CN110082325A - Fluorescence sense system and its construction method and application - Google Patents
Fluorescence sense system and its construction method and application Download PDFInfo
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- CN110082325A CN110082325A CN201910349341.2A CN201910349341A CN110082325A CN 110082325 A CN110082325 A CN 110082325A CN 201910349341 A CN201910349341 A CN 201910349341A CN 110082325 A CN110082325 A CN 110082325A
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- adenosine
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6432—Quenching
Abstract
The present invention discloses a kind of fluorescence sense system and its construction method and application.Fluorescence of the present invention is the carbon nano-particle-aptamers-adenosine fluorescence sense system being mixed to get by carbon nano-particle, aptamers and adenosine.The present invention can occur deamination reaction with adenosine using adenosine deaminase and generate inosine, and it is adapted to physical efficiency and is specifically bound by force with adenosine progress high-affinity, but the principle not having an effect with inosine, the fluorescence sense system can be used in the detection to adenosine deaminase, have the advantages that high specificity, anti-interference good, high sensitivity, easy to operate.
Description
Technical field
The present invention relates to detection technique fields, especially for detecting the fluorescence sense system and its building of adenosine deaminase
Method and detection method.
Background technique
Adenosine deaminase (adenosine deaminase, ADA) is the important nucleic acid decomposition metabolic enzyme of human body, can be special
Property catalysis adenosine generate irreversible deamination reaction, generate inosine, be finally oxidized to uric acid and excrete.It
Be distributed widely in tissue, it is most with content in caecum, spleen, exist in fibrocyte, amniocyte and liver, kidney, lung,
In skeletal muscle.Blood rbc, leucocyte, lymphocyte, ADA content is 40-70 times in serum in granulocyte.In recent years,
ADA is taken seriously in the study of pathogenesis of the early diagnosis of some diseases, treatment and dysimmunity, ADA in body fluid
It measures more and more extensive applied to clinic.For example, ADA shortage is the one of the major reasons for causing severe immune deficiency disease；
ADA is excessive to cause lung cancer, breast cancer, colorectal cancer etc. again.Therefore, detection ADA has important clinical meaning.
Currently, the method for detection ADA has high performance liquid chromatography, colorimetric method etc..But most of method has operation multiple
The miscellaneous disadvantage low with sensitivity.
Summary of the invention
Based on this, it is necessary to for the technical problems such as complicated for operation existing for existing ADA detection technique, sensitivity is low,
A kind of new fluorescence sense system based on aptamers and carbon nano-particle is provided.
Aptamers (Aptamer) are with index concentration method (systematic evolution of ligands by
Exponential enrichment, SELEX) one kind for screening from random oligonucleotide library can be with target molecule spy
The oligonucleotide sequence that the opposite sex combines.The sequence can be RNA, ssDNA or dsDNA.The target molecule combined with aptamers can
To be protein and other, it can also be the small molecules such as dyestuff, drug, amino acid, or even virus, spore, complete
Cell etc. can also be used as the target molecule of aptamers.
Carbon nano-particle (carbon nanoparticles, CNPs) is a kind of carbon nanomaterial risen in recent years.Mesh
The preceding research based on carbon nano-particle performance, is concentrated mainly on the optical property of smaller size of carbon nano-particle (carbon quantum dot)
On, and the report about large-sized carbon nano-particle is less.Since carbon nano-particle has carbon material property and nanometer simultaneously
Dimensional effect, it is stronger with the dye molecule effect with planar structure, and can be waited by electronics transfer or energy transfer
Cheng Yinqi fluorescent quenching, therefore using carbon nano-particle as nanometer quencher, it will be provided for biological Molecular fluorimetric recognition potential
Platform.
Therefore, the present invention provides a kind of fluorescence sense system, is mixed to get by carbon nano-particle, aptamers and adenosine
Carbon nano-particle-aptamers-adenosine fluorescence sense system.
Further, the adaptation that the aptamers in the fluorescence sense system are FAM label, concentration is 5-50nM
Body；The average grain diameter of the carbon nano-particle is 20-70nm, and concentration is 30-75 μ g/mL；The concentration of the adenosine is 5-9mM.
Further, the concentration of the carbon nano-particle is 37.5 μ g/mL；The concentration of the adenosine is 5mM.
The present invention provides the construction method of above-mentioned fluorescence sense system, includes the following steps；
S1 prepares adaptation liquid solution, carbon nano-particle solution and adenosine solution respectively；
S2 is first added the carbon nano-particle solution reaction in the adaptation liquid solution, adds the adenosine solution
Hybrid reaction, the adaptation liquid solution: the carbon nano-solution: the volume ratio of the adenosine solution is 1:50:60-1:30:30,
It is eventually adding buffer, obtains carbon nano-particle-aptamers-adenosine fluorescence sense system.
The building principle of fluorescence sense system of the present invention is as follows: deamination reaction can occur with adenosine for adenosine deaminase, raw
At inosine.Existing there are one section of aptamers (ACC TGG GGG AGT ATT GCG GAG GAA GGT), can carry out with adenosine high
The combination of affinity and strong specificity, without having an effect with inosine.Based on above principle, the present invention is constructed to be received based on carbon
Rice grain-aptamers-adenosine fluorescence sense system realizes the detection to adenosine deaminase (detection process is as shown in Figure 1).Such as
When not having adenosine deaminase shown in Figure 1A, in system, adenosine is closely linked with FAM label aptamers, carbon nano-particle
FAM fluorescence cannot be quenched；And inosine is generated when there are when adenosine deaminase, adenosine deaminase can be reacted with adenosine in system,
In conjunction with inosine can not mark aptamers with FAM.At this point, carbon nano-particle and FAM label aptamers have pi-pi accumulation effect and
Electrostatic repulsion, FAM label aptamers are adsorbed by carbon nano-particle, and FAM fluorescence is quenched by carbon nano-particle, and system has low
Fluorescence background (as shown in Figure 1B).Based on this, the detection to adenosine deaminase is realized.
Further, the carbon nano-particle is the carbon ash being prepared according to the method for burning candle, through concentrated nitric acid acid
Change, be heated to reflux to obtain.Since carbon ash cannot be soluble in water well, in order to realize the present invention, spy is with the following method to carbon
Ash is handled: carbon ash is dissolved in DMF solution, it is found that it has dispersibility well, concentrated nitric acid stirring is then added,
45-65 DEG C of heated overnight at reflux, is cooled to room temperature, and is centrifuged at a high speed to obtain lower black solid particle, be repeated several times, removal
Nitric acid and DMF, the black solid particle obtained at this time (carbon nano-particle i.e. of the present invention) can be very good to be dispersed in water,
Building for subsequent fluorescence sense system.
Further, the reaction time that the carbon nano-particle solution is added in adaptation liquid solution is 1-5min,
It is preferred that 5min.
Further, the time that adenosine solution hybrid reaction is added is 1-5min, preferably 5min.
Further, the buffer is the Tris-HCl buffer that pH is 8.0.
The present invention provides application of the above-mentioned fluorescence sense system in the detection of adenosine deaminase.
Specifically, measurement fluorescence is strong by the way that sample to be tested to be added in fluorescence sense system after reaction 20-30min by the present invention
Degree contains adenosine deaminase in sample to be tested if fluorescence intensity reduces.According to the numerical value that fluorescence intensity reduces, can calculate
Adenosine deaminase content in sample to be tested out.
Further, preferably sample to be tested is added in fluorescence sense system after reacting 20min and carries out the survey of fluorescence intensity
It is fixed.
Compared with prior art, the invention has the following beneficial effects:
The present invention using adenosine deaminase can with adenosine occur deamination reaction generate inosine, and be adapted to physical efficiency and adenosine into
Row high-affinity is specifically bound by force, the principle not having an effect with inosine but, can be by the fluorescent quenching type fluorescence sense system
For in the detection to adenosine deaminase, having the advantages that high specificity, anti-interference good, high sensitivity, easy to operate.
Detailed description of the invention
Fig. 1 is for the building process of fluorescence sense system of the present invention and its to the corresponding schematic diagram of the fluorescence of adenosine deaminase；
Fig. 2 is the TEM figure and grain size distribution of the carbon nano-particle in fluorescence sense system of the present invention；
Fig. 3 is in adaptation liquid solution, and carbon nano-particle concentration changes the influence schematic diagram to fluorescence intensity；
Fig. 4 is the fluorescence intensity versus time curve figure in adaptation liquid solution, after carbon nano-particle addition；
Fig. 5 is influence schematic diagram of the adenosine concentration variation to fluorescence intensity；
Fig. 6 is the fluorescence intensity versus time curve figure after adenosine is added；
Fig. 7 is selection specificity comparison diagram of the fluorescence sense system of the present invention to adenosine deaminase；
Fig. 8 A and Fig. 8 B are in fluorescence sense system of the present invention, to system fluorescence when the adenosine deaminase of various concentration is added
Linear relationship chart between the influence schematic diagram and fluorescence intensity and adenosine deaminase concentration of intensity；
Fig. 9 is the dynamic curve diagram that the adenosine deaminase reaction of various concentration is added in fluorescence sense system of the present invention；
Figure 10 is to be taken off using mice serum as practical sample to be tested using fluorescence sense system practical measurement adenosine of the present invention
The correction graph of adnosine deaminase.
Specific embodiment
The present invention will be further described in detail in the following with reference to the drawings and specific embodiments.Unless otherwise instructed, this hair
Experimental material used in bright is commercially available commercially available material, and method used in the present invention is respective name in this field
Represented conventional method, all experiments of the present invention are all reacted at 25 DEG C.The present invention experiment used in water be
Milli-Q ultrapure water (conductivity is more than 18M Ω cm), other reagents used are that analysis is pure.
Embodiment 1
1. the preparation of carbon nano-particle
Carbon nano-particle (carbon nanoparticles, CNPs) is prepared by the method for burning candle.Carbon nanometer
The acidification of grain: it weighs the dry carbon ash of 3.0mg-5.0mg and puts in the round-bottomed flask of 25mL, straight condensation is connected on round-bottomed flask
Pipe, straight cold finger one balloon of upper termination (prevent reaction in nitric acid decompose generate gas formed air pressure and make condenser pipe with
Round-bottomed flask is detached from or explosion).The concentrated nitric acid of 2mL and the DMF of 2mL is added, it is small to be heated to reflux 11 in 45-65 DEG C of oil bath pan
When -12 hours, obtain reaction solution；Then reaction solution is poured into 5mL centrifuge tube and is centrifuged, is divided into after centrifugation two layers, upper layer is yellowish-brown
Color liquid, lower layer are black solids, add secondary water after removing supernatant liquid, mixing is centrifuged again removes supernatant liquid, so
Repeated multiple times (this purpose is as far as possible except remaining nitric acid in dereaction, in order to avoid interfere subsequent application) is to get the carbon of oxidation
Nanoparticles solution contains water-soluble carbon nano-particle in solution.Solid carbon nano-particle is obtained after freeze-drying.Experiment
In, 0.3mg carbon nano-particle is dispersed in well in 1mL high purity water, so that the concentration of carbon nano-particle stock solution is 0.3mg/
mL。
2. the preparation of aptamers stock solution
The aptamers (5 '-FAM-ACCTGGGGGAGTATTGCGGAGGAAGGT-3 ') for taking the FAM of 1OD to mark are centrifuged 5 points
Clock is dissolved in 366 μ L three times in water, and obtaining Stock concentrations is 10 μM.Solution is encased with masking foil finally, is kept in dark place in 4-
8℃。
3. the preparation of adenosine stock solution
360mg adenosine is weighed, 40mL Tris-HCl buffer (20mM, pH 8.0,100mM NaCl, 5mM are dissolved in
MgCl2), ultrasound 30 minutes, the concentration for obtaining stock solution is 34mM, and 4-8 DEG C saves backup.
4. the building of fluorescence sense system
The preparation of carbon nano-particle-aptamers-adenosine fluorescence sense system is pressed: the aptamers of 5 10 μM of μ L lay in liquor
The middle carbon nano-particle solution reaction that 250 μ L 0.3mg/mL are added adds the adenosine deposit liquor of 294 μ L 34mM, so
Tris-HCl buffer (pH 8.0) is added afterwards, is settled to 2mL, is uniformly mixed, it is glimmering to obtain carbon nano-particle-aptamers-adenosine
Light sensing system.Wherein, the final concentration of 25nM of aptamers, the final concentration of 37.5 μ g/mL of carbon nano-particle, the end of adenosine are dense
Degree is 5mM.
5. the detection of adenosine deaminase
Sample to be tested is added in aforementioned resulting fluorescence sense system and is uniformly mixed, is reacted after ten minutes, entirely at 25 DEG C
Portion's reaction solution is transferred to the quartz sample pool of 1cm light path, and fluorescence spectrum (λ is measured in F-2500 type Fluorescence Spectrometerex/em
=492/522nm).
Embodiment 2TEM characterization test
1 step 1 gained carbon nano-particle of Example carry out TEM phenetic analysis it is found that carbon nano-particle average grain diameter
Range is 20 between 70nm, and wherein most of sizes are 30-40nm or so, and size distribution is uniform, has good
Dispersed (as shown in Figure 2).
The optimization of 3 system construction condition of embodiment
1. dosage and the reaction time of carbon nano-particle
From figure 3, it can be seen that the fluorescence intensity of aptamers is stronger when not having carbon nano-particle.With carbon nanometer
The increase of grain dosage, fluorescence intensity is rapidly decreased to minimum and reaches a platform, when carbon nano-particle dosage reaches 37.5 μ g/mL
When, fluorescence intensity reduces general 43% (illustration in such as Fig. 3), this shows there is interaction between carbon nano-particle and aptamers,
The fluorescence of FAM can be quenched in carbon nano-particle.This may be because the electronics between carbon nano-particle and FAM label aptamers turns
Caused by shifting.
After carbon nano-particle is added, fluorescence intensity versus time curve is as shown in Figure 4.It is received the result shows that carbon is added
After rice grain, fluorescence intensity is rapidly minimized, and reaches a platform, probably needs 1 minute time.This explanation, carbon
Nano particle is a cracking process to the suction-operated of aptamers.In order to minimize fluorescence and reach a stable shape
State, preferentially selecting the reaction time of addition carbon nano-particle is 5 minutes.In addition, select the concentration of aptamers for 25nM in experiment,
The dosage of carbon nano-particle is 37.5 μ g/mL, and system is reacted in the Tris-HCl buffer solution of pH 8.0.
2. dosage and the reaction time of adenosine
Influence result of the variation of adenosine concentration to system fluorescence intensity is as shown in Figure 5.When not having adenosine, fluorescence is strong
It spends lower.With the increase of adenosine concentration, fluorescence intensity is gradually increased, and when adenosine concentration reaches 5mM, fluorescence intensity reaches most
(illustration in such as Fig. 5) by force.Finally, it is preferred that be 5mM with the optium concentration of adenosine being best experimental concentration.
As shown in fig. 6, the fluorescence intensity of system is rapidly increased to highest, and reaches a platform after adenosine is added, probably need
Want 1 minute time.In order to make the fluorescence of system be raised to highest and reach a stable state, adenosine system is added in preferential selection
Reaction time be 5 minutes.
4 photoluminescent property of embodiment and selection Journal of Sex Research
It is selectivity of the evaluation system of the present invention to ADA, the present embodiment compares its reacting with various common enzymes.It is surveyed
The species of examination include fibrin ferment, trypsase, esterase, nitroreductase, L- lactase dehydrogenase, glucose dehydrogenase, L- paddy ammonia
Acidohydrogenase.As shown in fig. 7, these enzymes almost do not have fluorescence response to system, but ADA but has apparent fluorescence to ring to system
It answers, illustrates that the system shows high selectivity to ADA.This is because caused by the specific reaction of adenosine and ADA, and it is other
Enzyme cannot interact with adenosine.
Influence of the ADA of various concentration to carbon nano-particle-aptamers-adenosine fluorescence sense system fluorescence property is as schemed
Shown in 8A, when not having ADA, system has strong fluorescence background, however after ADA is added, under identical condition, body
The fluorescence intensity of system weakens with the increase of ADA concentration, this is because ADA reacted with adenosine generate inosine, and inosine be adapted to
Body is not specifically bound, so as to cause the decrease of system fluorescence intensity.As can be seen from Figure 8B, when the concentration of ADA reaches
When 3.13U/mL, the fluorescence of system reaches a platform.In addition, being added not in only carbon nano-particle-aptamers solution
ADA with concentration is as control experiment.The result shows that the fluorescence intensity of ADA fore-and-aft architecture is added when not having adenosine in system
It does not change.This shows that adenosine plays very important effect during constructing secondary sensing system.Above-mentioned optimal
Under the conditions of obtain fluorescence intensity (F0-F)/F0With the linear relationship of ADA concentration.Linear equation is (F0-F)/F0=0.0823
[ADA] (U/mL)+0.0205 (γ=0.98), range of linearity 0.25-3.13U/mL.(3S/m, S are reagent skies to the detection limit of ADA
White standard deviation, n=11, m are the slopes in linear equation) it is 0.23U/mL.
The experiment of 5 enzyme kinetics of embodiment
The kinetic curve that experimental system is reacted with various concentration ADA.As shown in figure 9, the fluorescence intensity of system is with ADA
The increase of concentration and gradually decrease, the reaction rate of system is also faster, and reaction in 20 minutes or so reaches platform.In experiment, selection
It is used as optimum reacting time within 20 minutes.
6 interference experiment of embodiment
It is coexisted by Common materials in analysis carbon nano-particle-aptamers-adenosine system, 0.5U/mL ADA and serum
Solution tests influence of these substances to fluorescence system detection ADA.When the relative error that certain substance generates analysis system exists
When in the range of 10%, concentration is largest tolerable concentration.The results are shown in Table 1, it is shown that the good anti-interference of system.
Influence of the Common materials to fluorescence system detection ADA in 1 serum of table
The analysis of 7 actual sample of embodiment
Constructed fluorescence system is carried out into practical measurement adenosine deaminase, mice serum is as actual sample to be tested.Blood
It is different from buffer solution clearly, the complex matrices that the inside is contained may generate strong background fluorescence.Therefore, selection is diluting ten
Calibration curve is done in serum again, correction equation is (F0-F)/F0=[ADA] 0.0994 (U/mL)+0.061 (γ=0.98), line
Property range be 0.25-3.13U/mL.As shown in Figure 10, in addition, determining the content of adenosine deaminase in actual sample.Table 2 can
To see, when ADA concentration is low, yield good result.
The measurement of ADA in 2 mice serum sample of table
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
Limitations on the scope of the patent of the present invention therefore cannot be interpreted as.It should be pointed out that for those of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to guarantor of the invention
Protect range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Claims (10)
1. a kind of fluorescence sense system, which is characterized in that it is that the carbon being mixed to get by carbon nano-particle, aptamers and adenosine is received
Rice grain-aptamers-adenosine fluorescence sense system.
2. fluorescence sense system according to claim 1, which is characterized in that the adaptation in the fluorescence sense system
Body is the aptamers 5 '-FAM-ACCTGGGGGAGTATTGCGGAGGAAGGT-3 ' that concentration is 5-50nM；The carbon nano-particle
Average grain diameter be 20-70nm, concentration be 30-75 μ g/mL；The concentration of the adenosine is 5-9mM.
3. fluorescence sense system according to claim 2, which is characterized in that the concentration of the carbon nano-particle is 37.5 μ
g/mL；The concentration of the adenosine is 5mM.
4. the construction method of claims 1 or 2 or the 3 fluorescence sense systems, which is characterized in that include the following steps；
S1 prepares adaptation liquid solution, carbon nano-particle solution and adenosine solution respectively；
The carbon nano-particle solution reaction is first added in the adaptation liquid solution in S2, adds the adenosine solution mixing
Reaction, the adaptation liquid solution: the carbon nano-solution: the volume ratio of the adenosine solution is 1:50:60-1:30:30, finally
Buffer is added, obtains carbon nano-particle-aptamers-adenosine fluorescence sense system.
5. the construction method of fluorescence sense system according to claim 4, which is characterized in that the carbon nano-particle is basis
The carbon ash that the method for burning candle is prepared.
6. the construction method of fluorescence sense system according to claim 4, which is characterized in that described to add in adaptation liquid solution
The reaction time for entering the carbon nano-particle solution is 1-5min.
7. the construction method of fluorescence sense system according to claim 4, which is characterized in that the addition adenosine solution is mixed
The time for closing reaction is 1-5min.
8. the construction method of fluorescence sense system according to claim 4, which is characterized in that the buffer is that pH is
8.0 Tris-HCl buffer.
9. the application of claims 1 or 2 or the 3 fluorescence sense systems in the detection of adenosine deaminase.
10. the application of fluorescence sense system according to claim 9, which is characterized in that fluorescence sense is added in sample to be tested
Fluorescence intensity is measured after reacting 20-30min in system, if fluorescence intensity reduces, contains adenosine deaminase in sample to be tested；Root
According to fluorescence decreasing value, it can be deduced that the concrete content of adenosine deaminase.
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