CN104250662B - A kind of nano-probe and preparation method thereof of in situ detection cell telomerase activity - Google Patents
A kind of nano-probe and preparation method thereof of in situ detection cell telomerase activity Download PDFInfo
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- CN104250662B CN104250662B CN201310256710.6A CN201310256710A CN104250662B CN 104250662 B CN104250662 B CN 104250662B CN 201310256710 A CN201310256710 A CN 201310256710A CN 104250662 B CN104250662 B CN 104250662B
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- G01N2333/9125—Nucleotidyltransferases (2.7.7) with a definite EC number (2.7.7.-)
- G01N2333/9128—RNA-directed DNA polymerases, e.g. RT (2.7.7.49)
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Abstract
The present invention relates to a kind of nano-probes and preparation method thereof of in situ detection cell telomerase activity.Obtained nano-probe is with porous silicon nano (MSN) for carrier, fluorescence quencher BHQ, filling fluorescein are covalently fixed in its duct, and fluorescein is blocked in duct in the DNA chain O1 of its surface package segment containing telomerase substrate (TP) by Electrostatic Absorption.The presence of BHQ has quenched the fluorescence of fluorescein, and the fluorescent state of nano-probe is "Off".Under the action of Telomerase, the 3 ' ends of detecting probe surface O1 are extended, and generate multiple TTAGGG segments complementary with 5 ' ends, both ends are caused to hybridize to form cyclic structure and be detached from detecting probe surface, to open duct, the fluorescein in probe is released, fluorescent state is made to be converted to "ON".By probe and cell culture, after probe enters cytoplasm, probe duct is opened under the effect of cytoplasm telomerase, the fluorescence generated using the fluorescein of release realizes the in situ detection of cell telomerase activity.
Description
One, technical field
The present invention relates to a kind of nano-probes and preparation method thereof of in situ detection cell telomerase activity.
Two, background technique
Telomerase is a kind of ribonucleoprotein complex being made of RNA and protein, is one kind of reverse transcriptase.Telomere
Enzyme use in cell in it RNA carry out reverse transcription as template, increase telomere by TTAGGG repetitive sequence to maintain
Telomere length.In normal human body cell, the activity of Telomerase is by quite strict regulation, only in hematopoietic cell, dry
It just can detecte telomerase activation in these cells that must constantly divide of cell and reproduction cell.In tumour cell, end
The activity of granzyme is activated, thus it is the important evidence of diagnosis and prediction cancer.
Currently, most popular activity test method of telomerase is telomeric repeat amplification program (TRAP).TRAP is with people
For the sequence without telomere of work synthesis as base band, the Telomerase in sample keeps base band elongated, it is elongated after product pass through polymerase
Chain reaction (PCR) is detected.This method detection sensitivity is very high, but since PCR product length is uncertain, time-consuming, limitation
The practical application of TRAP technology.Recently, some telomere enzyme biologic sensors and telomerase activation based on TRAP improved technology
Direct analytical technology be reported.These technologies include Telomerase method of purification, optical detection, array and biosensor core
Piece, electrochemical methods, bio-barcode technology, they all need to carry out cell liquid extraction in advance, can not direct in-situ detection work
Intracellular telomerase activation.
The present invention is directed to this problem, utilizes the chemistry of porous silicon nano (MSN) and thermodynamic stability, good
Biocompatibility and mammalian cell are to its efficient absorption ability, by cellular structure and with the biomolecule knot of identification function
It closes, by fixed in duct or filling fluorescent quenching molecule BHQ and fluorescein, wraps up segment containing telomerase substrate on its surface
DNA chain O1, have developed a kind of nano-probe of in situ detection cell telomerase activity.The probe can urging by Telomerase
3 ' the ends for changing prolonged action O1, generate multiple TTAGGG segments complementary with 5 ' ends, the both ends O1 hybridize to form cyclic structure and take off
From detecting probe surface, to open duct, the fluorescein in probe is released, fluorescence probe state is made to be converted to "ON".By probe
With cell culture, after probe enters cytoplasm, the in situ detection of cell telomerase activity is realized.
Three, summary of the invention
The purpose of the present invention is: using MSN as carrier, in hole, inner wall covalently fixes fluorescence quencher BHQ, fills fluorescence in hole
Element, and the biological function based on Telomerase, building include the DNA chain O1 of telomerase substrate segment, are adsorbed on the surface MSN,
Obtaining can be with the nano-probe of in situ detection cell telomerase activity.
Nano-probe proposed by the present invention is achieved through the following technical solutions:
1) with the DNA chain O1 sequence of Telomerase responsiveness are as follows: (5 '-(CCC TAA)6 AAT CCG TCGAGC AGA GTT-3').3 ' part of the ends with underscore are telomere zymolyte TP, and extending product can be with 5 ' end Complementary hybridization.
2) preparation of the nano-probe of in situ detection cell telomerase activity is first using MSN as carrier, in duct inner wall
It is covalently attached BHQ, fills fluorescein in duct, O1 is wrapped in by nanoparticle surface by Electrostatic Absorption, is formed as shown in Figure 1
Nano-probe.
The working principle of the invention:
The working principle of the invention is as shown in Fig. 2, the 3 ' ends of the nano-probe surface O1 are that Telomerase identifies substrate
TP forms multiple TTAGGG segments under the action of Telomerase.In the presence of Granzyme, the fluorescein in nano-probe is by O1
It blocks in duct, the BHQ that fluorescence is connected by cell walls is quenched, and fluorescent state is "Off".Containing in nano-probe and cell
After incubating in the cell culture fluid of deoxynucleoside triphosphate (dNTP), probe enters cytoplasm by cell endocytic, in cytoplasm
Under the action of Telomerase, the O1 of detecting probe surface extends, and the product head and the tail Complementary hybridization after extension forms hair fastener shape rigid structure,
To be detached from nanoparticle surface, duct is opened, discharge the fluorescein in duct, realizes the original position of cell telomerase activity
Imaging analysis.
Compared with prior art, the invention has the characteristics that:
It is glimmering by fixing or filling in duct by the cellular structure of MSN in conjunction with the biomolecule with identification function
Optical quenching molecule BHQ and fluorescein are developed into a kind of inspection in situ in the DNA chain O1 of its surface package segment containing telomerase substrate
Survey the nano-probe of cell telomerase activity.Relative to existing Telomerase activity method, have the following characteristics that
1. probe of the present invention is directly acted on living cells, the in situ imaging of cellular enzymes telomerase activation is realized,
It relative to existing method, can reflect telomerase activation in real time, without carrying out broken or other pretreatments to cell, have bright
Aobvious advantage.
2. DNA chain zymolyte containing telomere segment of the present invention can extend and produce in the downward length of telomere enzyme effect
Object and 5 ' end Complementary hybridizations, form hair fastener shape rigid structure and O1 are made to be detached from detecting probe surface, realize the realizing controlled-release of signaling molecule
It puts.
3. the present invention is detected glimmering compared to the existing Telomerase activity method based on gene promoter and mRNA
Optical signal is directly related with the activity of Telomerase, to realize the in situ imaging analysis of cell telomerase activity.
Four, Detailed description of the invention
The nano-probe structure and synthesis schematic diagram of Fig. 1 in situ detection cell telomerase activity.
The working principle diagram of Fig. 2 nano-probe in situ detection cell telomerase activity: a) O1 is under telomere enzyme effect
Extension, disengaging and fluorescein release；B) probe and cytosis schematic illustration.
Five, specific embodiment
Embodiment 1: in conjunction with Fig. 1, the nano-probe of in situ detection cell telomerase activity is synthesized
1mg carbodiimide (EDC), 2.5mgN- HOSu NHS (NHS) and 0.1mg carboxyl BHQ are added in
In 1mL1mg/mL amination MSN dispersion liquid, magnetic agitation 4 hours at room temperature.It is centrifugated and obtains MSN-BHQ after washing.It will
MSN-BHQ is dispersed in 1mL water and 0.1mg fluorescein is added, under room temperature continued stirring overnight.It is centrifugated and uses ultrapure
It is dried after water washing precipitate, obtains fluorescein@MSN-BHQ.1mg fluorescein@MSN-BHQ is dispersed in 1mL hybridization buffer
(Tri(Hydroxymethyl) Amino Methane Hydrochloride containing 10mM, 1mM ethylenediamine tetra-acetic acid, 50mM NaCl and 10mM MgCl2) in, it is added 10
μ L (100 μM) O1 is centrifugally separating to obtain MSN probe after persistently stirring 1 hour at 37 DEG C.Probe is dispersed in 1mLHB, in 4 DEG C
Under the conditions of save.
Embodiment 2: in conjunction with Fig. 2, the in situ detection of cell telomerase activity is carried out with nano-probe
Using HeLa cervical cancer cell as model, first by HeLa cell (0.5mL, 1 × 106mL-1) plant in the burnt training of 20-mm copolymerization
It supports in ware, is cultivated 24 hours at 37 DEG C.15 μ L MSN probes (1mg/mL) are added in the culture dish again, incubate 1.5 at 37 DEG C
After hour, detected with laser confocal fluorescence microscope.In the HeLa cell of Telomerase positive, the duct of MSN probe
It is opened, releases fluorescein, occur fluorescence signal in cytoplasm, realize the in situ imaging of cell telomerase activity.With end
The drugs point such as granzyme sense oligonucleotides (60 μ L, 10 μM), antisense oligonucleotides (60 μ L, 10 μM) and catechin (125 μ g)
Not with cell (0.5mL, 1 × 106mL-1) co-culture 48 hours, 15 μ L MSN probe (1mg/ are then added in the culture dish
ML), after incubating 1.5 hours at 37 DEG C, the variation of telomerase activation after drug effect is observed under Laser Scanning Confocal Microscope.
Claims (4)
1. a kind of nano-probe for detecting unicellular telomerase activity into cell in-situ, it is characterised in that with porous silicon nanometer
Particle is carrier, and cell walls are covalently attached fluorescent quenching molecule BHQ, and fluorescein is filled in duct, and particle surface package contains telomere
DNA chain O1, the O1 sequence of zymolyte segment are as follows: 5 '-(CCC TAA)6 AAT CCG TCG AGC AGA GTT- 3 ', the O1 sequence
Column have Telomerase responsiveness, and nano-probe can be made to have " on/off " transformational of fluorescein release.
2. nano-probe according to claim 1, it is characterised in that the 3 ' ends of O1 are telomerase substrate segment, can held
Granzyme effect is lower to extend, and generates multiple TTAGGG segments complementary with 5 ' ends, both ends is caused to hybridize to form cyclic structure and be detached from
Detecting probe surface, to open duct.
3. nano-probe according to claim 1, it is characterised in that fluorescence " on/off " the state conversion of nano-probe is logical
Duct " closing-opening " control is crossed to realize, when potential close, the fluorescence of fluorescein is quenched by BHQ in duct；It is beaten in duct
After opening, the fluorescein in probe is released, generates fluorescence.
4. nano-probe according to claim 1, it is characterised in that with after cell culture, probe enters cell, thin
Its duct is opened under the action of cytoplasm telomerase, discharges fluorescein, and the fluorescence generated using the fluorescein of release realizes cell
The in situ detection of telomerase activity.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105154563B (en) * | 2015-09-30 | 2018-10-23 | 陕西师范大学 | A method of based on the homogeneous nonstandard detection telomerase activation of triple amplifying techniques |
| CN107723339A (en) * | 2017-11-14 | 2018-02-23 | 山东师范大学 | Detect the nano-sensor and its detection method of O acetylglucosamine transferases |
| CN109897887B (en) * | 2019-01-04 | 2023-03-14 | 中国科学院苏州生物医学工程技术研究所 | Telomerase activity detection method based on FRET |
| CN110699353A (en) * | 2019-07-26 | 2020-01-17 | 安徽大学 | Nucleic acid probe targeting telomere |
| CN113782095B (en) * | 2020-06-10 | 2023-11-28 | 香港城市大学深圳研究院 | Method for detecting cell state in real time at high flux |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2007070982A1 (en) * | 2005-12-23 | 2007-06-28 | Sienna Cancer Diagnostics Ltd | Assay for detection of telomerase activity |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2007070982A1 (en) * | 2005-12-23 | 2007-06-28 | Sienna Cancer Diagnostics Ltd | Assay for detection of telomerase activity |
Non-Patent Citations (2)
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| 基于响应性聚合物组装体的生物医用与检测功能材料;万学娟;《中国博士学位论文全文数据库工程科技Ⅰ辑》;20110915(第9期);B014-75 * |
| 肿瘤细胞及端粒酶检测的生物传感技术新方法的研究;高庆;《中国优秀硕士学位论文全文数据库信息科技辑》;20130215(第2期);I140-233 * |
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