CN114002200A - Near-infrared two-region activated probe and application thereof - Google Patents
Near-infrared two-region activated probe and application thereof Download PDFInfo
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Abstract
The invention discloses a near-infrared two-region activated probe and application thereof. The near-infrared two-region activated probe comprises a detection system and an amplification system, wherein the detection system consists of a first strand probe, a second strand probe and a third strand probe, and the amplification system consists of a target sequence to be detected, the second strand probe, the third strand probe and a fourth strand probe; the second strand probe is completely complementary with the third strand probe, and the first strand probe and the fourth strand probe are partially complementary with the third strand probe; and partial sequence in the third strand probe is completely complementary with the target sequence to be detected, and partial sequence in the first strand and the fourth strand is partially identical with the target sequence to be detected. According to the invention, the toe-hold strand displacement reaction and the near-infrared two-region fluorescence are combined, and the constructed near-infrared two-region activated probe can be used for carrying out high-sensitivity, high-specificity and rapid detection on sentinel lymph node metastasis of breast cancer at the living body level, and has important significance for early diagnosis of breast cancer metastasis.
Description
Technical Field
The invention relates to a molecular probe, in particular to a near-infrared two-region activated probe and application thereof in detection of metastasis of sentinel lymph nodes of tumors, belonging to the technical field of nano biosensing.
Background
In recent years, the morbidity and mortality of malignant tumors are increasing, and the discovery of late stage, easy metastasis and difficult cure is a challenging problem in cancer treatment. The breast cancer is the most serious malignant tumor in Chinese women, and the mortality rate of the breast cancer is the fourth place. Metastasis of breast cancer is a major cause of mortality in patients, with lymphatic metastasis being the most prominent pathway for breast cancer spread. Sentinel lymph node positive identification is the main means for defining lymphatic metastasis and also provides guidance for subsequent treatment. The existing identification methods comprise ultrasonic, X-ray photographic examination, puncture biopsy and the like, and have certain limitations: firstly, the in-vivo detection method mainly adopts morphological detection and cannot be qualitative; secondly, biopsy is easy to miss due to limited material taking by combining biopsy puncture with pathological analysis. Therefore, a novel in-vivo in-situ, high-sensitivity and high-specificity detection method is developed, accurate detection of the tumor sentinel lymph node is realized, and the method has great significance for prevention and control of breast cancer.
miRNA is a kind of endogenous non-coding single-stranded short-molecule RNA (18-22nt), plays an important role in regulation and control in a series of biological processes, and the abnormal expression level of miRNA is related to various diseases, in particular to human cancers. In clinical applications, mirnas are becoming potential biomarkers for cancer diagnosis and prognosis. Research shows that miRNA such as miR-10b/21/135a/155/221/222/224/373/520c and the like are over-expressed in breast cancer cells. For example, miR-21 is in progressive up-regulation from normal mammary gland to tumor with high tumor stage. The miR-155 is over-expressed in the breast cancer cells, so that the cell proliferation and the growth of the xenograft tumor are promoted, and the migration and invasion of mammary epithelial cells are promoted. The miRNA plays an important role in the aspects of occurrence, development and the like of breast cancer, is widely involved in malignant biological behaviors such as proliferation, invasion and metastasis of breast cancer cells, and is related to key prognostic indexes such as clinical stages, molecular subtypes, histological grading, lymph node metastasis, key receptor expression, distant metastasis and the like of breast cancer patients.
In the aspect of imaging, fluorescence imaging has the unique advantages of high sensitivity, real-time dynamics, multiple parameters, green economy and the like, particularly near-infrared two-region fluorescence (1000 + 1700nm) overcomes the defect of insufficient tissue penetrability of the traditional fluorescence imaging, and meanwhile, due to the reduced biological tissue scattering effect and no autofluorescence interference, high-resolution and high-sensitivity detection of tiny hidden lesions of tumors in deep tissues of living bodies can be realized.
Therefore, how to construct a near-infrared two-region activated probe aiming at miRNA specific fluorescence activation response of specific expression in breast cancer cells is expected to provide a new approach for accurate diagnosis and treatment of breast cancer.
Disclosure of Invention
The invention mainly aims to provide a near-infrared two-region activated probe and application thereof, so that high sensitivity, high specificity and rapid detection on a sentinel lymph node of a tumor are realized, and the defects of the prior art are overcome.
In order to achieve the aim of the invention, the invention adopts the following scheme:
the embodiment of the invention provides a near-infrared two-region activated probe, which comprises a detection system and an amplification system, wherein the detection system consists of a first strand probe, a second strand probe and a third strand probe, and the amplification system consists of a target sequence to be detected, the second strand probe, the third strand probe and a fourth strand probe;
wherein the first strand probe comprises 1-sequence and 2-sequence, and 2*The 5' end of the sequence is connected with a fluorescence donor;
the second strand probe comprises 3*Sequence, and 3*The 3' end of the sequence is connected with a fluorescent acceptor;
the third strand probe comprises a sequence 1, a sequence 2, a sequence 3, a sequence 4 and a sequence 5;
the fourth strand probe comprises 1x sequence, 2 x sequence, 3 x sequence, and 4 x sequence;
the second strand probe is completely complementary with the third strand probe, and the first strand probe and the fourth strand probe are partially complementary with the third strand probe; the third strand probe has 1 sequence complementary to the target sequence to be detected, 1 sequence and 1 sequence partially complementary to 1 sequence, 2*Sequences and 2**The sequence is completely complementary to the sequence 2, 3*Sequences and 3**The sequence is completely complementary to 3 sequences, 4**The sequence is not complementary to the 4 sequence, and the 2 sequence and the 5 sequence are toe-binding sites for hold strand displacement;
the first strand probe, the second strand probe and the third strand probe can be paired under an annealing condition to form a ternary complex, so that a fluorescence acceptor quenches a fluorescence signal of a fluorescence donor;
in a complex formed by the target sequence to be detected, the second strand probe and the third strand probe, the fourth strand probe can be competitively combined with the target sequence to be detected to the third strand probe, so that the target sequence to be detected is dissociated, the dissociated target sequence is continuously competitively combined with the first strand probe to the third strand probe, the first strand probe is dissociated, and the fluorescence donor is far away from the fluorescence acceptor and emits a fluorescence signal.
In some embodiments, the near-infrared two-zone activated probe further comprises a delivery vehicle, and the first strand probe, the second strand probe, the third strand probe, and the fourth strand probe are loaded in the delivery vehicle.
In some embodiments, the delivery vehicle further has a targeting peptide bound thereto, the targeting peptide being capable of specifically binding to a target cell providing the target sequence.
The embodiment of the invention also provides application of the near-infrared two-region activated probe in preparing a product with the function of detecting metastasis of the tumor sentinel lymph node.
The embodiment of the invention also provides a product, which is applied to a detection method of target cells, and the detection method comprises the following steps:
providing the near-infrared second-region activated probe, and loading a ternary complex formed by annealing the first strand probe, the second strand probe and the third strand probe and a fourth strand probe into a delivery carrier; and
and delivering the delivery vector loaded with the near-infrared two-region activated probe into target cells containing target sequences to realize the detection of the target cells.
In some embodiments, the target cell comprises a tumor cell.
Compared with the prior art, the invention has the following advantages:
1) the near-infrared two-region activated probe provided by the invention can provide higher detection sensitivity from 'no' to 'present' fluorescence signals;
2) compared with the traditional fluorescence imaging, the near-infrared two-region fluorescence imaging of the near-infrared two-region activated probe has the advantages of high tissue penetration, high space-time resolution, low tissue autofluorescence and the like, can realize the accurate positioning of the tiny hidden metastasis of the tumor under the deep tissue of the living body, and can realize the noninvasive, comprehensive and accurate detection of multiple sites and multiple levels of tumor metastasis lymph nodes under the state of the living body;
3) the constructed near-infrared two-region activated probe combines the toe-hold strand displacement reaction with the near-infrared two-region fluorescence, so that the characteristic that the strand displacement process is accompanied by the near-infrared fluorescence activation can be used for carrying out high-sensitivity, high-specificity and rapid detection on the sentinel lymph node transfer of the breast cancer on the living body level, so as to provide guidance for the formulation of a surgical scheme and have important significance on the early diagnosis of the breast cancer transfer;
4) the near-infrared two-region activated probe constructed by the invention can detect whether the tumor sentinel lymph node is metastasized in real time in vivo, provides accurate in-vivo image information for staging and typing of breast cancer, and can further guide a subsequent treatment scheme.
Drawings
For a clearer explanation of the embodiments of the present application or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments described in the present application, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a schematic representation of a near-infrared two-zone activation probe for detecting metastasis in a sentinel lymph node of a tumor in an exemplary embodiment of the invention;
FIG. 2 is a graph of polyacrylamide electrophoresis characterization of the fourth chain amplification effect at different concentrations in an exemplary embodiment of the invention;
FIG. 3 is a representation of polyacrylamide electrophoresis of a near-infrared two-region activated probe detection system for detecting analytes of different concentrations in an exemplary embodiment of the invention;
FIG. 4 is a graph showing the result of fluorescence activation experiments performed by an analyte with respect to a near-infrared two-region fluorescence probe according to an exemplary embodiment of the present invention;
FIG. 5 is a graph showing the results of sentinel node imaging of a tumor in a mouse before and after subcutaneous injection of a near-infrared two-zone activated probe in an exemplary embodiment of the invention.
Detailed Description
In view of the defects in the prior art, the inventor of the present invention provides a technical scheme of the present invention through long-term research and a large amount of practice, and mainly constructs a near-infrared two-region activation type probe aiming at the miRNA specific fluorescence activation response of the specific expression in breast cancer cells, so as to realize accurate, rapid and noninvasive comprehensive detection in vivo, and provide guidance for the accurate detection of breast cancer tumor metastasis and the formulation of a treatment scheme. The technical solution, its implementation and principles, etc. will be further explained as follows.
One aspect of the embodiments of the present invention provides a near-infrared two-region activated probe, which includes a detection system and an amplification system, where the detection system includes a first strand probe, a second strand probe, and a third strand probe, and the amplification system includes a target sequence to be detected, and the second strand probe, the third strand probe, and a fourth strand probe;
wherein the first strand probe comprises 1-sequence and 2-sequence, and 2*The 5' end of the sequence is connected with a near-infrared two-region fluorescent dye as a fluorescence donor;
the second strand probe comprises 3*Sequence, and 3*The 3' end of the sequence is connected with a micromolecule quencher as a fluorescence receptor;
the third strand probe comprises a sequence 1, a sequence 2, a sequence 3, a sequence 4 and a sequence 5;
the fourth strand probe comprises 1x sequence, 2 x sequence, 3 x sequence, and 4 x sequence;
the second strand probe is completely complementary to the third strand probe, and the first strand probe, the fourth strand probe and the third strand probe are completely complementary to each otherSequencing complementation; the third strand probe has 1 sequence complementary to a target sequence to be detected (such as miRNA to be detected), 1 sequence and 1 sequence partially complementary to the 1 sequence, and 2*Sequences and 2**The sequence is completely complementary to the sequence 2, 3*Sequences and 3**The sequence is completely complementary to 3 sequences, 4**The sequences are not complementary to the 4 sequences, wherein the 2 and 5 sequences are binding sites for toe-hold strand displacement.
The first strand probe, the second strand probe and the third strand probe can be matched under an annealing condition to form a ternary complex, so that the near-infrared fluorescent dye and the small molecule quencher are close to each other, the spatial distance is less than 10nm, and the fluorescent acceptor quenches a fluorescent signal of a fluorescence donor; and under the condition that the miRNA to be detected reaches a certain concentration, the miRNA to be detected and the first strand are combined with the third strand in a competitive manner, so that the first strand is dissociated, the fluorescence donor is far away from the fluorescence acceptor and emits a fluorescence signal, and the effect of fluorescence activation is achieved.
In a complex formed by a target sequence to be detected (such as miRNA to be detected), a second strand probe and a third strand probe, a fourth strand probe can be competitively combined with the target sequence to be detected to the third strand probe, so that the target sequence to be detected is dissociated, the dissociated target sequence is continuously competitively combined with the first strand probe to the third strand probe, the first strand probe is dissociated, and a fluorescence donor is far away from a fluorescence acceptor and emits a fluorescence signal, so that the effect of signal amplification is achieved.
In some embodiments, the target sequences to be detected include all miRNA sequences highly expressed in breast cancer, the 1 sequence of the third strand probe is adjusted according to different miRNA sequences, and 1 complementary thereto*Sequences and 1**And (4) sequencing.
In some embodiments, the near-infrared two-region activated probes include tumor-targeting vesicles as delivery vehicles and activated probes for detection.
Further, the activated probe comprises a detection system and an amplification system, wherein the detection system comprises a first strand probe, a second strand probe and a third strand probe, and the amplification system comprises the miRNA to be detected, the second strand probe, the third strand probe and a fourth strand probe.
In some embodiments, the near-infrared two-region activated probes include tumor cell targeting vesicles, first strand probes linked to a fluorescence donor, second strand probes linked to a fluorescence acceptor, third and fourth strand probes, and the like; wherein the second strand probe is completely complementary to the third strand probe, and the first strand probe and the fourth strand probe are partially complementary to the third strand probe. And a part of sequences in the third strand probe are completely complementary with the miRNA sequence to be detected, and a part of sequences in the first strand probe and the fourth strand probe are partially identical with the miRNA sequence to be detected.
In some embodiments, the fluorescence donor comprises a near-infrared two-region fluorescent dye, and further, the fluorescence donor comprises Ag2Se、Ag2S、Ag2Te, 00-81, and the like.
In some embodiments, the fluorescent acceptor comprises a small molecule quencher, and further, the fluorescent acceptor comprises carboxyl modified A1094, Cu2Se or CuS, etc.
Wherein, the fluorescence donor and the fluorescence acceptor are both positioned in a near-infrared two-zone window, and compared with visible light, the fluorescence donor and the fluorescence acceptor have deeper penetration depth and higher spatial resolution, lower tissue scattering and lower tissue autofluorescence and can provide better biological imaging effect.
In some embodiments, the near-infrared two-region-activated probe further comprises a delivery vehicle, and the first strand probe, the second strand probe, the third strand probe, and the fourth strand probe are loaded (packaged) in the delivery vehicle.
Further, the delivery vehicle comprises vesicles of selected cellular origin, preferably comprising cell membranes, exosome membranes or platelet membranes of macrophages, stem cells or T-cells. Wherein vesicles of different cell sources are used as targeted delivery carriers, and comprise cell membranes or exosome membranes of macrophages, stem cells, T cells and the like, platelet membranes and the like. And a targeting peptide that specifically binds to the target cell is incorporated or chemically coupled to the surface of the delivery vehicle.
In some embodiments, the delivery vehicle further has a targeting peptide bound thereto, the targeting peptide being capable of specifically binding to a target cell providing the target sequence.
Furthermore, the delivery carrier is combined with targeting peptides such as RGD, HER-2, ER, PR and the like which can be specifically combined with target cells with high expression target miRNA sequences.
In some embodiments, the working concentration of each of the first strand probe, the second strand probe, and the third strand probe is 0.1 to 10. mu.M, and the working concentration of the fourth strand probe is 0.1 to 100. mu.M.
The embodiment of the invention also provides application of the near-infrared two-region activated probe in preparing a product with the function of detecting metastasis of a tumor sentinel lymph node.
Accordingly, another aspect of the embodiments of the present invention provides a product, which is applied to a method for detecting target cells, the method comprising:
providing the near-infrared second-region activated probe, and loading a ternary complex formed by annealing the first strand probe, the second strand probe and the third strand probe and a fourth strand probe into a delivery carrier; and
and delivering the delivery vector loaded with the near-infrared two-region activated probe into target cells containing target sequences to realize the detection of the target cells.
Further, the target sequence includes miRNA-155, microRNA-10b, microRNA-18a, microRNA-181, microRNA-221, microRNA-222 and the like, but is not limited thereto.
Further, the target cells include tumor cells, MDA MB 231, MCF-7, BT-474, etc., but are not limited thereto.
Further, the method can be used for detecting whether the breast cancer has the metastasis of a sentinel lymph node of the tumor.
Wherein, the fluorescence donor and the fluorescence acceptor are both positioned in a near-infrared two-zone window, and compared with visible light, the fluorescence donor and the fluorescence acceptor have deeper penetration depth and higher spatial resolution, lower tissue scattering and lower tissue autofluorescence and can provide better biological imaging effect.
In another aspect of the embodiments of the present invention, there is provided an activated probe, wherein the first strand probe, the second strand probe and the third strand probe are base complementary paired to each other, the fluorescence donor and the fluorescence acceptor are close to each other, and a fluorescence signal of the fluorescence donor is absorbed by the fluorescence acceptor, and the fluorescence signal is "none" when the fluorescence signal is "none"; when the miRNA sequence to be detected reaches a certain concentration, the miRNA to be detected and the first strand probe are combined with the third strand probe in a competitive manner, so that the first strand probe is dissociated, the fluorescence donor is far away from the fluorescence acceptor and emits a fluorescence signal, and the fluorescence signal is 'available'. Fluorescence signals from "no" to "present" can provide higher detection sensitivity.
In some embodiments, the difference between the specific miRNA expression levels of the target cell and the background cell can be determined, and the detection system can only respond to miRNA with a certain concentration by adjusting the chain length and the base sequence of each detection system, so that the background signal can be reduced, and the overall signal-to-noise ratio can be improved.
In conclusion, the toe-hold strand displacement reaction and the near-infrared two-zone fluorescence are combined, so that the characteristic that the strand displacement process is activated along with the near-infrared fluorescence can be used for carrying out high-sensitivity, high-specificity and rapid detection on the sentinel lymph node metastasis of the breast cancer on a living body level, so that guidance is provided for the formulation of a surgical plan, and the method has important significance for the early diagnosis of the breast cancer metastasis.
Furthermore, compared with the traditional fluorescence imaging, the near-infrared two-region fluorescence imaging has the advantages of high tissue penetration, high space-time resolution, low tissue autofluorescence and the like, and can realize the accurate positioning of the tiny tumor occult metastasis under the deep tissue of the living body. The near-infrared two-region activated probe can realize noninvasive, comprehensive and accurate detection of multiple sites and multiple levels of tumor metastasis lymph nodes in an in-vivo state.
The technical solutions of the present invention will be described in further detail below with reference to several preferred embodiments and accompanying drawings, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention. The conditions used in the following examples may be further adjusted as necessary, and the conditions used in the conventional experiments are not generally indicated.
Example 1
The present embodiment provides an activation probe for detecting tumor sentinel lymph node metastasis of miRNA 155 with high expression of breast cancer, which comprises: the tumor targeting peptide loaded on the delivery carrier has the targeting property, imaging and detecting functions. In an embodiment, the fluorescent material and the nucleic acid sequence are both wrapped in a targeting functionalized modified stem cell vesicle to form the near-infrared two-region activated probe.
1. Preparation of near-infrared activated probe
(1) DNA chain design: using IDT-OligoAnalyzerTMTool design each nucleic acid sequence: fluorescent dye chain (F-DNA), quencher chain (Q-DNA), Linker-DNA, Fuel chain (Fuel-DNA) and microRNA 155 (the sequences are respectively shown as SEQ ID No: 1-SEQ ID No: 5), all designed nucleic acid sequences are synthesized by the committee of Biotechnology engineering (Shanghai) GmbH, the modification of amino, sulfydryl and other groups is completed, the designed nucleic acid sequences are ensured not to form hairpin structures or dimers by themselves, and the stability is maintained at 37 ℃. The interaction of each nucleic acid sequence is analyzed by utilizing NUPACK software, so that the ternary complex (L/F/Q) formed by annealing F-DNA, Q-DNA and Linker-DNA is ensured to be stable at 37 ℃ and can not be automatically melted. When the target sequence is not detected, the ternary complex stably exists, and the fluorescence keeps a quenching state; when a detection target sequence exists, a toe-hold strand displacement reaction can be triggered, F-DNA is displaced, a fluorescent signal is recovered, and Fuel-DNA can release the detection target sequence through the toe-hold strand displacement reaction, so that the detection target sequence can continuously participate in the reaction, and the amplification of the fluorescent signal is realized.
In vitro experiments, all sequences were dissolved in secondary deionized water, and the reaction system was PBS buffer (containing 10mmol/L Mg)2+pH7.4) in a molar ratio ofLinker-DNA: F-DNA: Q-DNA ═ 1: (1-2): (1-2) after uniformly mixing the Linker-DNA, the F-DNA and the Q-DNA, annealing in a PCR instrument under the condition that the temperature is firstly 95 ℃ for 3 min; the temperature was then lowered by 1 ℃ every 1min until it was lowered to 4 ℃ to allow formation of a paired ternary complex.
Note: the nucleic acid sequence is mainly used for detecting tumor cell specific miRNA, aiming at different detected miRNA sequences, adjusting the 1 sequence of the third chain and the 1 complementary to the 1 sequence*Sequences and 1**And (4) sequencing.
(2) The connection of 00-81 (near infrared two-region fluorescent small molecules) on F-DNA and carboxyl modified A1094 on Q-DNA: the above 00-81 is used as a near infrared two-region fluorescent molecule, an amino group is modified at the 5' end of F-DNA, carboxyl on the surface of the 00-81 is activated by using 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) in the presence of N-hydroxy thiosuccinimide (sulfo-NHS), and then the F-DNA with the amino group is added into a reaction system to form the F-DNA-00-81 with a stable structure. The quencher A1094 has carboxyl groups, and as in the above step, the carboxyl groups on the surface of A1094 are activated with EDC in the presence of sulfo-NHS, and then Q-DNA having amino groups at the 3' -end is added to form Q-DNA-A1094. Finally F-DNA with fluorescent group and Q-DNA with quenching agent are obtained.
(3) Preparation of a delivery vector: and culturing and collecting the mesenchymal stem cells, and expressing the azide groups on the surfaces of the stem cells by using a metabolic engineering method. The cell is broken by hypotonic, and is ground and broken, and the suspension of the cell membrane of the mesenchymal stem cell with high expression azide group is obtained by a mode of centrifugally separating organelles and other substances in the cell by different parameters step by step, and the cell membrane solid powder can also be obtained by continuous freeze-drying. Mixing the probe and the cell membrane according to a ratio of 1:10, extruding the mixed suspension respectively through porous filter membranes with the aperture of 1 mu m, 500nm, 200nm and 100nm, or coating the cell membrane on the surface of the probe in an ultrasonic mode to obtain a delivery carrier with uniform appearance and size and stable property, and modifying the RGD peptide on the surface of the cell membrane through click reaction.
2. In vitro validation experiment
(1) Non-denaturing polyacrylamide gel electrophoresis experiment verifies: 10 μ M Linker-DNA, molar ratio Linker-DNA: F-DNA: Q-DNA ═ 1: (1-2): and (1-2) uniformly mixing the Linker-DNA, the F-DNA and the Q-DNA, annealing to form a ternary compound L/F/Q, wherein the reaction system is 50 mu L. Adding the annealed product and Fuel-DNA (1 mu mol/L-10 mu mol/L) with different concentrations into 50 mu L PBS buffer solution at the same time, adding a certain amount of miRNA 155 (concentration: 1nmol/L-500nmol/L), incubating the ternary complex in the reaction system for 1 mu M at 37 ℃ for different reaction times (1-6h), verifying the strand displacement reaction result by non-denaturing polyacrylamide gel electrophoresis, and proving that the ideal strand displacement effect can be obtained when the molar ratio of L/F/Q to Fuel-DNA is 1:2 and the incubation time is 1.5 h.
(2) In-vitro fluorescence activation experiment results show that when the molar ratio of L/F/Q to Fuel-DNA is 1:2 and the incubation time at 37 ℃ is 1.5h, the designed activation type probe can be used for detecting miRNA 155 with high sensitivity, high specificity and high signal-to-noise ratio.
3. In vivo level verification
1x10 of high expression miRNA 1555The MDA MB 231 cells are inoculated to the forelimb lymph node part of a mouse in a lymph injection mode, when tumors grow to a certain size, a ternary complex formed by annealing a vesicle-wrapped first chain probe, a second chain probe and a third chain probe and a fourth chain probe are injected to the sentinel lymph node of the mouse in a lymph injection mode, miRNA 155 highly expressed by the tumor cells starts a chain displacement reaction, a fluorescence signal is activated, and noninvasive, accurate and comprehensive detection of the living body level can be realized.
FIG. 1 is a schematic representation of a near-infrared two-zone activation probe for detecting metastasis in a sentinel lymph node of a tumor; FIG. 2 is a graph showing the effect of amplifying the fourth strand probe in different concentrations; FIG. 3 is a representation of polyacrylamide electrophoresis of a near-infrared two-region activated probe detection system for detecting analytes of different concentrations; FIG. 4 is a graph showing the result of a fluorescence activation experiment of an analyte with respect to a near-infrared two-region fluorescent probe; FIG. 5 is a graph showing the results of developing sentinel lymph nodes of a tumor in a mouse before and after subcutaneous injection of a near-infrared two-zone activation type probe.
While the invention has been described with reference to illustrative embodiments, it will be understood by those skilled in the art that various other changes, omissions and/or additions may be made and substantial equivalents may be substituted for elements thereof without departing from the spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from its scope. Therefore, it is intended that the invention not be limited to the particular embodiment disclosed for carrying out this invention, but that the invention will include all embodiments falling within the scope of the appended claims.
Sequence listing
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Claims (10)
1. A near-infrared two-region activated probe is characterized by comprising a detection system and an amplification system, wherein the detection system consists of a first strand probe, a second strand probe and a third strand probe, and the amplification system consists of a target sequence to be detected, the second strand probe, the third strand probe and a fourth strand probe;
wherein the first strand probe comprises 1-sequence and 2-sequence, and 2*The 5' end of the sequence is connected with a fluorescence donor;
the second strand probe comprises 3*Sequence, and 3*The 3' end of the sequence is connected with a fluorescent acceptor;
the third strand probe comprises a sequence 1, a sequence 2, a sequence 3, a sequence 4 and a sequence 5;
the fourth strand probe comprises 1x sequence, 2 x sequence, 3 x sequence, and 4 x sequence;
the second strand probe is completely complementary with the third strand probe, and the first strand probe and the fourth strand probe are partially complementary with the third strand probe; the third strand probe has 1 sequence complementary to the target sequence to be detected, 1 sequence and 1 sequence partially complementary to 1 sequence, 2*Sequences and 2**The sequence is completely complementary to the sequence 2, 3*Sequences and 3**The sequence is completely complementary to 3 sequences, 4**The sequence is not complementary with the sequence 4, and the sequence 2 and the sequence 5 are binding sites of toe-hold strand displacement;
the first strand probe, the second strand probe and the third strand probe can be paired under an annealing condition to form a ternary complex, so that a fluorescence acceptor quenches a fluorescence signal of a fluorescence donor;
in a complex formed by the target sequence to be detected, the second strand probe and the third strand probe, the fourth strand probe can be competitively combined with the target sequence to be detected to the third strand probe, so that the target sequence to be detected is dissociated, the dissociated target sequence is continuously competitively combined with the first strand probe to the third strand probe, the first strand probe is dissociated, and the fluorescence donor is far away from the fluorescence acceptor and emits a fluorescence signal.
2. The near-infrared two-zone activated probe according to claim 1, characterized in that: the fluorescence donor comprises a near-infrared two-zone fluorescent dye, preferably comprising Ag2Se、Ag2S、Ag2Te or 00-81.
3. The near-infrared two-zone activated probe according to claim 1, characterized in that: the fluorescence acceptor comprises a micromolecular quencher, preferably carboxyl modified A1094 and Cu2Se or CuS.
4. The near-infrared two-region-activated probe according to claim 1, further comprising a delivery vehicle, wherein the first strand probe, the second strand probe, the third strand probe, and the fourth strand probe are loaded in the delivery vehicle.
5. The near-infrared two-zone activated probe according to claim 4, characterized in that: the delivery vehicle comprises vesicles of selected cellular origin, preferably comprising cell membranes, exosome membranes or platelet membranes of macrophages, stem cells or T cells.
6. The near-infrared two-zone activated probe according to claim 4, characterized in that: a targeting peptide is also bound to the delivery vector, and the targeting peptide can be specifically bound with target cells providing a target sequence; preferably, the targeting peptide comprises RGD, HER-2, ER or PR.
7. The near-infrared two-zone activated probe according to claim 1, characterized in that: the working concentration of the first strand probe, the working concentration of the second strand probe and the working concentration of the third strand probe are all 0.1-10 mu M, and the working concentration of the fourth strand probe is 0.1-100 mu M.
8. Use of the near-infrared two-region activated probe according to any one of claims 1 to 7 for preparing a product having a function of detecting metastasis of a tumor sentinel lymph node.
9. A product applied to a detection method of target cells, which is characterized in that the detection method comprises the following steps:
providing the near-infrared two-region activated probe of any one of claims 1-7, and loading the ternary complex formed after annealing of the first strand probe, the second strand probe, the third strand probe, and the fourth strand probe into a delivery vehicle; and
and delivering the delivery vector loaded with the near-infrared two-region activated probe into target cells containing target sequences to realize the detection of the target cells.
10. The product of claim 9, wherein: wherein the target sequence comprises microRNA-155, microRNA-10b, microRNA-18a, microRNA-181, microRNA-221 or microRNA-222; and/or, the target cell comprises a tumor cell, MDA MB 231, MCF-7, or BT-474.
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