CN107044888B - The construction method of thermometer based on fluorescent dye ThT, RET gene - Google Patents
The construction method of thermometer based on fluorescent dye ThT, RET gene Download PDFInfo
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- CN107044888B CN107044888B CN201710159912.7A CN201710159912A CN107044888B CN 107044888 B CN107044888 B CN 107044888B CN 201710159912 A CN201710159912 A CN 201710159912A CN 107044888 B CN107044888 B CN 107044888B
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- temperature
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- ret gene
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01K—MEASURING TEMPERATURE; MEASURING QUANTITY OF HEAT; THERMALLY-SENSITIVE ELEMENTS NOT OTHERWISE PROVIDED FOR
- G01K11/00—Measuring temperature based upon physical or chemical changes not covered by groups G01K3/00, G01K5/00, G01K7/00 or G01K9/00
- G01K11/12—Measuring temperature based upon physical or chemical changes not covered by groups G01K3/00, G01K5/00, G01K7/00 or G01K9/00 using changes in colour, translucency or reflectance
- G01K11/16—Measuring temperature based upon physical or chemical changes not covered by groups G01K3/00, G01K5/00, G01K7/00 or G01K9/00 using changes in colour, translucency or reflectance of organic materials
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Abstract
The present invention relates to a kind of buildings of thermometer for causing fluorescent dye thioflavin T fluorometric Strength Changes based on the variation of RET gene constellation, specifically: fluorescent dye thioflavine T (ThT) and RET are mixed by the molar ratio of 1:1, Tris-HCl buffer solution is added, adjusts pH=7,8 and 9.The system that the present invention constructs obvious responses to pH and temperature, especially when pH is 7,8,9, increases with temperature, the regular decline of the fluorescence intensity of ThT may be designed to thermometer accordingly.Because pH is 7 or so in human body, " clinical thermometer " is designed between 25-40 DEG C for pH=7.
Description
Technical field
The present invention relates to the construction methods of the thermometer based on fluorescent dye ThT, RET gene, are essentially based on RET gene
Conformation change causes fluorescent dye ThT fluorescence intensity change to indicate the construction method of the thermometer of temperature change.
Background technique
Thermometer is the general name of temperature measuring device, can accurately judge and measure temperature.Usually utilize solid, liquid, gas
The phenomenon that body is influenced by temperature and expands with heat and contract with cold etc. is the foundation of design.There are kerosene thermometer, alcohol thermometer, mercury temperature
Meter, gas thermometer, resistance thermometer, thermoelectric thermometer, radiation thermometer and flash ranging thermometer, bimetallic thermometer etc.
Various temperature meter.It is come design temperature meter mostly using the physical property of substance in different environment.
RET albumen is a kind of receptor tyrosine kinase (receptor tyrosine kinase), a kind of proto-oncogene,
There is response to hydrogen ion, in weakly acidic condition, i-motif structure can be folded into.There is the deoxidation rich in cytimidine (C) in people's telomere
Nucleotide (DNA) sequence, these sequences can pass through cytimidine (C) and protonation cytimidine CH in acidic environment+Between hydrogen
Key interacts to form parallel double-strand, and antiparallel be mutually inserted of two double-strands is formed by four chain non-covalent complexes, referred to as ties
Structure.Structure is present in the promoter region of proto-oncogene more, thin such as vascular endothelial growth factor, proto-oncogene and at retina
Born of the same parents' tumor gene etc. becomes the target spot of gene regulation and Anti-Cancer Drug Design.
Thioflavine T (ThT) is also known as basic yellow 1,2- [4- (dimethylamino) phenyl] -3,6- dimethyl-benzothiazole chlorine
Compound, molecular formula are C17H19ClN2S is a kind of chemical dyestuff.When RET is hairpin structure, ThT has fluorescence hair in conjunction with RET
It penetrates, when RET becomes i-motif structure, ThT is separated with RET, unstressed configuration.
Summary of the invention
The object of the invention is in order to provide a kind of building side of thermometer based on fluorescent dye ThT, RET gene
Method, efficient and sensible, simple, quick changed based on RET gene constellation cause fluorescent dye ThT fluorescence intensity change to indicate temperature
Spend the construction method of the thermometer of variation.
The purpose of the present invention can be achieved through the following technical solutions: a kind of based on fluorescent dye ThT, RET gene
The construction method of thermometer, in the steps below:
First, fluorescent dye ThT and RET gene molar ratio is 1:1, and culture forms body in Tris-HCl buffer solution
System, by changing the pH value of Tris-HCl buffer solution, the pH value of control system is 7,8 and 9 respectively；
Second, within the scope of 4-55 DEG C of temperature, at interval of 3 DEG C constant temperature incubation 30 minutes；
Third, detects fluorescence, and parameter is set as slit 10nm, voltage 500V, obtains each temperature gradient fluorescence intensity parameter.
Mechanism of the present invention is: under weak acid environment, RET gene can be folded into i-motif structure, when RET gene is hair
When card structure, fluorescent dye ThT is in conjunction with RET gene in system, and in the void area in hairpin structure, ThT has fluorescence hair
It penetrates.
The construction method of the thermometer based on fluorescent dye ThT, RET gene utilizes under weak acid environment
RET gene can be folded into i-motif structure, RET gene at pH=7,8,9 with temperature and the temperature of occurred conformation variation is Tm,
When RET gene is hairpin structure, fluorescent dye ThT is in conjunction with RET gene, in the void area in hairpin structure, causes glimmering
The variation of photoinitiator dye ThT fluorescence intensity, obvious responses to temperature.
When temperature increases the temperature more than its conformation changeWhen, ThT fluorescence intensity decline, thus it is speculated that hairpin structure unlocks change
At single-stranded structure, ThT is separated with RET at this time.
The present invention can design a kind of thermometer based on fluorescent dye ThT, RET gene using above-mentioned construction method.
In addition, pH value is adjusted to 7 according to the characteristic of the pH value 7 or so of human body, at 25-40 DEG C, per once constant temperature incubation 30
Minute.PH value is adjusted to, design temperature clinical thermometer at 25-40 DEG C close with the pH value of human body, fluorescence intensity, to per once
It obvious responses to and can distinguish, retest more than three times, takes the average value of every degree fluorescence intensity, is designed to commercially available
" clinical thermometer ".
And the present invention has mainly probed into the conformation change that RET gene occurs at pH=7,8,9 with temperature, so as to cause
The variation of fluorescent dye ThT fluorescence intensity, discovery obvious response to temperature, devise thermometer with this.
The present invention utilizes RET genetic characteristics, cultivates in Tris-HCl buffer solution with fluorescent dye ThT and forms fluorophor
System is used for thermometer.
Detailed description of the invention
When Fig. 1 is pH=7, the fluorescence emission curves figure of 4-55 DEG C of section (be divided into 3 DEG C)；
When Fig. 2 is pH=7, the linear graph of 4-55 DEG C of section (be divided into 3 DEG C)；
When Fig. 3 is pH=8, the fluorescence emission curves figure of 4-50 DEG C of section (be divided into 3 DEG C)；
When Fig. 4 is pH=8, the linear graph of 4-55 DEG C of section (be divided into 3 DEG C)；
When Fig. 5 is pH=9, the fluorescence emission curves figure of 4-55 DEG C of section (be divided into 3 DEG C)；
When Fig. 6 is pH=9, the linear graph of 4-55 DEG C of section (be divided into 3 DEG C)；
When Fig. 7 is pH=7, the fluorescence emission curves figure of 25-40 DEG C of section (be divided into 1 DEG C)；
When Fig. 8 is pH=7, the linear graph of 25-40 DEG C of section (be divided into 1 DEG C)；
When Fig. 9 is pH=7, the circulation figure in 25-40 DEG C of section；
Figure 10 principle schematic diagram of the present invention.
Specific embodiment
A kind of construction method of the thermometer based on RET conformation change, under weak acid environment, RET gene can be folded into
I-motif structure, when RET gene is hairpin structure, fluorescent dye ThT is in conjunction with RET gene, the hole in hairpin structure
In area, ThT has fluorescent emission, is illustrated in fig. 10 shown below the structural schematic diagram in left side
RET gene is affected by temperature meeting occurred conformation and changes, when temperature increases the temperature (T more than its conformation changem) when,
Speculate that hairpin structure is unlocked and become single-stranded structure, ThT is separated with RET at this time, the decline of ThT fluorescence.Structure on the right side of following Figure 10
Signal.
One, the preparation of system construction:
1.Tris-HCl buffer (Tris:10 mM, pH=7,8,9) preparation: really weigh 0.6057 g of Tris in
It in 500 mL beakers, is completely dissolved with 400 mL triple distillation water, adjusts pH respectively to 7,8,9 with dilute hydrochloric acid solution, move
Liquid is to 500 mL volumetric flasks.Three times with triple distillation water washing, rinse liquid is transferred in volumetric flask beaker, constant volume, is uniformly mixed
It is spare.
2. RET DNA Tris-HCl buffer solution is dissolved, concentration is 100 μM.
The preparation of 3.ThT storage liquid (50 mL, 100 μM): accurately weighing ThT 1.6mg, and 50mL triple distillation water is added
It is dissolved, is uniformly mixed, 4 DEG C are kept in dark place, and spare, concentration is 100 μM.
Embodiment 1
Example thermometer building based on fluorescent dye ThT, RET gene system:
1. 10 μ L RET DNA(100 μM are taken respectively), 10 μ L ThT (100 μM), 980 μ L 20mM Tris-HCl (pH=7,
8,9), constant temperature incubation 30 minutes at a temperature of being spaced 3 DEG C.
2. detecting fluorescence, excites under 425nm, have obvious fluorescence at 525nm, system varies with temperature in 4-55 DEG C of section
Fluorescence intensity change it is as shown in Figures 1 to 6.It can be used for making thermometer using architectural characteristic.
Embodiment 2
Under neutral environment (pH=7), respectively at 25-40 DEG C per once constant temperature incubation 30 minutes, inspection follows same system
Ring.
In 25-40 DEG C of fluorescence intensity, to per once obvious responsing to and can distinguish, retest three times with
On, as shown in Figs. 7-9, the present embodiment retest six times, the average value of every degree fluorescence intensity is taken, is designed to " clinical thermometer ".
Claims (4)
1. a kind of construction method of the thermometer based on fluorescent dye ThT, RET gene, in the steps below:
First, fluorescent dye ThT and RET gene molar ratio is 1:1, and culture forms system in Tris-HCl buffer solution, is led to
The pH value for changing Tris-HCl buffer solution is crossed, the pH value of control system is 7,8 and 9 respectively；
Second, within the scope of 4-55 DEG C of temperature, at interval of 3 DEG C constant temperature incubation 30 minutes；
Third, detects fluorescence, and parameter is set as slit 10nm, voltage 500V, obtains each temperature gradient fluorescence intensity parameter.
2. the construction method of the thermometer according to claim 1 based on fluorescent dye ThT, RET gene, feature exist
In:
Under weak acid environment, i-motif structure can be folded into using RET gene, RET gene at pH=7,8,9 with temperature and
The temperature of occurred conformation variation is Tm, and when RET gene is hairpin structure, fluorescent dye ThT is in conjunction with RET gene, in hair
In the void area of card structure, causes the variation of fluorescent dye ThT fluorescence intensity, temperature is obvious responsed to；
When temperature increases the temperature more than its conformation changeWhen, thus it is speculated that hairpin structure, which is unlocked, becomes single-stranded structure, at this time ThT with
RET separation, the decline of ThT fluorescence intensity.
3. designing a kind of thermometer based on fluorescent dye ThT, RET gene using construction method described in as claimed in claim 1 or 22.
4. thermometer according to claim 3, it is characterised in that: control system pH value is adjusted to 7, it is each at 25-40 DEG C
Degree constant temperature incubation 30 minutes.
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| WO2015009988A1 (en) * | 2013-07-19 | 2015-01-22 | The Johns Hopkins University | Cardiac imaging methods and use in diagnosis and treatment of heart failure and related disease |
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