CN104818325A - Nanoprobe for acute promyelocytic leukemia fluorescence detection and preparing method thereof - Google Patents
Nanoprobe for acute promyelocytic leukemia fluorescence detection and preparing method thereof Download PDFInfo
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Abstract
The invention relates to a nanoprobe for acute promyelocytic leukemia fluorescence detection and a preparing method thereof. The preparing method includes the steps that an up-conversion luminescence nanocrystalline (CS-UCNPs for short) of core-shell structure is firstly synthesized, sodium citrate serves as a ligand exchange agent, a ligand exchange method is adopted, functionalization is carried out on carboxyl on the surface of the CS-UCNPs, and a water solubility nanocrystalline Cit-UCNPs; and covalent bond assembling is carried out on DNA molecules of a functional modification specific recognition PML/RARalpha fusion gene segment and the Cit-UCNPs, a nanocrystalline Cit-UCNPs-ssDNA is obtained, and finally a single wall carbon nanohom or graphene oxide and Cit-UCNPs-ssDNA are combined together through the pi-pi mutual effect, and the nanoprobe Cit-UCNPs-ssDNA-SWCNHs and Cit-UCNPs-ssDNA-GO is constructed. Fluorescence detection on the PML/RARalpha fusion gene segment in an aqueous solution is achieved. The method has the beneficial effects of being simple in process, convenient to operate and simple in structure control. The prepared nano fluorescence probe has the beneficial effects of being uniform in size, stable in structure, low in toxin and good in biocompatibility, and the nanoprobe has the potential application value in the fields of cell genetics and molecular biology study
Description
Technical field
The present invention relates to a kind of nano-probe for acute promyelocytic leukemia fluoroscopic examination and preparation method thereof.
Background technology
Acute promyelocytic leukemia (APL) is a kind of specific type of acute myelocytic leukemia, accounts for 10% of Aduit Acute Myeloid Leukemia, and most APL patients exists typical t (15; 17) (q22; Q21) chromosome translocation, positive rate accounts for 98%, Retinoic acid receptor α gene (RAR α) and No. 15 chromosomal promyelocytic leukemia (PML) genes form PML-RAR alpha fusion gene, the downstream albumen molecular complex of this fusion gene is for transcribing Co inhibitor, transcribed by suppressor gene thus the differentiation of T suppression cell and apoptosis, this is the specificity marker of APL and the important molecule basis of targeted therapy, it can not only help the diagnosis of APL, and can as the leading indicator of the state of an illness and examination of curative effect in APL therapeutic process.So the detection of PML/RAR alpha fusion gene plays an important role in the Diagnosis and Treat process of APL.
The detection of PML/RAR alpha fusion gene is one of the emphasis and focus in cytogenetics and molecular biology research field always, and detection method conventional at present mainly contains: chromosome analysis, fluorescence in situ hybridization (FISH), flow cytometry (FCM), Real-time quantity polymerase-chain reaction (RT-PCR) etc.Although chromosome analysis method in structure and the exception quantitatively analyzing single cell chromosome, but can will spend longer time and efforts, thus reduce detection efficiency.Fluorescence in situ hybridization is non-radioassay system, fluorescent reagent and probe economy, safety, stable, and highly sensitive, but can not reach 100% hybridization, particularly when applying shorter complementary DNA probe, efficiency obviously declines.Although flow cytometry can high speed analysis art up to ten thousand cells, be only limitted to the pattern, the size and photoluminescent property etc. that identify cell on a cellular level.RT-PCR method has very high sensitivity, but usually there will be false positive and false negative result, and in addition, the method is difficult to quantitatively.Therefore, exploitation highly sensitive, highly selective, the nano-probe of PML/RAR alpha fusion gene fluoroscopic examination that what background influence was little can be used for has important theory significance and the using value of reality.
Summary of the invention
An object of the present invention is for the deficiencies in the prior art, provides a kind of nano-probe for acute promyelocytic leukemia fluoroscopic examination, thus realizes the detection to PML/RAR alpha fusion gene.
Two of object of the present invention is the preparation method providing this nano-probe.
For achieving the above object, technical scheme provided by the present invention is:
For a nano-probe for acute promyelocytic leukemia fluoroscopic examination, it is characterized in that this nano-probe is made up of energy donor and energy acceptor, described energy donor and energy acceptor are interacted by pi-pi bond and combine; Described energy donor is one end with the DNA probe of amino by the up-conversion luminescence nanometer crystal with nucleocapsid structure after surface carboxyl groups of the mode grafting of covalent bonding is formed, and described energy donor and the mass ratio of energy acceptor are 20:1 ~ 24:1; Described energy acceptor is: single angle, graphene oxide or carbon nanotube; The described surface carboxyl groups rate with the up-conversion luminescence nanometer crystal of nucleocapsid structure is: 30% ~ 60%; Described DNA probe is: 5 '-NH
2-TCT CAA TGG CTG CCT CCC-3 '; The described DNA probe with amino and the mol ratio of described up-conversion luminescence nanometer crystal are: 1:2000 ~ 1:2500.
The above-mentioned up-conversion luminescence nanometer crystal with nucleocapsid structure is: NaYF
4: Yb, Er@NaYF
4, NaYF
4: Yb, Er@NaYF
4: Yb, Er, NaYF
4: Yb, Er@NaGdF
4or NaYF
4: Yb:Er@NaGdF
4: Yb, Er.
Prepare the above-mentioned method for acute promyelocytic leukemia fluoroscopic examination, it is characterized in that the concrete steps of the method are:
A. by ligand exchange method by there is nucleocapsid structure up-conversion luminescence nanometer crystal finishing on carboxyl, obtain Cit-UCNPs; By this Cit-UCNPs through N-hydroxy-succinamide and the activation of 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide, obtain the Cit-UCNPs after activating;
B. the Cit-UCNPs after being activated by step a gained and DNA probe DNA molecular are dissolved in deionized water according to the ratio of mass ratio 260:1 ~ 290:1, and 4 DEG C are stirred 10 ~ 12 hours; Energy donor and Cit-UCNPs-ssDNA is obtained through separating-purifying;
C. be dissolved in deionized water by step b gained energy donor and energy acceptor according to the ratio that mass ratio is 20:1 ~ 24:1,4 DEG C are stirred 1 ~ 2 hour, finally obtain the nano-probe of acute promyelocytic leukemia fluoroscopic examination.
The concrete grammar of above-mentioned step a is:
(1) be dispersed in by the up-conversion luminescence nanometer crystal with nucleocapsid structure in the mixed solvent of toluene and chloroform, wherein, the volume ratio of toluene and chloroform is 1:2 ~ 2:3; The mass volume ratio of up-conversion luminescence nanometer crystal and mixed solvent is: 1mg/mL ~ 3mg/mL;
(2) under inert atmosphere, Citric Acid, usp, Anhydrous Powder sodium is dissolved in Diethylene Glycol and is mixed with the solution that concentration is 0.1388M ~ 0.1735M, at 100 DEG C ~ 120 DEG C, keep 30 ~ 40min, cooling;
(3) step (1) gained nanocrystal solution is slowly added drop-wise in the solution that step (2) obtains, be heated to 130 DEG C, keep 40 ~ 50min, toluene, chloroform are removed in evaporation, are heated to 180 DEG C, under ar gas environment, keep 1 ~ 2 hour, cooling, centrifugal, with ethanol and water washing, namely obtained water miscible Cit-UCNPs is nanocrystalline.
(4) N-hydroxy-succinamide and 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide are joined in 1 ~ 3mL deionized water according to mass ratio 1:1 ~ 3:1, then step (3) gained Cit-UCNPs is added nanocrystalline, 4 DEG C are stirred 2 ~ 4 hours, by centrifugal for obtained mixture, with deionized water wash 3 times, the Cit-UCNPs obtaining activating is nanocrystalline;
(5) Cit-UCNPs step (4) obtained is nanocrystalline to be dissolved in deionized water with DNA probe molecule according to the ratio of mass ratio 260:1 ~ 290:1, and 4 DEG C are stirred 10 ~ 12 hours; By centrifugal for obtained mixture, after washing 3 times with deionized water, obtain energy donor and Cit-UCNPs-ssDNA.
The invention has the advantages that: construct nano-probe using rare earth up-conversion luminescence nanometer crystal as energy donor, its excitation light source is positioned at the 980nm of near-infrared region, and the light loss that effectively prevent high-energy light injures the strong shortcoming of biological context fluorescence.Secondly, synthesized nano-probe has good biocompatibility and low bio-toxicity, can realize the detection of fusion gene.In addition, the nano-probe uniform particle diameter prepared by the inventive method, pattern are good, Stability Analysis of Structures, and experiment condition is gentle, and repetition rate is high.
Accompanying drawing explanation
Fig. 1 is UCNPs (A) in the embodiment of the present invention 1; CS-UCNPs (B); Cit-UCNPs (C); The TEM figure of Cit-UCNPs-ssDNA (D).
Fig. 2 is the up-conversion fluorescence spectrogram after the target dna adding different concns in the embodiment of the present invention 2 in nano-probe Cit-UCNPs-ssDNA-SWCNHs under 980nm excitation light irradiation.
Fig. 3 is the up-conversion fluorescence spectrogram after the target dna adding different concns in the embodiment of the present invention 2 in nano-probe Cit-UCNPs-ssDNA-GO under 980nm excitation light irradiation.
Fig. 4 is that in the embodiment of the present invention 3, nano-probe Cit-UCNPs-ssDNA-SWCNHs adds the up-conversion fluorescence spectrogram after different concns single base mismatch DNA under 980nm excitation light irradiation.
Fig. 5 is that in the embodiment of the present invention 3, nano-probe Cit-UCNPs-ssDNA-SWCNHs adds the up-conversion fluorescence spectrogram after different concns incomplementarity DNA under 980nm excitation light irradiation.
Fig. 6 is that in the embodiment of the present invention 3, nano-probe Cit-UCNPs-ssDNA-GO adds the up-conversion fluorescence spectrogram after different concns single base mismatch DNA under 980nm excitation light irradiation.
Fig. 7 is that in the embodiment of the present invention 3, nano-probe Cit-UCNPs-ssDNA-SWCNHs adds the up-conversion fluorescence spectrogram after different concns incomplementarity DNA under 980nm excitation light irradiation.
Embodiment
For making the present invention easier to understand, describe in detail below in conjunction with drawings and Examples.These embodiments only play illustrative effect, are not limited to range of application of the present invention.
The preparation method with the up-conversion luminescence nanometer crystal of nucleocapsid structure refers to document:
Li, Z.; Zhang, Y., An efficient and user-friendly method for the synthesis of hexagonal-phase NaYF
4:Yb,Er/Tm nanocrystals with controllable shape and upconversion fluorescence.
Nanotechnology 2008,
19 (34), 345606.
Embodiment 1:
The present embodiment provides the synthesis of a routine nano-probe Cit-UCNPs-ssDNA-SWCNHs, and it comprises the following steps:
(1) utilize solvent structure to have upper conversion nano crystalline substance and the CS-UCNPs of nucleocapsid structure, and be distributed in cyclohexane solution, namely the upper conversion nano of obtained nucleocapsid structure is brilliant;
(2) utilize ligand exchange method to be transferred to from cyclohexane solution in aqueous phase by the upper conversion nano crystalline substance of nucleocapsid structure, obtain the up-conversion luminescence nanometer crystal of good aqueous solubility, now, nanocrystal surface contains carboxyl, i.e. obtained Cit-UCNPs;
(3) surface is contained the upper conversion nano crystalline substance of carboxyl and Cit-UCNPs and one end to react with the DNA probe of amino, combined by the mode of covalent bonding, as energy donor, i.e. obtained Cit-UCNPs-ssDNA;
(4) the single angle of Cit-UCNPs-ssDNA and purchase is interacted by π-π combine, construct containing a pair energy donor and energy acceptor, and effectively can there is the nano-probe of transmission ofenergy, namely prepare Cit-UCNPs-ssDNA-SWCNHs nano-probe.
Described step (1) specifically comprises the following steps:
(1.1) utilize solvent-thermal method to prepare and change nanocrystalline NaYF
4: Yb, Er, and be dispersed in cyclohexane solution;
(1.2) use above-mentioned nanocrystalline, on the CS-UCNPs utilizing solvent structure to have a nucleocapsid structure, conversion nano is brilliant, and wherein the Shell Materials of nanoparticle is six side phase NaYF
4, the thickness of shell is about 2.5nm.
Described step (2) specifically comprises the following steps:
(2.1) 8 ~ 10mgCS-UCNPs, 3 ~ 5mL toluene, 3 ~ 5mL chloroform, 15 ~ 20mL Diethylene Glycol, 0.4 ~ 0.6g Citric Acid, usp, Anhydrous Powder sodium is prepared;
(2.2) be dispersed in by the up-conversion luminescence nanometer crystal of the nucleocapsid structure of preparation in the 5mL toluene of preparation and the mixing solutions of chloroform, wherein, the volume ratio of toluene and chloroform is 1:2 ~ 2:3;
(2.3) the Citric Acid, usp, Anhydrous Powder sodium of preparation is dissolved in 15mL Diethylene Glycol, under ar gas environment, is heated to 110 DEG C, and keeps 30 ~ 40min, cooling;
(2.4) be slowly added drop-wise in the solution that step (2.3) obtains by nanocrystalline for step (2.2) gained, be heated to 130 DEG C, keep 40 ~ 50min, toluene, chloroform are removed in evaporation, are heated to 180 DEG C, under ar gas environment, keep 1 ~ 2 hour, cooling, centrifugal, with ethanol and water washing, namely obtained Cit-UCNPs is nanocrystalline.
Described step (3) specifically comprises the following steps:
(3.1) the nanocrystalline 3 ~ 4mg of Cit-UCNPs, 3 ~ 4mg N-hydroxy-succinamide prepared by above-mentioned steps (2.4) is prepared; i.e. NHS, 2 ~ 3mg 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide; i.e. EDC, 20 ~ 30 μ L, 100 μMs of DNA probe molecules, 1 ~ 3mL deionized waters;
(3.2) NHS and EDC of preparation is joined in 1 ~ 3mL deionized water according to mass ratio 1:1 ~ 3:1, then pre-prepared Cit-UCNPs is added nanocrystalline, 4 DEG C are stirred 2 ~ 4 hours, by centrifugal for obtained mixture, with deionized water wash 3 times, the Cit-UCNPs obtaining activating is nanocrystalline;
(3.3) mixture above-mentioned steps obtained and DNA probe molecule are dissolved in deionized water according to the ratio of mass ratio 260:1 ~ 290:1, and 4 DEG C are stirred 10 ~ 12 hours; By centrifugal for obtained mixture, after washing 3 times with deionized water, obtain energy donor and Cit-UCNPs-ssDNA.
Described step (4) specifically comprises the following steps:
(4.1) nanocrystalline 2 ~ 5mg, the 0.1mg/mL SWCNHs of Cit-UCNPs-ssDNA 3 ~ 7mL prepared by above-mentioned steps (3.3) is prepared, deionized water 1 ~ 3mL;
(4.2) be dissolved in 1 ~ 3mL deionized water with single wall nano angle according to the ratio that mass ratio is 20:1 ~ 24:1 by nanocrystalline for the Cit-UCNPs-ssDNA of preparation, 4 DEG C are stirred 1 ~ 2 hour, finally obtain the Cit-UCNPs-ssDNA-SWCNHs nano-probe of acute promyelocytic leukemia fluoroscopic examination.
Embodiment 2:
The present embodiment provides nano-probe Cit-UCNPs-ssDNA-SWCNHs or Cit-UCNPs-ssDNA-GO to detect the spectrogram of PML/RAR alpha fusion gene fragment and target dna, and it comprises the following steps:
(1) 0.5 ~ 1mL, 1 μM of target dna (5 '-GGG AGG CAG CCA TTG AGA-3 ') is prepared with deionized water.
(2) prepare nano-probe Cit-UCNPs-ssDNA-SWCNHs or Cit-UCNPs-ssDNA-GO, be then diluted to desired concn with deionized water.
(3) fluorescence recovers in experiment, 1.0cm × 1.0cm quartz colorimetric utensil is placed in pipette, extract 2.0mL nano-probe Cit-UCNPs-ssDNA-SWCNHs, in this solution, target dna solution is dropwise added with microsyringe, after often adding the target dna solution of certain volume, then stirred at ambient temperature 30 ~ 50min carries out spectrum test, and result as shown in Figure 2; Equally, in nano-probe Cit-UCNPs-ssDNA-GO, add target dna, operation steps is constant, and result as shown in Figure 3.
Embodiment 3:
In order to prove that this nano-probe can specific recognition PML/RAR alpha fusion gene fragment, spectrogram after the present embodiment provides nano-probe Cit-UCNPs-ssDNA-SWCNHs or Cit-UCNPs-ssDNA-GO and adds single base mismatch DNA, incomplementarity DNA respectively, it comprises the following steps:
(1) 0.5 ~ 1mL, 1 μM of single base mismatch DNA (5 '-GGG A
ag CAG CCA TTGAGA-3 '), 0.5 ~ 1mL, 1 μM of incomplementarity DNA (5 '-AGT TCA TCC TGC GCT CTT-3 ') is prepared with deionized water.
(2) prepare nano-probe Cit-UCNPs-ssDNA-SWCNHs or Cit-UCNPs-ssDNA-GO, be then diluted to desired concn with deionized water.
(3) 1.0cm × 1.0cm quartz colorimetric utensil is placed in pipette, extract 2.0mL nano-probe Cit-UCNPs-ssDNA-SWCNHs, in this solution, single base mismatch DNA is dropwise added with microsyringe, after often adding the target dna solution of certain volume, then stirred at ambient temperature 30 ~ 50min carries out spectrum test, and result as shown in Figure 4; 1.0cm × 1.0cm quartz colorimetric utensil is placed in pipette, extract 2.0mL nano-probe Cit-UCNPs-ssDNA-SWCNHs, in this solution, incomplementarity DNA is dropwise added with microsyringe, after often adding the target dna solution of certain volume, then stirred at ambient temperature 30 ~ 50min carries out spectrum test, and result as shown in Figure 5;
(3) 1.0cm × 1.0cm quartz colorimetric utensil is placed in pipette, extract 2.0mL nano-probe Cit-UCNPs-ssDNA-GO, in this solution, single base mismatch DNA is dropwise added with microsyringe, after often adding the target dna solution of certain volume, then stirred at ambient temperature 30 ~ 50min carries out spectrum test, and result as shown in Figure 6; 1.0cm × 1.0cm quartz colorimetric utensil is placed in pipette, extract 2.0mL nano-probe Cit-UCNPs-ssDNA-GO, in this solution, incomplementarity DNA is dropwise added with microsyringe, after often adding the target dna solution of certain volume, then stirred at ambient temperature 30 ~ 50min carries out spectrum test, and result as shown in Figure 7;
Fig. 1 is UCNPs (A) in the embodiment of the present invention 1; CS-UCNPs (B); Cit-UCNPs (C); The TEM figure of Cit-UCNPs-ssDNA (D).As can be seen from the figure, the good and size uniformity of synthesized UCNPs nanoparticle dispersion, the size of nanoparticle is at 30nm; CS-UCNPs size is at 35nm; Good and the size uniformity of nanoparticle dispersion.Cit-UCNPs, Cit-UCNPs-ssDNA nanoparticle, compared with the particle diameter of CS-UCNPs, changes not quite and still has good dispersiveness.
Fig. 2 be add different concns in the embodiment of the present invention 2 in nano-probe Cit-UCNPs-ssDNA-SWCNHs target dna after up-conversion fluorescence spectrogram under 980nm excitation light irradiation; Fig. 3 is the up-conversion fluorescence spectrogram after the target dna adding different concns in nano-probe Cit-UCNPs-ssDNA-GO under 980nm excitation light irradiation.As can be seen from the figure, add the target dna of different concns respectively in solution after, under 980nm exciting light, along with the increase of target DNA concentration, the fluorescence intensity level that 545nm goes out strengthens gradually, illustrate that first probe ssDNA enough carries out base pair complementarity with target dna and form stable DNA double spirane structure, that is alternately occur, the skeleton of the chain that hydrophilic deoxyribosyl and phosphate are formed, be positioned at double-helical outside, hydrophobic base pair is then inner at helical molecule, this result finally can cause SWCNHs or GO away from the surface of nano particle, energy transfer process is prohibited, fluorescence can recover again, thus the detection realized the PML/RARA fusion gene fragment of acute promyelocytic leukemia (APL).
Fig. 4 is that in the embodiment of the present invention 3, nano-probe Cit-UCNPs-ssDNA-SWCNHs adds the up-conversion fluorescence spectrogram after different concns single base mismatch DNA under 980nm excitation light irradiation; Fig. 5 is nano-probe Cit-UCNPs-ssDNA-
SWCNHs adds the up-conversion fluorescence spectrogram after different concns incomplementarity DNA under 980nm excitation light irradiation; Fig. 6 is the up-conversion fluorescence spectrogram (C) that nano-probe Cit-UCNPs-ssDNA-GO adds after different concns single base mismatch DNA under 980nm excitation light irradiation; Fig. 7 is the up-conversion fluorescence spectrogram that nano-probe Cit-UCNPs-ssDNA-GO adds after different concns incomplementarity DNA under 980nm excitation light irradiation.As can be seen from the figure, under 980nm exciting light, along with the increase of single base mismatch DNA or incomplementarity DNA concentration, the fluorescence intensity at 545nm place does not almost change, instruction book base mispairing DNA or incomplementarity DNA can not carry out base pair complementarity with DNA probe makes SWCNHs or GO away from the surface of nano particle, therefore fluorescence does not recover, thus proves that this probe specificity can detect PML/RARA fusion gene fragment.
According to provided by the present invention, DNA probe is modified at rare earth up-conversion luminescence nanometer crystal surface, the product obtained has size homogeneous and favorable dispersity, Stability Analysis of Structures, the advantages such as up-conversion fluorescence is stronger, particularly gained nano-probe can be applicable to specificity and detect PML/RARA fusion gene fragment.It is simple that the inventive method has technique, and easy to operate, the advantage of easy control of structure, has potential using value in the field such as cytogenetics and molecular biology research.
As described in the above embodiment the present invention, other nano-probe for specificity detection PML/RARA fusion gene fragment adopting method same or similar with it to obtain, all in scope.
Claims (4)
1. for a nano-probe for acute promyelocytic leukemia fluoroscopic examination, it is characterized in that this nano-probe is made up of energy donor and energy acceptor, described energy donor and energy acceptor are interacted by pi-pi bond and combine; Described energy donor is one end with the DNA probe of amino by the up-conversion luminescence nanometer crystal with nucleocapsid structure after surface carboxyl groups of the mode grafting of covalent bonding is formed, and described energy donor and the mass ratio of energy acceptor are 20:1 ~ 24:1; Described energy acceptor is: single angle, graphene oxide or carbon nanotube; The described surface carboxyl groups rate with the up-conversion luminescence nanometer crystal of nucleocapsid structure is: 30% ~ 60%; Described DNA probe is: 5 '-NH
2-TCT CAA TGG CTG CCT CCC-3 '; The described DNA probe with amino and the mol ratio of described up-conversion luminescence nanometer crystal are: 1:2000 ~ 1:2500.
2. the preparation method of the nano-probe for acute promyelocytic leukemia fluoroscopic examination according to claim 1, is characterized in that the described up-conversion luminescence nanometer crystal with nucleocapsid structure is: NaYF
4: Yb, Er@NaYF
4, NaYF
4: Yb, Er@NaYF
4: Yb, Er, NaYF
4: Yb, Er@NaGdF
4or NaYF
4: Yb:Er@NaGdF
4: Yb, Er.
3. prepare the method for acute promyelocytic leukemia fluoroscopic examination according to claim 1 and 2, it is characterized in that the concrete steps of the method are:
A. by ligand exchange method by there is nucleocapsid structure up-conversion luminescence nanometer crystal finishing on carboxyl, obtain Cit-UCNPs; By this Cit-UCNPs through N-hydroxy-succinamide and the activation of 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide, obtain the Cit-UCNPs after activating;
B. the Cit-UCNPs after being activated by step a gained and DNA probe DNA molecular are dissolved in deionized water according to the ratio of mass ratio 260:1 ~ 290:1, and 4 DEG C are stirred 10 ~ 12 hours; Energy donor and Cit-UCNPs-ssDNA is obtained through separating-purifying;
C. be dissolved in deionized water by step b gained energy donor and energy acceptor according to the ratio that mass ratio is 20:1 ~ 24:1,4 DEG C are stirred 1 ~ 2 hour, finally obtain the nano-probe of acute promyelocytic leukemia fluoroscopic examination.
4. the preparation method for acute promyelocytic leukemia fluoroscopic examination according to claim 3, is characterized in that the concrete grammar of described step a is:
(1) be dispersed in by the up-conversion luminescence nanometer crystal with nucleocapsid structure in the mixed solvent of toluene and chloroform, wherein, the volume ratio of toluene and chloroform is 1:2 ~ 2:3; The mass volume ratio of up-conversion luminescence nanometer crystal and mixed solvent is: 1mg/mL ~ 3mg/mL;
(2) under inert atmosphere, Citric Acid, usp, Anhydrous Powder sodium is dissolved in Diethylene Glycol and is mixed with the solution that concentration is 0.1388M ~ 0.1735M, at 100 DEG C ~ 120 DEG C, keep 30 ~ 40min, cooling;
(3) step (1) gained nanocrystal solution is slowly added drop-wise in the solution that step (2) obtains, be heated to 130 DEG C, keep 40 ~ 50min, toluene, chloroform are removed in evaporation, are heated to 180 DEG C, under ar gas environment, keep 1 ~ 2 hour, cooling, centrifugal, with ethanol and water washing, namely obtained water miscible Cit-UCNPs is nanocrystalline;
(4) N-hydroxy-succinamide and 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide are joined in 1 ~ 3mL deionized water according to mass ratio 1:1 ~ 3:1, then step (3) gained Cit-UCNPs is added nanocrystalline, 4 DEG C are stirred 2 ~ 4 hours, by centrifugal for obtained mixture, with deionized water wash 3 times, the Cit-UCNPs obtaining activating is nanocrystalline;
(5) Cit-UCNPs step (4) obtained is nanocrystalline to be dissolved in deionized water with DNA probe molecule according to the ratio of mass ratio 260:1 ~ 290:1, and 4 DEG C are stirred 10 ~ 12 hours; By centrifugal for obtained mixture, after washing 3 times with deionized water, obtain energy donor and Cit-UCNPs-ssDNA.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ES2580138A1 (en) * | 2016-05-24 | 2017-05-09 | Universidad Complutense De Madrid | Biosensor for the detection of nucleic acids, method of preparation and use (Machine-translation by Google Translate, not legally binding) |
| CN107267149A (en) * | 2016-04-08 | 2017-10-20 | 中国科学院苏州纳米技术与纳米仿生研究所 | Red up-conversion luminescence nanomaterial and preparation method thereof |
| CN111410950A (en) * | 2020-02-25 | 2020-07-14 | 常州诺达生化科技有限公司 | Double-block DNA modified upconversion nanoparticle and preparation method and application thereof |
| CN114058721A (en) * | 2021-11-18 | 2022-02-18 | 江苏大学 | Preparation method of up-conversion fluorescent recognition probe, product and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101935529A (en) * | 2010-07-13 | 2011-01-05 | 中国科学院长春光学精密机械与物理研究所 | Multifunctional nano-composite having up-conversion luminescence, oxygen sensing and biological connectivity and preparation method thereof |
| CN103468260A (en) * | 2013-08-08 | 2013-12-25 | 南京邮电大学 | pH nanometer sensing material with upconversion luminescence property, and preparation method thereof |
| CN103540310A (en) * | 2013-10-11 | 2014-01-29 | 上海大学 | Surface direct mesoporous modification method used for multi-morphology rear earth-doped up-conversion luminescent nanocrystallines |
| CN103756668A (en) * | 2014-01-08 | 2014-04-30 | 上海大学 | Core-shell type rare earth up-conversion composite nanoparticle and preparation method thereof |
-
2015
- 2015-04-16 CN CN201510178685.3A patent/CN104818325A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101935529A (en) * | 2010-07-13 | 2011-01-05 | 中国科学院长春光学精密机械与物理研究所 | Multifunctional nano-composite having up-conversion luminescence, oxygen sensing and biological connectivity and preparation method thereof |
| CN103468260A (en) * | 2013-08-08 | 2013-12-25 | 南京邮电大学 | pH nanometer sensing material with upconversion luminescence property, and preparation method thereof |
| CN103540310A (en) * | 2013-10-11 | 2014-01-29 | 上海大学 | Surface direct mesoporous modification method used for multi-morphology rear earth-doped up-conversion luminescent nanocrystallines |
| CN103756668A (en) * | 2014-01-08 | 2014-04-30 | 上海大学 | Core-shell type rare earth up-conversion composite nanoparticle and preparation method thereof |
Non-Patent Citations (4)
| Title |
|---|
| CHENGHUI LIU等: "Efficient fluorescence resonance energy transfer between upconversion nanophosphors and graphene oxide: a highly sensitive biosensing platform", 《CHEMCOMM》 * |
| FENG WANG ET AL: "Upconversion nanoparticles in biological labeling, imaging, and therapy", 《ANALYST》 * |
| 于文强等: "PML-RARa基因在早幼粒白血病发病中的作用研究", 《中国肿瘤生物治疗杂志》 * |
| 陈庆涛等: "NaYF4:Yb/Er-碳纳米管功能纳米材料的制备、表征及其在肿瘤细胞的靶向检测和治疗中的应用", 《中国科学: 化学》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107267149A (en) * | 2016-04-08 | 2017-10-20 | 中国科学院苏州纳米技术与纳米仿生研究所 | Red up-conversion luminescence nanomaterial and preparation method thereof |
| CN107267149B (en) * | 2016-04-08 | 2020-03-27 | 中国科学院苏州纳米技术与纳米仿生研究所 | Red up-conversion luminescent nano material and preparation method thereof |
| ES2580138A1 (en) * | 2016-05-24 | 2017-05-09 | Universidad Complutense De Madrid | Biosensor for the detection of nucleic acids, method of preparation and use (Machine-translation by Google Translate, not legally binding) |
| CN111410950A (en) * | 2020-02-25 | 2020-07-14 | 常州诺达生化科技有限公司 | Double-block DNA modified upconversion nanoparticle and preparation method and application thereof |
| CN111410950B (en) * | 2020-02-25 | 2023-04-07 | 常州诺达生化科技有限公司 | Double-block DNA modified upconversion nanoparticle and preparation method and application thereof |
| CN114058721A (en) * | 2021-11-18 | 2022-02-18 | 江苏大学 | Preparation method of up-conversion fluorescent recognition probe, product and application thereof |
| CN114058721B (en) * | 2021-11-18 | 2024-04-16 | 江苏大学 | Preparation method of up-conversion fluorescent identification probe, and product and application thereof |
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