CN102260745A - Fluorescent biological probe of upconversion nano particle labeled aptamer - Google Patents
Fluorescent biological probe of upconversion nano particle labeled aptamer Download PDFInfo
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- CN102260745A CN102260745A CN2011102124611A CN201110212461A CN102260745A CN 102260745 A CN102260745 A CN 102260745A CN 2011102124611 A CN2011102124611 A CN 2011102124611A CN 201110212461 A CN201110212461 A CN 201110212461A CN 102260745 A CN102260745 A CN 102260745A
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Abstract
The invention relates to a fluorescent biological probe of an upconversion nano particle labeled aptamer, belonging to the technical field of biomolucule detection and overcoming the technical problems existing in the existing biological probe of a semiconductor quantum dots (QDs) labeled aptamer. The technical problems comprise injury of organism nucleic acid in which a target molecule is located and high background signal and potential toxicity, 'blinking' effect and chemical instability of QDs. The fluorescent biological probe of the upconversion nano particle labeled aptamer provided by the invention comprises an aptamer labeled by an upconversion nano particle as a fluorescent donor and a DNA chain complementary with the aptamer, wherein a pigment molecule which can generate an FRET (fluorescence resonance energy transfer) effect with the fluorescent donor and is used as a fluorescent receptor is connected to the DNA chain. According to the invention, the used fluorescent donors are UCNPs (upconversion nano particles) which have the advantages of low background signal, no toxicity, no 'blinking' effect and strong chemical stability.
Description
Technical field
The invention belongs to the biomolecule detection technical field, particularly, the present invention relates to the biological probe of the adaptive son of up-conversion nanoparticles (UCNPs) mark.
Background technology
Adaptive son is the special nucleotide chain of a class, and it has high specific and high affinity at most target molecules, such as organic dye molecule, amino acid, microbiotic, albumen, even whole cell and microorganism cause of disease bacterium.Adaptive son is by in a synthetic random sequence oligonucleotides storehouse, with the phyletic evolution procedural mode of index concentration aglucon through wash-out, collection repeatedly with increase again and screen and prepare.With traditional antibody Comparatively speaking, adaptive son has the characteristic of a lot of excellences as the bio-sensing recognition component., preparation process little such as size is simple, long preservative period, regeneration and chemically modified are easy.The most important thing is that wherein adaptive son can be folded into changeable tertiary structure, effect by hydrogen bond, Van der Waals force and base stacking force, interact with conformation complementary mode and target molecule, the characteristics of this process are by the target molecule conformational change, realize that target molecule detects.These characteristics make the flexible design of adaptive sub-bioprobe and easy.
At present, be used for the adaptive son of mark and mainly be divided into two classes as the material of fluorescence donor: fluoresceins are (as FAM, FITC), the rhodamine class is (as TRITC, TAMRA), flower cyanines class (as Cy3, Cy5) waits dye molecule and semiconductor-quantum-point.
Fluoresceins are (as FAM, FITC), the rhodamine class is (as TRITC, TAMRA) and flower cyanines class (as Cy3, dye molecule such as Cy5), as the suitable gametogenic bioprobe of fluorescence donor mark, be by the change of fluorescence anisotropy between the adaptive son that is labeled and the target molecule, disclose the interaction process between adaptive son and the target molecule.But the influence of solution environmental and other molecule caused the photoluminescent property instability near the dye molecule photoluminescent property that this class is used for the adaptive son of mark very easily was subjected to, and can influence its detection sensitivity.In addition, the excitation band of luminescent dye molecule is narrow, the emitting fluorescence bands of a spectrum are wide, and the fluorescence instability, and these defectives make adaptive sub-bioprobe be subjected to considerable restraint in all many-sides in actual applications.
People used semiconductor-quantum-point (QDs) to replace dye molecule as the fluorescence donor afterwards, adopted the adaptive son preparation of QDs mark bioprobe.Its concrete structure of prepared bioprobe is as follows: nucleus is QDs, QDs is connected adaptive son with avidin with vitamin H, be connected with complementary DNA chain with it on the adaptive son, on the DNA chain, be connected with and the dye molecule of FRET effect take place as fluorescent receptor with QDs.But QDs needs in the biologic applications of reality with short wavelength's excitation such as ultraviolets, and this exciting light can cause the damage of target molecule place organism nucleic acid.And also there are deficiencies such as high background signal, genotoxic potential, " blinking " effect and chemical instability in QDs.
Summary of the invention
In order to overcome the existing technical problem of bioprobe of the existing adaptive son of QDs mark, comprise the damage that causes target molecule place organism nucleic acid, the high background signal of QDs, genotoxic potential, " blinking " effect and chemical instability provide the biological probe of the adaptive son of a kind of up-conversion nanoparticles mark.
The biological probe of the adaptive son of up-conversion nanoparticles mark, comprise adaptive son by fluorescence donor mark, with with adaptive sub-complementary DNA chain, be connected with on the DNA chain and can the dye molecule of FRET effect as fluorescent receptor take place with the fluorescence donor, it is characterized in that, by up-conversion nanoparticles as the fluorescence donor.
Its effect of the present invention is, bioprobe of the present invention because that the UCNPs of the adaptive son of mark and dye molecule lean on is very near, satisfies the required distance of FRET effect.At this moment, under the exciting of 980nm light, the FRET effect takes place in UCNPs and dye molecule, and corresponding fluorescent signal can be detected.When adding sequence in the system can be with adaptive subsequence complementary target molecule the time, because the ability of adaptive sub-binding target molecule is better than complementary DNA chain with it, the complementary duplex structure will be destroyed, the DNA chain that connects dye molecule will break away from adaptive son, dye molecule is also just away from UCNPs, the FRET effect weakens like this, and corresponding fluorescent signal changes.
The present invention compares with the bioprobe of the adaptive son of existing QDs mark, has following advantage:
(1) utilize near infrared light (980nm) to excite the last convert light that obtains 535nm and 650nm, can avoid the damage of high-octane exciting light (purple light or blue light) to detected organism nucleic acid, simultaneously also can avoid the organism autofluorescence, this will improve the sharpness of detection sensitivity and image greatly.
(2) the fluorescence donor that uses of the present invention is UCNPs, its advantage for low background signal, nontoxic, do not have " blinking " effect and chemical stability strong.
(3) use the biological probe of the adaptive son of up-conversion nanoparticles mark of the present invention not need expensive ultrashort pulse laser.
(4) adopt UCNPs as the fluorescence donor, it is luminous to have characteristics such as optical stability is strong, luminescence spectrum is narrow, the luminous life time of the level is long.
(5) lack and the resolution instrument that does not need costliness for the analyzing and testing time of fluorescent signal.
(6) compare with QDs, UCNPs has high stability, does not produce the singlet oxygen composition under situation about exciting, and can not cause the damage of detected biological specimen.
Description of drawings
Fig. 1 is the synoptic diagram of the adaptive sub-bioprobe of QDs mark in the prior art;
Fig. 2 is the structural representation of the biological probe of the adaptive son of up-conversion nanoparticles mark of the present invention;
Fig. 3 is the detection design sketch of the biological probe of the adaptive son of up-conversion nanoparticles mark of the present invention to ATP.
Embodiment
Embodiment one: present embodiment is described in conjunction with Fig. 2, the biological probe of the adaptive son of up-conversion nanoparticles mark, comprise adaptive son by fluorescence donor mark, with with adaptive sub-complementary DNA chain, be connected with on the DNA chain and can the dye molecule of FRET effect as fluorescent receptor take place with the fluorescence donor, as the fluorescence donor, the described UCNPs of present embodiment is NaYF by UCNPs
4: Yb, Er, NaYF
4: Yb, Tm, NaYF
4: Yb, Ho, LaF
3: Yb, Er, LaF
3: Yb, Tm or LaF
3: Yb, a kind of among the Ho, the described UCNPs of present embodiment is water miscible, the described UCNPs of present embodiment is connected with vitamin H with avidin with adaptive son.
Embodiment two: in conjunction with Fig. 2 and Fig. 3 present embodiment is described, present embodiment is the preparation method of the biological probe of the adaptive son of embodiment one described up-conversion nanoparticles mark:
(1) preparation UCNPs: with 0.272 gram CF
3COONa, 0.376 gram Y (CF
3COO)
3, 0.113 gram Yb (CF
3COO)
3With 0.055g gram Er (CF
3COO)
3Be dissolved in the oleyl amine of 10ml, insert in the three-necked bottle, under argon shield, 100 ℃ were stirred 30 minutes, progressively were warming up to 320 ℃ and kept 1 hour.The product centrifugation, and use the hexanaphthene supersound washing, obtain the cyclohexane solution of UCNPs.
(2) UCNPs that utilizes phosphoramidic acid that step (1) is prepared carries out the water transfer: take by weighing 0.13 gram phosphoramidic acid and be scattered in the 5ml ethanol, to the cyclohexane solution that wherein drips 3mlUCNPs, stirring at room 24 hours, product ethanol centrifuge washing, and be scattered in PBS solution, it is crystalline dispersion in the colloidal solution of PBS to obtain UCNPs.
(3) the affinity element is joined in the resulting product of step (2), generate the UCNPs be connected with avidin: get the resulting colloidal solution of 2mL step (2), and to add final concentration be 5% glutaraldehyde, stirring reaction 12 hours; After the product eccentric cleaning removed excessive glutaraldehyde three times, be scattered in again in the PBS damping fluid.
(4) adaptive son that will be connected with vitamin H and the UCNPs coupling that is connected with avidin generate the adaptive son by the UCNPs mark: add the plain molecule of 1mg affinity in the product that step (3) obtains, continue oscillatory reaction 48 hours down at 4 ℃; With the resultant centrifugal purification, 4 ℃ are stored in the test signal damping fluid (pH 8.2,20mM Tris-HCl, 5mM KCl, 1mM MgCl
2) in.
(5) in the product that obtains by step (4), add by the dye molecule mark and adaptive sub-complementary DNA chain, generate the bioprobe of the adaptive son of UCNPs mark: add in the product that step (4) obtains by the dye molecule mark and adaptive sub-complementary DNA chain, as shown in table 1, table 1 is for making up the used dna sequence dna of fluorescent probe of the adaptive son of UCNPs mark, vibration is heated to 70 ℃ earlier in the signal damping fluid, keep 3min, and then solution is reduced to room temperature slowly, get product.
Table 1
In conjunction with Fig. 3, in the product that step (5) is obtained, constantly splash into a series of concentration ATP molecules shown in the figure X-coordinate, hatch 1h under 37 ℃, carry out spectrum test then.Make relation curve with I540/I577 value and adding ATP concentration.Do not have ATP to divide the period of the day from 11 p.m. to 1 a.m in system, fluorescence donor and fluorescent receptor form duplex structure by base complementrity paired principle.So stable can being pulled to the fluorescence donor and the very near position of fluorescent receptor distance that this structure is enough causes producing between fluorescence donor and the fluorescent receptor fluorescent energy resonance efficiently and transmits.Divide the period of the day from 11 p.m. to 1 a.m when in system, adding ATP, because adaptive subsequence more is inclined to the ATP molecule and combines.This dynamic (dynamical) trend is greater than base complementrity paired trend, and this just forces fluorescent receptor to disintegrate down from the dna double chain, and the distance of fluorescent receptor and UCNPs becomes far away.And then be accompanied by the change of fluorescent signal.When the concentration of ATP reaches certain value, the I540/I577 value state that almost reaches capacity.This is because UCNPs to adaptive sub-molecule space steric effect, has stoped more ATP molecule and adaptive sub the combination.In ATP concentration is between the 0.05-0.75mM, and the I540/I577 value has good linear corresponding relation with it.
Claims (4)
1. the biological probe of the adaptive son of up-conversion nanoparticles mark, comprise adaptive son by fluorescence donor mark, with with adaptive sub-complementary DNA chain, be connected with on the DNA chain and can the dye molecule of FRET effect take place as fluorescent receptor with the fluorescence donor, it is characterized in that, by up-conversion nanoparticles as the fluorescence donor.
2. the biological probe of the adaptive son of up-conversion nanoparticles mark according to claim 1 is characterized in that described up-conversion nanoparticles is water miscible.
3. the biological probe of the adaptive son of up-conversion nanoparticles mark according to claim 1 is characterized in that up-conversion nanoparticles is connected with vitamin H with avidin with adaptive son.
4. the biological probe of the adaptive son of up-conversion nanoparticles mark according to claim 1 is characterized in that described up-conversion nanoparticles is NaYF
4: Yb, Er, NaYF
4: Yb, Tm, NaYF
4: Yb, Ho, LaF
3: Yb, Er, LaF
3: Yb, Tm or LaF
3: Yb, a kind of among the Ho.
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Cited By (8)
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| CN105086997A (en) * | 2015-09-08 | 2015-11-25 | 上海海事大学 | Fluorescent probe and preparation method thereof |
| CN106086173A (en) * | 2016-06-14 | 2016-11-09 | 西安交通大学 | A kind of quick bacteria detection method based on up-conversion fluorescence Resonance energy transfer |
| CN107011905A (en) * | 2017-05-12 | 2017-08-04 | 南方科技大学 | Temperature sensing and multichannel imaging method based on up-conversion material |
| CN110746963A (en) * | 2019-09-18 | 2020-02-04 | 广西师范大学 | Near-infrared luminous biomass quantum dot and intracellular mRNA ratio fluorescence imaging nano probe and preparation method and application thereof |
| CN111426658A (en) * | 2019-09-23 | 2020-07-17 | 湖北大学 | Method for detecting thrombin by using quantum dot sensitized up-conversion nano material |
| CN111789961A (en) * | 2020-08-26 | 2020-10-20 | 西南大学 | Nano probe for nucleolin cross-linking induction of tumor cell apoptosis and preparation method and application thereof |
| CN112649605A (en) * | 2020-12-14 | 2021-04-13 | 哈尔滨理工大学 | Based on NaBiF4ECL biosensor of up-conversion nano-particles |
| CN112945923A (en) * | 2021-02-03 | 2021-06-11 | 长沙理工大学 | Interface sensitization type detection reagent and preparation method and application thereof |
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Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105086997A (en) * | 2015-09-08 | 2015-11-25 | 上海海事大学 | Fluorescent probe and preparation method thereof |
| CN106086173A (en) * | 2016-06-14 | 2016-11-09 | 西安交通大学 | A kind of quick bacteria detection method based on up-conversion fluorescence Resonance energy transfer |
| CN106086173B (en) * | 2016-06-14 | 2019-12-24 | 西安交通大学 | Rapid bacteria detection method based on up-conversion fluorescence resonance energy transfer |
| CN107011905A (en) * | 2017-05-12 | 2017-08-04 | 南方科技大学 | Temperature sensing and multichannel imaging method based on up-conversion material |
| CN107011905B (en) * | 2017-05-12 | 2020-05-05 | 南方科技大学 | Temperature sensing and multichannel imaging method based on up-conversion material |
| CN110746963A (en) * | 2019-09-18 | 2020-02-04 | 广西师范大学 | Near-infrared luminous biomass quantum dot and intracellular mRNA ratio fluorescence imaging nano probe and preparation method and application thereof |
| CN111426658A (en) * | 2019-09-23 | 2020-07-17 | 湖北大学 | Method for detecting thrombin by using quantum dot sensitized up-conversion nano material |
| CN111789961A (en) * | 2020-08-26 | 2020-10-20 | 西南大学 | Nano probe for nucleolin cross-linking induction of tumor cell apoptosis and preparation method and application thereof |
| CN111789961B (en) * | 2020-08-26 | 2022-03-29 | 西南大学 | Nano probe for nucleolin cross-linking induction of tumor cell apoptosis and preparation method and application thereof |
| CN112649605A (en) * | 2020-12-14 | 2021-04-13 | 哈尔滨理工大学 | Based on NaBiF4ECL biosensor of up-conversion nano-particles |
| CN112649605B (en) * | 2020-12-14 | 2022-11-01 | 哈尔滨理工大学 | Based on NaBiF4ECL biosensor for up-conversion of nanoparticles |
| CN112945923A (en) * | 2021-02-03 | 2021-06-11 | 长沙理工大学 | Interface sensitization type detection reagent and preparation method and application thereof |
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Application publication date: 20111130 |