CN112725414A - Long-afterglow molecular beacon probe, and construction method and application thereof - Google Patents
Long-afterglow molecular beacon probe, and construction method and application thereof Download PDFInfo
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- CN112725414A CN112725414A CN202011516896.0A CN202011516896A CN112725414A CN 112725414 A CN112725414 A CN 112725414A CN 202011516896 A CN202011516896 A CN 202011516896A CN 112725414 A CN112725414 A CN 112725414A
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- molecular beacon
- afterglow
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Abstract
The invention discloses a long-afterglow molecular beacon probe, and a construction method and application thereof. The long persistence molecular beacon probe comprises: a molecular beacon; fluorescent donor materials and energy acceptor materials respectively modified at two ends of the molecular beacon; the fluorescence donor material comprises a rare earth-based long-afterglow luminescent material, phosphorescent/long-afterglow luminescent carbon quantum dots, phosphorescent semiconductor quantum dots and the like, the energy acceptor material is a quencher, and the quencher comprises an optical material with quenching long-afterglow performance. The long afterglow luminescent material adopted by the invention has simpler preparation steps, good water solubility, more stable luminescence and longer emission life, and the constructed time-resolved molecular beacon probe based on long afterglow luminescence has higher signal-to-noise ratio and sensitivity, is applied to the high sensitivity detection of micro RNA in an in vitro biological sample, and provides a new strategy for the design of the molecular beacon probe.
Description
Technical Field
The invention relates to a molecular beacon probe, in particular to a time-resolved molecular beacon probe based on rare earth long afterglow luminescence and a construction method thereof, which are applied to the high-sensitivity detection of micro RNA or other nucleic acids in-vitro biological samples and belong to the technical field of nano fluorescence detection.
Background
Molecular Beacons (MBs) are a fluorescently labeled oligonucleotide chain, generally composed of three parts: loop region: consists of 15-30 nucleotides which can be specifically combined with target molecules; stem area: generally consists of 5-8 base pairs which can be reversibly dissociated; ③ fluorescence donor-acceptor: the two ends of the molecular beacon are respectively marked with a fluorescent dye and a quenching dye. When no target molecule exists, the fluorescence donor and the quenching group of the molecular beacon are close to each other, energy transfer occurs, and fluorescence is quenched; upon binding to the target molecule, the spatial configuration of the molecular beacon changes, and the energy transfer process is inhibited, resulting in a recovery of fluorescence. The molecular beacon technology has extremely high specificity and sensitivity, and is widely applied to aspects such as clinical diagnosis, gene detection, environmental monitoring, living cell imaging, gene chips, biosensing and the like.
However, most of the existing molecular beacons use organic small molecules such as fluorescein, rhodamine, or cyanine as fluorescence donors, the excitation wavelength of such organic dyes is usually in the visible light region, and under the excitation, the sample matrix brings optical interference such as background fluorescence and scattered light, and the sensitivity is reduced. The discovery of materials with special luminescent properties brings new opportunities for the development of molecular beacon technology. The two-photon material with near-infrared excitation and the rare earth up-conversion luminescent material are better applied to the design of molecular beacons, the near-infrared excitation obviously overcomes the interference of background fluorescence and scattered light, and the signal-to-noise ratio is improved. However, the two-photon excitation requires a special femtosecond pulse laser, and the quantum efficiency of the rare earth up-conversion luminescence is low, so that the application is limited to a certain extent. Phosphorescent materials (such as rare earth metal coordination compounds) and long-afterglow luminescent materials with time resolution performance are special materials which can still emit light for a period of time after an excitation light source is closed, the interference of background fluorescence and scattered light in a substrate is completely overcome, the signal-to-noise ratio is highest during imaging or detection, and the long-afterglow luminescent materials are widely applied to the fields of biochemical sensing, biological imaging and the like.
The literature (anal. chem.2011,83, 1356-3+The complex is an organic micromolecule phosphorescence donor, and the BHQ-2 is a time resolution molecular beacon probe of an energy acceptor, and is applied to high-sensitivity detection of target DNA.
The above prior art mainly has the following problems: 1. when the organic dye molecule is used as a fluorescence donor to construct a molecular beacon, the visible light excitation characteristic of the organic dye molecule can cause optical interference such as matrix background fluorescence, scattered light and the like, so that the sensitivity is reduced; 2. rare earth Eu with phosphorescence characteristics3+The synthesis steps of the complex are complex and tedious, and the water solubility of the complex is usually poor, so that the complex is not beneficial to constructing a molecular beacon.
Disclosure of Invention
The invention mainly aims to provide a long-afterglow molecular beacon probe to overcome the defects in the prior art.
The invention also aims to provide a construction method and application of the long-afterglow molecular beacon probe.
In order to achieve the purpose, the technical scheme adopted by the invention comprises the following steps:
the embodiment of the invention provides a long afterglow molecular beacon probe, which comprises:
a molecular beacon;
fluorescent donor materials and energy acceptor materials respectively modified at two ends of the molecular beacon;
the fluorescence donor material comprises any one or combination of more than two of rare earth-based long-afterglow luminescent materials, phosphorescent/long-afterglow luminescent carbon quantum dots and phosphorescent semiconductor quantum dots, the energy acceptor material is a quencher, and the quencher comprises an optical material with the property of quenching long afterglow.
In some embodiments, the rare earth-based long-lasting phosphor is a polyacrylic acid-modified ZnGa2O4:Cr3+Long persistence nanocrystalline.
In some embodiments, the quencher comprises any one or a combination of two or more of an organic quenching dye, a noble metal nanomaterial, a two-dimensional layered material, a polymer nanoparticle, and the like.
In some embodiments, the molecular beacon comprises a nucleic acid molecular beacon and/or a molecular beacon in which the stem-loop portion is an aptamer.
Further, the nucleic acid molecular beacon is a molecular beacon for detecting target DNA or RNA, and has a sequence shown as SEQ ID NO.1 or a sequence shown as SEQ ID NO. 2.
Further, the stem part is a molecular beacon of the aptamer, which is a molecular beacon for targeting recognition of thrombin and has a sequence shown in SEQ ID No. 3.
The embodiment of the invention also provides a construction method of the long afterglow molecular beacon probe, which comprises the following steps:
and reacting the fluorescent donor material with the molecular beacon by an EDC/NHS activation coupling method to obtain the long-afterglow molecular beacon probe.
In some embodiments, the method of making comprises:
dissolving a fluorescent donor material in an MES buffer solution, adding EDC and NHS, and carrying out oscillation reaction at room temperature for 10-120 min;
and adjusting the pH value of the obtained reaction liquid to 7.4 by adopting alkali liquor, then adding 0.5-3 OD molecular beacon, and oscillating and reacting at room temperature for 2-24 h to obtain the long-afterglow molecular beacon probe.
The embodiment of the invention also provides application of the long-afterglow molecular beacon probe in preparing products with nucleic acid, protein or small molecule detection functions.
The embodiment of the invention also provides the application of the long-afterglow molecular beacon probe in the detection field of nucleic acid, protein or small molecules and the like.
The embodiment of the invention also provides a detection method of a target substance, which comprises the following steps:
providing the long-afterglow molecular beacon probe;
establishing a standard curve between the concentration of a target substance and the luminous intensity at the strongest emission wavelength of the long-afterglow molecular beacon probe;
and mixing the long-afterglow molecular beacon probe with a sample containing a target substance to be detected, reacting at 60-95 ℃ for 2-8 min, annealing to 20-37 ℃, reacting for 0.5-2 h, carrying out phosphorescence detection on a product, and contrasting with the standard curve to realize the detection of the target substance.
Compared with the prior art, the invention has the advantages that:
the invention provides a time-resolved molecular beacon probe based on rare earth long afterglow luminescence, which is applied to the high-sensitivity detection of micro RNA in an in vitro biological sample, mainly solves the spectral interference problems of background fluorescence, scattered light and the like caused by fluorescence excitation in the existing molecular beacon probe, improves the detection signal-to-noise ratio, and provides a new strategy for the design of the molecular beacon probe; meanwhile, the long-afterglow luminescent material adopted by the invention has simpler preparation steps, good water solubility, more stable luminescence, longer emission life, and higher signal-to-noise ratio and sensitivity of the constructed molecular beacon probe.
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 diagram illustrating a process of constructing a near-infrared long-afterglow molecular beacon probe and a principle of detecting miRNA21 in an exemplary embodiment of the invention;
FIGS. 2A and 2B are ZnGa diagrams in an exemplary embodiment of the invention2O4:Cr3+A Transmission Electron Microscope (TEM) picture and an X-ray powder crystal diffraction (XRD) crystal phase representation result picture of the long-afterglow nano particles;
FIG. 3 shows ZnGa in an exemplary embodiment of the invention2O4:Cr3+The optical characterization result graph of the long-afterglow luminescent material, wherein a is an absorption spectrum curve, b is an excitation spectrum curve, and c is an emission spectrum curve;
FIGS. 4A and 4B are schematic views of water-soluble ZnGa according to an exemplary embodiment of the present invention2O4:Cr3+TEM image and Fourier infrared spectrum characterization result image of the long afterglow nano particle;
fig. 5 is a schematic diagram of the result of applying the near-infrared long-afterglow molecular beacon probe to miRNA21 detection in an exemplary embodiment of the invention.
Detailed Description
The technical solution of the present invention will be explained in more detail below. It is to be understood, however, that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with one another to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
In view of the defects in the prior art, the inventor of the present invention has made long-term research and a great deal of practice to provide a technical scheme of the present invention, which mainly provides a time-resolved molecular beacon probe based on rare earth long afterglow luminescence, and applies the time-resolved molecular beacon probe to the high-sensitivity detection of micro RNA in an in vitro biological sample, mainly solves the problem of spectral interference of background fluorescence, scattered light and the like caused by fluorescence excitation in the existing molecular beacon probe, improves the detection signal-to-noise ratio, and provides a new strategy for the design of the molecular beacon probe. The technical solution, the implementation process and the principle thereof will be further explained with reference to the drawings.
One aspect of the embodiments of the present invention provides a long persistence molecular beacon probe, which includes:
a molecular beacon;
fluorescent donor materials and energy acceptor materials respectively modified at two ends of the molecular beacon;
the fluorescence donor material comprises any one or combination of more than two of rare earth-based long-afterglow luminescent materials, phosphorescent/long-afterglow luminescent carbon quantum dots, phosphorescent semiconductor quantum dots and the like, the energy acceptor material is a quencher, and the quencher comprises an optical material with the property of quenching long afterglow.
In some embodiments, the rare earth-based long-lasting phosphor is preferably polyacrylic acid-modified ZnGa2O4:Cr3 +Long persistence nanocrystalline.
The key point of the invention is how to construct a time-resolved molecular beacon probe system based on long-afterglow luminescence, wherein the important point is the selection of the long-afterglow material, including but not limited to the rare earth long-afterglow luminescence material ZnGa mentioned in the invention2O4:Cr3+And the material can be any other kind of long-life luminescent material, including other rare earth based afterglow luminescent materials, phosphorescent/long afterglow luminescent carbon dots, phosphorescent semiconductor quantum dots, and the like, but is not limited thereto.
In some embodiments, the quencher includes any one or a combination of two or more of an organic quenching dye, a noble metal nanomaterial, a two-dimensional layered material, a polymer nanoparticle, and the like, but is not limited thereto.
Further, the organic quenching dye is BHQ, but is not limited thereto.
The energy acceptor material of the molecular beacon of the invention includes but is not limited to BHQ and other organic quenching dyes, and can also be any other optical material with quenching long afterglow performance. For example, BHQ may be replaced with other quenchers, such as: noble metal nanometer materials, two-dimensional layered materials, polymer nanoparticles and the like can be regarded as the modified design of the invention, and the design concept and the technical principle of the invention are the same as those of the scheme.
In some embodiments, the molecular beacon comprises a nucleic acid molecular beacon, a molecular beacon in which the stem-loop portion is an aptamer, or the like. Wherein, the nucleic acid molecular beacon can be replaced by a nucleic acid aptamer for detecting corresponding protein, small molecule or metal ion and the like.
Further, the nucleic acid molecular beacon is a molecular beacon for detecting target DNA or RNA, and has a sequence shown as SEQ ID NO.1, and specifically, the molecular beacon sequence is designed as follows: NH (NH)2-C6-CGATGCGTCAACATCAGTCTGATAAGCTATCCATCG-BHQ3。
Further, the nucleic acid molecular beacon has a sequence shown as SEQ ID NO.2, and specifically, the molecular beacon sequence is designed as follows: NH (NH)2-C6-GCTACGATGCGTCAACATCAGTCTGATAAGCTATCCATCGAGCA-SH。
In some embodiments, the stem-loop portion aptamer-targeted molecular beacon is a thrombin-targeted molecular beacon having a sequence shown in SEQ ID No.3, specifically, the thrombin-targeted molecular beacon has a sequence: NH (NH)2-C6-CGATGCGTGGTTGGTGTGGTTGGATCCATCG-BHQ3。
Another aspect of the embodiments of the present invention further provides a method for constructing the aforementioned long-afterglow molecular beacon probe, which includes: and reacting the fluorescent donor material with the molecular beacon by an EDC/NHS activation coupling method to obtain the long-afterglow molecular beacon probe.
In some embodiments, the construction method comprises:
dissolving a fluorescent donor material in an MES buffer solution, adding EDC and NHS, and carrying out oscillation reaction at room temperature for 10-120 min;
and adjusting the pH value of the obtained reaction liquid to 7.4 by adopting alkali liquor, then adding 0.5-3 OD molecular beacon, and oscillating and reacting at room temperature for 2-24 h to obtain the long-afterglow molecular beacon probe.
In some embodiments, the fluorescence donor material is polyacrylic acid modified ZnGa2O4:Cr3+Long afterglowNanocrystalline and, the polyacrylic acid modified ZnGa2O4:Cr3+The preparation method of the long afterglow nanocrystal comprises the following steps:
uniformly mixing an alkaline substance, oleic acid, water and ethanol to form a first mixed solution;
the Zn source, the Ga source and the Cr source are mixed according to a molar ratio of 1: 2: 0.01-0.3, and uniformly mixing to form a second mixed solution;
dropwise adding the second mixed solution into the first mixed solution, uniformly stirring at room temperature for 10-60 min, and carrying out hydrothermal reaction on the formed hydrothermal reaction system at 180-220 ℃ for 8-24 h to obtain ZnGa2O4:Cr3+Long afterglow nanocrystals; and the number of the first and second groups,
polyacrylic acid is adopted to react the ZnGa2O4:Cr3+Modifying the long afterglow nanocrystal to obtain the ZnGa modified by the polyacrylic acid2O4:Cr3+Long persistence nanocrystalline.
Further, the alkaline substance is NaOH, but is not limited thereto.
Further, the Zn source is Zn (NO)3)2But is not limited thereto.
Further, the Ga source is Ga (NO)3)3But is not limited thereto.
Further, the Cr source is Cr (NO)3)3But is not limited thereto.
In some embodiments, the method of making comprises:
reacting the ZnGa with a catalyst2O4:Cr3+Dissolving the long afterglow nanocrystal in an ethanol-hydrochloric acid solution with the pH value of 1-5, stirring for 0.5-6 h, centrifuging, and dispersing in water to obtain ZnGa2O4:Cr3+Long afterglow nanocrystalline aqueous solutions;
dissolving polyacrylic acid in water, and reacting with ZnGa2O4:Cr3+Mixing the long afterglow nanocrystalline aqueous solution, stirring for 4-24 h at room temperature, and centrifuging the obtained reaction liquid to obtain the ZnGa modified by polyacrylic acid2O4:Cr3+Long persistence nanocrystalline.
Referring to fig. 1, the preparation of the long afterglow luminescent material and the construction of the molecular beacon probe of the present invention comprise the following steps:
the first step is as follows: preparation of oleic acid coated oil-soluble long-afterglow luminescent material ZnGa2O4:Cr3+
The method comprises the following specific steps:
(1) adding 0.3g of NaOH into a mixed solution of 3mL of oleic acid, 4mL of deionized water and 10mL of absolute ethyl alcohol, and stirring for 15min until the NaOH is completely dissolved;
(2) 0.5mL (1M) of Zn (NO)3)2、1mL(1M)Ga(NO3)3And 10 to 100. mu.L (0.05M) of Cr (NO)3)3Mixing, performing ultrasonic treatment to obtain a transparent mixed aqueous solution, then dropwise adding the transparent mixed aqueous solution into the mixed solution in the step (1), and uniformly stirring at room temperature for 30 min;
(3) transferring the obtained mixed solution into a hydrothermal kettle (the inner lining is made of polytetrafluoroethylene) with the volume of 15-50 mL, sealing the hydrothermal kettle, and heating in an oven at the temperature of 180-220 ℃ for 8-24 hours;
(4) taking out the hydrothermal kettle naturally cooled to room temperature from the oven, centrifuging the white precipitate at the bottom of the kettle to obtain a product, adding a proper amount of cyclohexane as a solvent, and washing for 3 times by using a proper amount of organic solvent as a precipitant to remove a by-product of the reaction;
(5) drying the product in a vacuum oven at 60 ℃ for 12h to obtain ZnGa2O4:Cr3+White powder of long-afterglow nanocrystal.
The second step is that: polyacrylic acid modified water-soluble PLNPs
(1) Taking 15mL of absolute ethyl alcohol into a test tube, and then adding a proper amount of concentrated hydrochloric acid to make the pH value of the test tube equal to 1;
(2) weighing 40mg of ZnGa2O4:Cr3+Dissolving the long afterglow nanocrystal powder in absolute ethyl alcohol with the pH value of 1-5, and stirring vigorously for 0.5-6 h;
(3) centrifuging, washing with anhydrous ethanol for 3 times, and dispersing in deionized water;
(4) dissolving 10-100 mg of polyacrylic acid in 2mL of deionized water, mixing with the PLNPs aqueous solution, and stirring at normal temperature for 12 h;
(5) the reaction solution is centrifuged, and the precipitate is washed with deionized water for 2 times and finally dispersed in deionized water.
The third step: covalent coupling of PLNPs to molecular beacons
The reaction between PLNPs and molecular beacons was carried out by the usual EDC/NHS activated coupling method.
(1) 1mg of PLNPs was weighed out and dissolved in 1ml of MES buffer solution (pH.6.1,100mM), followed by addition of 10mg of EDC.HCl and 15mg of Sulfo-NHS and reaction with shaking at ordinary temperature for 30 minutes. Subsequently, the pH of the reaction solution was adjusted to 7.4 with NaOH solution (1M), and 0.1-5 OD molecular beacons (for example, miRNA21 was detected, the molecular beacon sequence was designed as NH)2-C6CGATGCGTCAACATCAGTCTGATAAGCTATCCATCG-BHQ3), reacted for 12 hours with shaking at normal temperature, followed by centrifugation, water washing 2 times, and finally stored in HEPES buffer (50mM, pH:7.4) in a refrigerator at 4 ℃ for later use.
The fourth step: application of PLNPs molecular beacon probe in detection of microRNA21
EP tubes containing PLNPs-MB (20. mu.g/mL) were arranged in parallel, then miRNA21 of 0,0.1,0.5,2.0,5.0,10,20,50nM,100nM was added, finally HEPES buffer (50mM, pH:7.4) was added to a constant volume of 1.2mL, then gradually heated to 95 ℃ for 5 minutes, then annealed to 37 ℃ for 1 hour of reaction, then phosphorescence detection was performed, the phosphorescence emission spectra of each sample were measured, and a calibration curve between the miRNA21 concentration and the emission intensity at the strongest emission wavelength of PLNPs was established.
The other aspect of the embodiment of the invention also provides the application of the long-afterglow molecular beacon probe in preparing products with the functions of detecting nucleic acid, protein or small molecules and the like.
Further, the nucleic acid is microRNA, but is not limited thereto.
Further, the protein is a thrombin protein, but is not limited thereto.
The other aspect of the embodiment of the invention also provides the application of the long-afterglow molecular beacon probe in the fields of nucleic acid, protein or small molecule detection and the like.
Further, the nucleic acid is microRNA, but is not limited thereto.
For example, another aspect of the embodiments of the present invention also provides a method for detecting a target substance, including:
providing the long-afterglow molecular beacon probe;
establishing a standard curve between the concentration of a target substance and the luminous intensity at the strongest emission wavelength of the long-afterglow molecular beacon probe;
and mixing the long-afterglow molecular beacon probe with a sample containing a target substance to be detected, reacting at 60-95 ℃ for 2-8 min, annealing to 20-37 ℃, reacting for 0.5-2 h, carrying out phosphorescence detection on a product, and contrasting with the standard curve to realize the detection of the target substance.
Further, the target substance includes nucleic acid, protein or small molecule (such as Glutathione (GSH)), and is particularly preferably microRNA21 or thrombin, etc., but is not limited thereto.
The detection object of the invention is not limited to miRNA, and can be any specific sequence nucleic acid, some proteins, small molecules, metal ions and the like with corresponding aptamers.
In conclusion, the invention provides a time-resolved molecular beacon probe based on rare earth long afterglow luminescence, and the time-resolved molecular beacon probe is applied to the high-sensitivity detection of micro RNA in an in vitro biological sample, mainly solves the problem of spectral interference of background fluorescence, scattered light and the like caused by fluorescence excitation in the existing molecular beacon probe, improves the detection signal-to-noise ratio, and provides a new strategy for the design of the molecular beacon probe; meanwhile, the long-afterglow luminescent material adopted by the invention has simpler preparation steps, good water solubility, more stable luminescence, longer emission life, and higher signal-to-noise ratio and sensitivity of the constructed molecular beacon probe.
In order to make the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Examples of these preferred implementations are illustrated in the accompanying drawings. The embodiments of the invention shown in the drawings and described in accordance with the drawings are exemplary only, and the invention is not limited to these embodiments.
It should be noted that, in order to avoid obscuring the present invention due to unnecessary details, only the structures and/or processing steps closely related to the scheme according to the present invention are shown in the drawings, and other details that are not relevant to the present invention are omitted.
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: construction of near-infrared long-afterglow molecular beacon probe and miRNA21 detection application
(1) Preparation and surface water-soluble modification of near-infrared long-afterglow luminescent material
Adding 0.3g of NaOH into a mixed solution of 3mL of oleic acid, 4mL of deionized water and 10mL of absolute ethyl alcohol, and stirring for 15min until the NaOH is completely dissolved; followed by the addition of 0.5mLZn (NO)3)2(1M)、2mLGa(NO3)3(2M) and 40. mu.L of Cr (NO)3)3(0.05M), and uniformly stirring at room temperature for 30 min; the mixed solution was then transferred to a 25mL capacity hydrothermal kettle (lined with Teflon) and heated in an oven at 210 ℃ for 16 hours. Naturally cooling to room temperature, centrifuging the reaction solution in the hydrothermal kettle to obtain a product, and washing and precipitating for 3 times by using a mixed solvent of cyclohexane and ethanol. Finally, the precipitate is dried in a vacuum oven at 60 ℃ for 12 hours to obtain oil-soluble ZnGa2O4:Cr3+White powder of long-afterglow nanocrystal. Weighing 40mg of the powder, adding 15ml of ethanol, adjusting the pH value to 1 with hydrochloric acid, fully stirring for 2 hours, centrifugally washing, adding 50mg of polyacrylic acid, stirring overnight, and finally washing with deionized water for 2 times to obtain the polyacrylic acid modified near-infrared ZnGa2O4:Cr3+Long persistence luminescent materials.
(2) Near-infrared long-afterglow molecular beacon probe construction and miRNA21 detection application
1mg of water-soluble PLNPs was weighed out and dissolved in 1ml of MES buffer solution (pH.6.1,100mM), followed by addition of 10mg of EDC.HCl and 15mg of Sulfo-NHS and shaking at room temperatureShould be 30 minutes. Subsequently, the pH of the reaction solution was adjusted to 7.4 with NaOH solution (1M), and 1OD molecular beacon (for detecting miRNA21, the molecular beacon sequence was designed as NH)2-C6CGATGCGTCAACATCAGTCTGATAAGCTATCCATCG-BHQ3), reacted for 12 hours with shaking at normal temperature, followed by centrifugation, water washing 2 times, and finally stored in HEPES buffer (50mM, pH:7.4) in a refrigerator at 4 ℃ for later use. EP tubes containing PLNPs-MB (20 mu g/mL) are arranged in parallel, then miRNA21 of 0,0.05, 0.1,0.5,2.0,5.0,10,20,50nM and 100nM is added, finally HEPES buffer (50mM, pH 7.4) is added to the volume of 1.2mL, then the temperature is gradually increased to 95 ℃ for 5 minutes, then the mixture is annealed to 37 ℃ and reacted for 1 hour, then phosphorescence detection is carried out, the phosphorescence emission spectrum of each sample is measured, a working curve between the concentration of miRNA21 and the emission intensity at the strongest emission wavelength of PLNPs is established, and the probe is found to have better linear relation at the concentration of miRNA21 between 0.1 and 20nM, and the detection limit is 37 pM.
Example 2: construction of near-infrared long-afterglow aptamer molecular beacon probe and thrombin detection application
(1) Preparation and surface water-soluble modification of near-infrared long-afterglow luminescent material
Adding 0.3g of NaOH into a mixed solution of 3mL of oleic acid, 4mL of deionized water and 10mL of absolute ethyl alcohol, and stirring for 15min until the NaOH is completely dissolved; followed by the addition of 0.5mL Zn (NO)3)2(1M)、2mL Ga(NO3)3(2M), and 8. mu.L (Cr (NO)3)3(0.01M), and uniformly stirring at room temperature for 20 min; the mixed solution was then transferred to a 25mL hydrothermal kettle (lined with Teflon) and heated in an oven at 220 ℃ for 8 hours. Naturally cooling to room temperature, centrifuging the reaction solution in the hydrothermal kettle to obtain a product, and washing and precipitating for 3 times by using a mixed solvent of cyclohexane and ethanol. Finally, the precipitate is dried in a vacuum oven at 60 ℃ for 12 hours to obtain oil-soluble ZnGa2O4:Cr3+White powder of long-afterglow nanocrystal. Weighing 40mg of the powder, adding 15ml of ethanol, adjusting the pH value to 3 by hydrochloric acid, fully stirring for 6 hours, centrifugally washing, adding 50mg of polyacrylic acid, stirring overnight, and finally washing for 2 times by deionized water to obtain the polyacrylic acid modified polyacrylic acidDecorated near-infrared ZnGa2O4:Cr3+Long persistence luminescent materials.
(2) Construction of near-infrared long-afterglow aptamer molecular beacon probe and application of probe in thrombin detection
1mg of water-soluble PLNPs was weighed out and dissolved in 1ml of MES buffer solution (pH.6.1,100mM), followed by addition of 10mg of EDC.HCl and 15mg of Sulfo-NHS and reaction with shaking at ordinary temperature for 120 minutes. Subsequently, the reaction solution was adjusted to pH 7.4 with NaOH solution (1M), and 3OD thrombin aptamer molecular beacons (sequence: NH) were added2-C6CGATGCGTGGTTGGTGTGGTTGGATCCATCG-BHQ3), reacted at room temperature with shaking for 2 hours, followed by centrifugation, washing with water for 2 times, and finally stored in HEPES buffer (50mM, pH:7.4) in a refrigerator at 4 ℃ for later use. EP tubes containing PLNPs-MB (20. mu.g/mL) were arranged in parallel, miRNA21 was added at 0,0.05, 0.1,0.5,2.0,5.0,10,20,50nM, finally HEPES buffer (50mM, pH:7.4) was added to 1.2mL, followed by shake incubation at 37 ℃ for 1 hour, followed by phosphorescence detection, phosphorescence emission spectra of each sample were measured, a working curve was established between thrombin concentration and luminescence intensity at the strongest emission wavelength of PLNPs, and it was found that the probe had a good linear relationship at thrombin concentration of 0.5-20nM with a detection limit of 0.13 nM.
Example 3: construction of nano gold rod super-quenching long-afterglow molecular beacon probe and application of nano gold rod super-quenching long-afterglow molecular beacon probe in Glutathione (GSH) detection
(1) Preparation and surface water-soluble modification of near-infrared long-afterglow luminescent material
Adding 0.3g of NaOH into a mixed solution of 3mL of oleic acid, 4mL of deionized water and 10mL of absolute ethyl alcohol, and stirring for 15min until the NaOH is completely dissolved; followed by the addition of 0.5mLZn (NO)3)2(1M)、1mLGa(NO3)3(2M), and 240. mu.L (Cr (NO)3)3(0.3M), and uniformly stirring at room temperature for 10 min; the mixed solution was then transferred to a 25mL capacity hydrothermal kettle (lined with Teflon) and heated in an oven at 180 ℃ for 24 hours. Naturally cooling to room temperature, centrifuging the reaction solution in the hydrothermal kettle to obtain a product, and washing and precipitating for 3 times by using a mixed solvent of cyclohexane and ethanol. Finally, the precipitate is dried in a vacuum oven at 60 ℃ for 12 hours to obtain oil-soluble ZnGa2O4:Cr3+White powder of long-afterglow nanocrystal. Weighing 40mg of the powder, adding 15ml of ethanol, adjusting the pH value to 5 with hydrochloric acid, fully stirring for 0.5 hour, centrifuging and washing, then adding 50mg of polyacrylic acid, stirring for 4-24 hours, and finally washing for 2 times with deionized water to obtain the polyacrylic acid modified near-infrared ZnGa2O4:Cr3+Long persistence luminescent materials.
(2) Construction of near-infrared long-afterglow aptamer molecular beacon probe and application of probe in GSH detection
1mg of water-soluble PLNPs was weighed out and dissolved in 1ml of MES buffer solution (pH.6.1,100mM), followed by addition of 10mg of EDC.HCl and 15mg of Sulfo-NHS and reaction with shaking at ordinary temperature for 10 minutes. Subsequently, the pH of the reaction solution was adjusted to 7.4 with NaOH solution (1M), and 0.5 OD molecular beacon (for detecting GSH, the molecular beacon sequence is shown in SEQ ID NO.4, and the sequence is NH) was added2-C6-GCT GGA CAG AGT AT-S-S-ATA TCA ATT TTT TTTAGT CCA GC-SH), reacted for 24 hours at normal temperature with shaking, followed by centrifugation, water washing 2 times, and finally stored in HEPES buffer (50mM, pH:7.4) in a refrigerator at 4 ℃ for later use. An EP tube containing PLNPs-MB (20 mu g/mL) is arranged in parallel, then a nanogold rod with the concentration of 1nM (the absorption peak is 680nM) is added, the reaction is shaken for 24h overnight, and then the ultraquenched molecular beacon probe is obtained by centrifugal separation. An EP tube of an ultraquenched PLNPs-MB-nanogold rod (20 mu g/mL) is arranged in parallel, then 0,0.05,0.2, 0.5,1.0,2.0,5.0 and 10mM GSH is added, finally HEPES buffer (50mM, pH:7.4) is added to the volume of 1.2mL, then the shaking incubation reaction is carried out for 1 hour at 37 ℃, then phosphorescence detection is carried out, the phosphorescence emission spectrum of each sample is measured, a working curve between the GSH concentration and the luminous intensity at the strongest emission wavelength of the PLNPs is established, and the probe is found to have a better linear relation at the GSH concentration of 0.05-5.0mM, and the detection limit is 15 mu M.
Example 4: construction of polydopamine super-quenching phosphorescent semiconductor quantum dot molecular beacon probe and nucleic acid detection application thereof
(1) Preparation of phosphorescent semiconductor quantum dots and molecular beacon modification
The preparation process of the Mn-doped ZnS semiconductor phosphorescent carbon dot comprises the following steps: 50mL of aqueous solution was added 0.02M mercaptopropionic acid, 5mL of ZnSO4(0.1M)，1.5mL of 0.01M MnCl2The pH was adjusted to 11, and the mixture was aerated for 30 minutes under an argon atmosphere. Then, 5mL of Na2And (3) quickly injecting the S solution (0.1M) into the solution, keeping quick stirring, reacting for 2 hours at 50 ℃, adding ethanol for precipitation, performing centrifugal separation, and finally storing in a water phase to obtain the mercaptopropionic acid modified Mn doped ZnS semiconductor quantum dot.
Modification of molecular beacons: 2mg of manganese-doped ZnS semiconductor quantum dot was dissolved in MES buffer solution (pH.6.1,100mM), followed by addition of 10mg of EDC.HCl and 15mg of Sulfo-NHS and reaction with shaking at ordinary temperature for 30 minutes. And then, adjusting the pH of the reaction solution to 7.4 by using NaOH solution (1M), adding 2 molecular beacons with OD (5' -end amino group modification), reacting for 2 hours, and purifying by using a 30K ultrafiltration tube to obtain the phosphorescent semiconductor molecular beacon probe.
(2) Construction of phosphorescent semiconductor quantum dot molecular beacon probe and application of target nucleic acid detection
An EP tube containing a quantum dot-molecular beacon conjugate (10 mug/mL) is arranged in parallel, polydopamine nano-ions (20 mug/mL) are added, shaking reaction is carried out for 1h, then 0,0.05,0.2, 0.5,1.0,2.0,5.0 and 10nM single-stranded complementary nucleic acids (target nucleic acids) are added, HEPES buffer (50mM, pH:7.4) is added to the volume of 1.2mL, then shaking incubation reaction is carried out for 1h at 37 ℃, then phosphorescence detection is carried out, phosphorescence emission spectra of each sample are measured, a working curve between the concentration of the target nucleic acids and the luminous intensity at the strongest emission wavelength of the quantum dots is established, and the probe has a better linear relation at the concentration of the target nucleic acids of 0.2-5.0nM and a detection limit of 0.08 nM.
Example 5: construction of graphene oxide super-quenching long-afterglow carbon dot molecular beacon probe and nucleic acid detection application
(1) Preparation of long afterglow carbon dots and molecular beacon modification
Preparation of long afterglow carbon dots: adding 1ml of ethylenediamine into 15ml of deionized water, fully stirring, then slowly adding 2ml of phosphoric acid, stirring for 30 minutes, adding into a microwave oven for reaction (750W, 2 minutes), cooling the reactant, adding 20ml of water, fully dissolving by ultrasonic, removing large particles at a low rotation speed of 6000 rpm, adjusting the pH value to be neutral by using sodium carbonate, and then dialyzing for 2 days by using a dialysis bag (molecular weight cut-off of 500), thus obtaining the carbon dots. Further, 1mg of the carbon dots is taken and dissolved in 25ml of aqueous solution, silane precursor TEOS (0.5ml) and silanization coupling agent APTES (30 mul) are added, after fully stirring for 30 minutes, 0.5ml of ammonia water is added, stirring reaction is continued for 8 hours at normal temperature, and finally the sample is purified by a microporous filter membrane to obtain the aminated long-afterglow carbon dots.
Modification of molecular beacons: 1OD molecular beacon (5' -terminal carboxyl group modification) was dissolved in MES buffer (pH.6.1,100mM), followed by addition of 10mg of EDC.HCl and 15mg of Sulfo-NHS, and the reaction was shaken at room temperature for 30 minutes. And then, adjusting the pH of the reaction solution to 7.4 by using NaOH solution (1M), adding 1mg of aminated long-afterglow carbon dots, reacting for 2 hours, and purifying by using a 10K ultrafiltration tube to obtain the phosphorescent semiconductor molecular beacon probe.
(2) The target nucleic acid detection application of the long afterglow carbon point molecular beacon probe comprises the following steps: an EP tube containing a long afterglow carbon dot-molecular beacon conjugate (10 mu g/mL) is arranged in parallel, then layered graphene oxide (30 mu g/mL) is added, shaking reaction is carried out for 30h, then 0,0.05,0.2, 0.5,1.0,2.0,5.0 and 10nM single-stranded complementary nucleic acid (target nucleic acid) is added, finally HEPES buffer (50mM, pH:7.4) is added for constant volume to 1.2mL, then shaking incubation reaction is carried out for 1h at 37 ℃, then phosphorescence detection is carried out, the phosphorescence emission spectrum of each sample is measured, a working curve between the concentration of the target nucleic acid and the luminous intensity at the strongest emission wavelength of the long afterglow carbon dot is established, and the probe is found to have a better linear relation at the concentration of the target nucleic acid of 0.5-10nM and the detection limit of 0.15 nM.
The inventors of the present invention, taking example 1 as an example, characterized and tested the prepared near-infrared long-afterglow luminescent material and the constructed near-infrared long afterglow molecular beacon probe, and the results are as follows:
FIGS. 2A and 2B respectively show ZnGa in an exemplary embodiment of the invention2O4:Cr3+Transmission Electron Microscope (TEM) image and X-ray powder crystal diffraction (XRD) crystal phase characterization result image of long-afterglow nano-particles, and FIG. 3 shows ZnGa2O4:Cr3+And (3) an optical characterization result graph of the long-afterglow luminescent material, wherein a is an absorption spectrum curve, b is an excitation spectrum curve, and c is an emission spectrum curve.
FIG. 4A and FIG. 4BFIG. 4B shows water-soluble ZnGa in an exemplary embodiment of the invention2O4:Cr3+TEM image and Fourier infrared spectrum characterization result image of the long afterglow nano particles.
Fig. 5 is a schematic diagram illustrating the result of applying the near-infrared long-afterglow molecular beacon probe to miRNA21 detection in an exemplary embodiment of the invention.
The aspects, embodiments, features and examples of the present invention should be considered as illustrative in all respects and not intended to be limiting of the invention, the scope of which is defined only by the claims. Other embodiments, modifications, and uses will be apparent to those skilled in the art without departing from the spirit and scope of the claimed invention.
The use of headings and chapters in this disclosure is not meant to limit the disclosure; each section may apply to any aspect, embodiment, or feature of the disclosure.
Throughout this specification, where a composition is described as having, containing, or comprising specific components or where a process is described as having, containing, or comprising specific process steps, it is contemplated that the composition of the present teachings also consist essentially of, or consist of, the recited components, and the process of the present teachings also consist essentially of, or consist of, the recited process steps.
It should be understood that the order of steps or the order in which particular actions are performed is not critical, so long as the teachings of the invention remain operable. Further, two or more steps or actions may be performed simultaneously.
In addition, the inventor also carries out corresponding tests by using other process conditions and the like listed in the foregoing to replace the corresponding process conditions in the examples 1 to 5, and the contents to be verified are similar to the products of the examples 1 to 5. Therefore, the contents of the verification of each example are not described herein one by one, and only examples 1 to 5 are used as representatives to describe the excellent points of the present invention.
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 long persistence molecular beacon probe, comprising:
a molecular beacon;
fluorescent donor materials and energy acceptor materials respectively modified at two ends of the molecular beacon;
the fluorescence donor material comprises any one or combination of more than two of rare earth-based long-afterglow luminescent materials, phosphorescent/long-afterglow luminescent carbon quantum dots and phosphorescent semiconductor quantum dots, the energy acceptor material is a quencher, and the quencher comprises an optical material with the property of quenching long afterglow.
2. The long persistence molecular beacon probe of claim 1, wherein: the rare earth-based long-afterglow luminescent material is ZnGa modified by polyacrylic acid2O4:Cr3+Long persistence nanocrystalline.
3. The long persistence molecular beacon probe of claim 1, wherein: the quencher comprises any one or the combination of more than two of organic quenching dye, precious metal nano material, two-dimensional layered material and polymer nano particles; preferably, the organic quenching dye is BHQ.
4. The long persistence molecular beacon probe of claim 1, wherein: the molecular beacon comprises a nucleic acid molecular beacon and/or a molecular beacon with a stem-loop part as an aptamer; preferably, the nucleic acid molecular beacon is a molecular beacon for detecting target DNA or RNA, and has a sequence shown as SEQ ID NO.1 or a sequence shown as SEQ ID NO. 2; preferably, the stem-loop part is a molecular beacon of the aptamer, which is a molecular beacon for targeting recognition of thrombin and has a sequence shown in SEQ ID NO. 3.
5. The method for constructing a long-afterglow molecular beacon probe as claimed in any one of claims 1 to 4, which comprises: and reacting the fluorescent donor material with the molecular beacon by an EDC/NHS activation coupling method to obtain the long-afterglow molecular beacon probe.
6. The building method according to claim 5, characterized by comprising:
dissolving a fluorescent donor material in an MES buffer solution, adding EDC and NHS, and carrying out oscillation reaction at room temperature for 10-120 min;
and adjusting the pH value of the obtained reaction liquid to 7.4 by adopting alkali liquor, then adding 0.5-3 OD molecular beacon, and oscillating and reacting at room temperature for 2-24 h to obtain the long-afterglow molecular beacon probe.
7. The method of claim 5, wherein the fluorescence donor material is ZnGa modified by polyacrylic acid2O4:Cr3+Long persistence nanocrystals, and, the polyacrylic acid modified ZnGa2O4:Cr3+The preparation method of the long afterglow nanocrystal comprises the following steps:
uniformly mixing an alkaline substance, oleic acid, water and ethanol to form a first mixed solution;
the Zn source, the Ga source and the Cr source are mixed according to a molar ratio of 1: 2: 0.01-0.3, and uniformly mixing to form a second mixed solution;
dropwise adding the second mixed solution into the first mixed solution, uniformly stirring at room temperature for 10-60 min, and carrying out hydrothermal reaction on the formed hydrothermal reaction system at 180-220 ℃ for 8-24 h to obtain ZnGa2O4:Cr3+Long afterglow nanocrystals; and the number of the first and second groups,
polyacrylic acid is adopted to react the ZnGa2O4:Cr3+Modifying the long afterglow nanocrystal to obtain the ZnGa modified by the polyacrylic acid2O4:Cr3+Long afterglow nanocrystals;
preferably, the alkaline substance is NaOH; preferably, the Zn source is Zn (NO)3)2(ii) a Preferably, the Ga source is Ga (NO)3)3(ii) a Preferably, the Cr source is Cr (NO)3)3；
Preferably, the preparation method comprises the following steps:
reacting the ZnGa with a catalyst2O4:Cr3+Dissolving the long afterglow nanocrystal in an ethanol-hydrochloric acid solution with the pH value of 1-5, stirring for 0.5-6 h, centrifuging, and dispersing in water to obtain ZnGa2O4:Cr3+Long afterglow nanocrystalline aqueous solutions;
dissolving polyacrylic acid in water, and reacting with ZnGa2O4:Cr3+Mixing the long afterglow nanocrystalline aqueous solution, stirring for 4-24 h at room temperature, and centrifuging the obtained reaction liquid to obtain the ZnGa modified by polyacrylic acid2O4:Cr3+Long persistence nanocrystalline.
8. Use of the long-afterglow molecular beacon probe of any one of claims 1 to 4 for preparing a product with a nucleic acid, protein or small molecule detection function; preferably, the nucleic acid is microRNA.
9. Use of the long persistence molecular beacon probe of any one of claims 1 to 4 in the field of nucleic acid, protein or small molecule detection; preferably, the nucleic acid is microRNA.
10. A method for detecting a target substance, characterized by comprising:
providing the long persistence molecular beacon probe of any one of claims 1 to 4;
establishing a standard curve between the concentration of a target substance and the luminous intensity at the strongest emission wavelength of the long-afterglow molecular beacon probe;
mixing the long-afterglow molecular beacon probe with a sample containing a target substance to be detected, reacting at 60-95 ℃ for 2-8 min, annealing to 20-37 ℃, reacting for 0.5-2 h, carrying out phosphorescence detection on a product, and contrasting with the standard curve to realize the detection of the target substance;
preferably, the target substance comprises a nucleic acid, a protein or a small molecule, and particularly preferably microRNA21, thrombin or glutathione.
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