3 as a solvent and tetramethylsilane (TMS) as an internal standard by Bruker Avance III 500HD spectrometer (Germany).
UV-vis absorption spectra are obtained by UV-3600 spectrophotometer (Shimadzu, Japan).
Photoluminescence (PL) spectra and fluorescence lifetimes are measured using FLS-980 fluorescence spectrophotometer (Edinburgh, U.K).
AFM is obtained in the ScanAsyst mode by an atomic force microscope (BRUKER Dimension Icon, Germany).
A 5-nm Cr layer and a 50-nm Au layer are deposited on an ITO glass sheet by PVD 75 electron beam deposition system (Kurt J. Lesker, USA).
ECL spectra are obtained by an ECL spectrometer produced by Prof. Guizheng Zhou's laboratory at Shandong University. The ECL spectrometer is composed of Acton SP 2300i monochromator including PyLoN 400BR-eXcelon digital CCD detector (Princeton, USA) to be cooled by liquid N2 and VersaSTAT3 electrochemical analyzer (Princeton, USA).
Cyclic voltammetry experiments are performed on CHI 630D electrochemical workstation (CHI instruments Inc., China).
Anodic and cathodic ECL experiments are performed in an in-house manufactured reaction vessel of an MPI-EII ECL analyzer (Xi'an Remex, China). The analyzer employs a three-electrode system.
ECL imaging is performed with an in-house manufactured ECL imaging system. The imaging system includes a focusing lens (EF 50 mm f/1.2 L USM, Canon), an electron multiplying charge coupled device (EMCCD, iXon Ultra, Andor, UK), and a CHI-660D electrochemical workstation. An Au/ITO electrode is used as a working electrode, Ag/AgCl wire is used as a reference electrode, and Pt wire is used as a counter electrode.
a. Synthesis of Hot Exciton Molecule BCzP-BT
First, 4-(9H-carbazol-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 mixed solution of 20 mL of toluene and 4 mL of water contained in a Schlenk tube, and then the resulting mixed solution was stirred in the Schlenk tube at 90° C. for 18 hours in an atmosphere of argon. Subsequently, the reacted mixed solution was extracted with dichloromethane by using petroleum ether and dichloromethane as eluents and purified by silica gel column chromatography, whereby 0.7512 g of a BCzP-BT product (that is, 4,7-bis(4-(9H-carbazol-9-yl)phenyl)benzo[c][1,2,5]thiadiazole (p-BCzP-BT), hereinafter abbreviated as “BCzP-BT”) was obtained and the yield thereof was 86%. The synthesis of the BCzP-BT molecule was verified by nuclear magnetism (1H NMR) and a mass spectrum. The results are illustrated in
b. Production of Hot Exciton Organic Nanorods (BB NRs)
First, 2.5 mL of a THF solution containing 50 μg/mL of BCzP-BT and 10 μg/mL of a polystyrene-maleic anhydride copolymer (PSMA, Mn=1700) was ultrasonically treated at room temperature for 20 minutes. The solution was then quickly added to 10 mL of water and ultrasonically treated for 30 seconds. By rotary evaporation at 35° C., the solvent THF was removed, and the resultant solution was further filtered through a polyethersulfone syringe filter to obtain BB NRs.
c. Excited State and Characteristics Analysis of BCzP-BT Molecule
An excited state of the BCzP-BT molecule produced in Example 1 was analyzed. The results of the analysis are illustrated in
First, normalized ultraviolet-visible absorbance and fluorescence (FL) spectra of the BCzP-BT molecule in different polar solvents were examined. The results are illustrated in
Subsequently, differences in FL spectra of CzP and BT in the BCzP-BT molecule and BCzP-BT in the 10−5M THF solution were analyzed. The results are illustrated in
Furthermore, to study the excited state properties of BCzP-BT, a variation curve of Stokes displacement (va-vf) and the orientation polarizability (Δf) of the solvent was drawn, based on a Lippert-Mataga solvent-induced color change model, and a dipole moment of S1 was calculated based on the slope of the curve. The results are illustrated in
In
Furthermore, the fluorescence lifetime of BCzP-BT was analyzed. The results are illustrated in
To verify the possibility of the hot exciton process, the energy levels of the lowest singlet and triplet excited states of BCzP-BT were calculated using TD-m062x/6-311g(d). The results are illustrated in
The results illustrated in
Furthermore, natural transition orbital (NTO) simulations were performed for the BCzP-BT molecule by using the Multiwfn software. The simulation results are illustrated in
d. Characteristics Analysis of BB NRs
The characteristics of the BB NRs produced in Example 1 of the present invention were analyzed. The results are illustrated in
First, the form of synthesized BB NRs was analyzed by TEM and AFM. The TEM and AFM images are illustrated in
e. ECL Characteristics of BB NRs
The ECL characteristics of the BB NRs produced in Example 1 of the present invention were analyzed.
First, the stability of a radical intermediate of the BB NRs was examined by conducting an annihilation ECL experiment. Note that specific conditions of the annihilation ECL experiment are as follows.
The results of the experiment are illustrated in
In addition, as seen from
At the same time, the annihilated state ECL of the BB NRs was detected using the pulse step method. The results are illustrated in
The ECL of the BB NRs in the presence of a coreactant was further analyzed. The results of the analysis are illustrated in
First, an anodic ECL behavior of BB NRs was examined using tripropylamine (TPrA) as a coreactant.
Specific experimental conditions for the anodic ECL experiment are as follows.
The results of the analysis are illustrated in
As seen from
As seen from
Furthermore, the ECL efficiency of BB NRs was verified by recording a current-time curve and ECL spectra of BB NRs by using 1 mM of Ru(bpy)32+ as a standard in the presence of 10 mM of TPrA· or 100 mM of K2S2O8. Note that specific experimental conditions are the same as for the anodic and cathodic ECL experiments described above. The results are illustrated in
In Table 3, relative ECL efficiencies of BB NRs/GCE and Ru(bpy)32+/GCE are illustrated.
Here, φ and φ°, I and I°, and Q and Q° respectively indicate ECL efficiencies, ECL intensity integrals (integrated ECL spectrum vs. wavelength), and charge consumption (integrated CV curve vs. time) of a sample and a standard.
As seen from
Furthermore, the ECL attribution of the BB NRs was analyzed. The results of the analysis are illustrated in
First, anodic and cathodic ECL and FL spectra of the BB NRs were tested. The test results are illustrated in
Furthermore, a CV curve in an organic phase of the BB NRs was tested. The test results are illustrated in
Here, specific test conditions for the organic phase CV are as follows.
1) BB NRs Probe Modified with Oligonucleotide
a. Production of ssDNA-BB NRs
First, 12 μL of polyethylene glycol (PEG, Mw=3350) and 12 μL of 1-M 4-(2-hydroxyethyl)−1-piperazineethanesulfonic acid (HEPES) were sequentially added to 600 μL of BB NRs, and then the pH of the resulting solution was adjusted to 7.4 with 0.1-M NaOH, and then single-stranded DNA (ssDNA-BHQ2, 100 μM) modified with 40 μL of a black hole quencher 2 (BHQ2) and 24 μL of just-prepared EDC (10 mg/mL) were added to the solution. The resulting solution was stirred at room temperature for 24 hours in a dark place, and then free ssDNA-BHQ2 was removed by centrifugation by ultrafiltration to obtain ssDNA-BB NRs. The ssDNA-BB NRs were diluted so as to have the same absorbance at 410 nm as that of the BB NRs, and stored away from light.
b. Characteristics Analysis of ssDNA-BB NRs
The ultraviolet absorption spectrum and Zeta potential of the ssDNA-BB NRs were measured. The results are illustrated in
As seen from the curve a in
As seen from
When the ssDNABB NRs were purified by ultrafiltration, a filtrate was collected each time and ultraviolet thereof was measured. As seen from
4) ECL Switch Based on ssDNA-BB NRs Nanoprobe
a. Principle of Signal Switch
The ssDNA-BB NRs nanoprobe will be described as a reporter probe. As illustrated in
b. Verification of Signal Switch
First, a switch response of the Cas12a enzyme to the ssDNA-BB NRs nanoprobe was analyzed by fluorescence.
Test conditions for a Cas enzyme-fluorescence test are as follows.
The results of the fluorescence test are illustrated in
After the 1-nM target molecule was added, the Cas12a enzyme was activated, the cleavage of BHQ-ssDNA on the BB NRs occurred, an ECL ON response was generated, and consequently the ECL intensity of the sensor electrode was recovered up to 75% (the curve c). The results indicated the feasibility of an ECL switch system based on the ssDNA-BB NRs nanoprobe.
In addition, as seen from
ECL Imaging Detection of Sensor Array Constructed Based on ssDNA-BB NRs
a. Design of Detection
The detection method is constructed based on the “ECL switch of ssDNA-BB NRs nanoprobe” (
b. Production of Detection Chip and Detection by Detection Chip: A Specific Detection Flow is Illustrated in
Specific operational steps are as follows.
Step 1: Indium tin oxide (ITO) glass was ultrasonically cleaned with toluene, acetone, ethanol, and ultrapure water for 5 minutes each, and then plated with a 5-nanometer thick chromium layer and a 50-nanometer thick gold layer by an electron beam evaporation sputtering apparatus to obtain an Au/ITO electrode.
Step 2: A porous seal (2 mm in diameter, 1 mm in depth, 4×7 well array) is attached to a surface of the Au/ITO electrode.
Step 3: 3 μL of the ssDNA-BB NRs was dropped into each well and dried at 37° C. for 30 minutes, and then each well was carefully washed with diethylpyrocarbonate (DEPC) water.
Step 4: 0.15 μL of LbCas12a (1 μM), 0.3 μL of crRNA (750 nM), 0.3 μL of a NE buffer (10×), and 1.95 μL of DEPC water were allowed to react at 37° C. for 10 minutes, and then 0.3 μL of HPV16 dsDNA or a sample having different concentrations was added thereto, and the resulting mixed solution was quickly dropped into each well and incubated at 37° C. for 40 minutes.
Step 5: Each well on the chip was washed three times with DEPC water to remove released BHQ2, and then the resulting was dried at 37° C. for 5 minutes. After that, the resulting was used for ECL imaging detection.
Step 6: ECL imaging detection was performed in a dark box by using an Au/ITO electrode as a working electrode, an Ag/AgCL electrode as a reference electrode, a platinum wire electrode as a counter electrode, an electron multiplying charge coupled device (EMCCD, iXon Ultra, Andor, UK) as a signal read-out device, and a 0.1-M pH 7.4 phosphate buffer solution of 5-mM tri-n-propylamine (TPrA) as an electrolyte. Cyclic voltammetry scanning was performed at 0 to +1.0 V, in which the scanning speed was 0.1 V/s and the exposure time was 20 seconds.
c. Optimization of ECL Detection Conditions
First, ECL detection according to the present invention was performed under different detection conditions. The results of the detection are illustrated in
Specifically, as illustrated in
In
In the detection, conditions are selected so that an ECL signal reaches the maximum platform period, and are set as optimal detection conditions. As illustrated in
Next, ECL images of ITO and Au/ITO electrodes modified with BB NR in 5-mM TPrA-containing 0.1 M, pH 7.4 PBS were compared by ImageJ software. The results are illustrated in
As seen from
d. Detection Performance
Detection Linearity and Sensitivity: ECL imaging detection was performed for a HPV16 DNA target molecule by a constructed sensor array under optimal detection conditions. The results are illustrated in
Test for detection selectivity: In the present example, detection specificity for the target nucleic acid HPV16 DNA was tested in the presence of interfering DNA having a 10-fold higher concentration. The test results are illustrated in
As seen from the above-described examples, by using BCzP-BT as a luminescent molecule, the present invention produced hot exciton organic nanorods (BB NRs) exhibiting sensitive annihilation ECL and the luminescence of a strong coreactant ECL for the first time. Because of the high ΦPL owing to the locally-excited state and high exciton utilization efficiency owing to the charge transfer state, the hot exciton organic nanorods are superior in ΦECL to conventional fluorescent materials and thermally activated delayed fluorescent materials. Furthermore, prompt and high-throughput detection of HPV16 DNA with a simplified operating procedure was achieved by using the ECL nanoprobe of the present invention, identifying a target nucleic acid by the CRISPR/Cas enzyme system, and switching the signal from off to on. Thus, according to the present invention, a highly sensitive ECL detection platform for detecting HPV16 DNA can be constructed. The detection effect of the platform is similar to an ECL detection method using Ru(bpy)32+ as an ECL standard. Thus, the ECL detection method according to the present invention is a feasible and reliable ECL detection method.
While certain embodiments have been described, these embodiments have been presented by way of example only, and are not intended to limit the scope of the inventions. Indeed, the novel embodiments described herein may be embodied in a variety of other forms; furthermore, various omissions, substitutions and changes in the form of the embodiments described herein may be made without departing from the spirit of the inventions. The accompanying claims and their equivalents are intended to cover such forms or modifications as would fall within the scope and spirit of the inventions.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202310682568.5 | Jun 2023 | CN | national |
