CN107192697B - A kind of fluorescence sense method detecting exonuclease I - Google Patents
A kind of fluorescence sense method detecting exonuclease I Download PDFInfo
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Abstract
The invention discloses a kind of fluorescence sense methods for detecting exonuclease I (Exo I), feature is that specific step is as follows: (1) mixing cationic conjugated polymer (CCP) with complex of iridium, then Exo I buffer is added, obtains the fluorescent optical sensor system based on CCP Yu complex of iridium fluorescence resonance energy transfer (FRET) effect for detecting Exo I.Fluorescence intensity；(2) single stranded DNA (ssDNA) is added to the system in step (1), the variation of fluorescence intensity；(3) the Exo I of various concentration is added in step (2), the activity of Exo I in test sample, advantage be for the first time invented a good species specificity, high sensitivity, detection speed it is fast, as a result accurately and reliably analysis method for sensing be used for Exo I detection.
Description
Technical field
The present invention relates to biological sensors, pass more particularly, to a kind of fluorescence for detecting exonuclease I (Exo I)
Sensing method.
Background technique
Exonuclease (Exonucleases) is one kind sequentially catalytic degradation nucleosides since one of polynucleotide chain
The enzyme of acid.Exonuclease is divided into: exonuclease I (Exo I) and exonuclease III (Exo III).Exo I is a kind of
It is only capable of from the 3 ' of single stranded DNA (ssDNA) ends to 5 ' ends, according to base sequence, the phosphodiester bond between catalyzing hydrolysis base one by one,
The gradually enzyme of degradation ssDNA.After Exo I hydrolyzes ssDNA, final product is deoxyribonucleotide one by one.Exo I is to ssDNA
Base sequence do not require, without hydrolysis double-stranded DNA ability.Exonuclease biology it is many during
It plays an important role, such as DNA replication dna, recombination, reparation.Most important one function is exactly to maintain the intracorporal gene of biology prominent
Become probability, to maintain the stability of hereditary information.In recent years, the quantitative detection of Exo III is reported in succession, however Exo I
Research it is also seldom.
At present to the Activity determination of Exo I general lack of quantitative description because the active method of traditional detection Exo I be by
Digestion effect is observed in the DNA of standard and Exonucleolytic enzyme reaction, gel electrophoresis, and this method gives up to semi-quantitative results, and
And troublesome in poeration, poor reproducibility, the Enzyme activities under different condition can not be provided in real time.Therefore, develop a kind of quick, quasi-
Really, the sensitive active method of quantitative detection Exo I, will there is important value.
With luminous efficiency, high, biggish Stokes is displaced complex of iridium, luminescent color can pass through change ligand structure
It is adjusted and has become a hot topic of research the advantages that good photostability.According to reports, complex of iridium is in bio-sensing
In terms of device using more and more extensive.Cationic conjugated polymer (CCP) has as a kind of novel water soluble fluorescence molecule
The advantages that molar absorption coefficient is big, fluorescence quantum yield is high.Meanwhile conjugated polymer has the function of molecular wire, Neng Goushi
The amplification of existing fluorescence signal.Fluorescence resonance energy transfer (FRET) phenomenon can occur for complex of iridium and CCP.
The present invention is based on the FRET phenomenons of complex of iridium and CCP, construct a kind of simple, highly sensitive, quick analysis sensing
Method, is used for quantitative detection Exo I, process and its simple, provides a kind of very promising detection means for clinical application.Mesh
Before, based on the FRET phenomenon of complex of iridium and CCP the detection active fluorescent optical sensor of Exo I, there is not been reported.
Summary of the invention
Good, high sensitivity that technical problem to be solved by the invention is to provide a species specificity, detection speed is fast, result is quasi-
The really fluorescence sense method of reliable detection Exo I.
The technical scheme of the invention to solve the technical problem is: a kind of fluorescence sense method for detecting Exo I,
Specific step is as follows:
It (1) be 3~4 μM of CCP by 85~95 μ L concentration with 0.5~2 μ L concentration is that 0.5~2mM complex of iridium mixes, so
Afterwards be added respective volume 10 × Exo I buffer, obtain 100 μ L for detect Exo I based on CCP and complex of iridium FRET
The fluorescent optical sensor system of effect.Fluorescence intensity calculates the fluorescence intensity and complex of iridium (530nm) of CCP (415nm)
Fluorescence intensity ratio.
(2) ssDNA of 5~15 μM of 1~3 μ L of addition is calculated to the system in step (1), the variation of fluorescence intensity
The fluorescence intensity of CCP (415nm) and the fluorescence intensity ratio of complex of iridium (530nm).
(3) the Exo I of various concentration is added in step (2), and 10 × Exo I buffer of respective volume is then added,
25~35min is incubated at 30~40 DEG C, the variation of fluorescence intensity calculates the fluorescence intensity and complex of iridium of CCP (415nm)
The fluorescence intensity ratio of (530nm) obtains a series of corresponding fluorescence intensity ratio of Exo I of various concentrations, it is strong to establish fluorescence
Spend the quantitative relationship between ratio and Exo I concentration；It can detecte the activity of Exo I in sample according to this quantitative relationship.
Using the above-mentioned method based on the biological sensor of complex of iridium and CCP detection Exo I, fluorescence point is carried out
Slit width is arranged in analysis are as follows: 10nm, excitation wavelength are 380nm, and scanning voltage is 700V, detect CCP/ssDNA/ complex of iridium
System calculates the fluorescence intensity (I of CCP (415nm) to the fluorescence intensity of the Exo I of different activities415) and complex of iridium
Fluorescence intensity ratio (the I of (530nm)530)。
Inventive principle: the present invention is based on ssDNA interference CCP and complex of iridium between FRET developed it is a kind of it is unmarked,
Simple and easy sensor is used to detect the activity of Exo I.Since CCP is positively charged, lead between the complex of iridium with negative electricity
It crosses electrostatic interaction and FRET occurs, when ssDNA is added, the fluorescence intensity ratio of the two is reduced；In the presence of having Exo I, due to Exo
I hydrolyzes ssDNA, so that the fluorescence intensity ratio of the two increases.Exo I concentration is bigger, the fluorescence intensity ratio I of the two530/I415
It is bigger, it is in a linear relationship between fluorescence intensity ratio and the logarithm of Exo I concentration.
Compared with the prior art, the advantages of the present invention are as follows: the present invention constructs a kind of fluorescence sense side for detecting Exo I
Method.Firstly, interfering the FRET between CCP and complex of iridium using ssDNA.Secondly, being hydrolyzed in the presence of Exo I using Exo I
SsDNA, so that I530/I415Increase.Exo I concentration is bigger, the fluorescence intensity ratio I of the two530/I415It is bigger.Experimental result table
It is bright, it is in a linear relationship between fluorescence intensity ratio and the logarithm of Exo I concentration, realize the detection to Exo I.The advantage is that:
(1) highly sensitive.In 0.01-2U/mL concentration range, I530/I415Between value and the logarithm of Exo I concentration (C)
Good linear relationship, linear equation I is presented530/I415=1.29+0.45 × 1gC (U/mL), linearly dependent coefficient R2=
0.9926, indicate that curve has good degree of fitting, detection is limited to 0.01U/mL.These results indicate that the sensor is to Exo I
Detection have lower detection limit and higher sensitivity.
(2) high specific.Exonuclease (Exo III), limitation restriction endonuclease (Hind III), exonuclease (λ-
Exo), limitation restriction endonuclease (EcoR I) is used as control substance of plant drug, is 0.1U/mL, detects noiseless.
(3) result is accurate.In 10% urine and 10% serum environment, the rate of recovery is between 90%~110%.
(4) preparation with detection method reagent dosage it is few, detect speed it is fast, at low cost.The present invention need to only consume a small amount of material
The highly sensitive detection to Exo I is achieved that with reagent.
In conclusion the present invention be interfered based on ssDNA the FRET between CCP and complex of iridium construct it is a kind of it is unmarked,
Simple and easy sensor is used to detect the activity of Exo I, have high sensitivity, selectivity be good, easy to operate, analysis quickly,
The advantages that easily operated, may be implemented the detection of low concentration Exo I, have a good application prospect.
Detailed description of the invention
Fig. 1 is the feasibility Experiment figure of inventive sensor；
Fig. 2 is fluorescence response figure of the inventive sensor to various concentration Exo I；
Fig. 3 is calibration graph of the inventive sensor to various concentration Exo I；
Fig. 4 is selective lab diagram of the inventive sensor to Exo I；
Fig. 5 is interference--free experiments figure of the inventive sensor to Exo I.
Specific embodiment
The present invention will be described in further detail below with reference to the embodiments of the drawings.
One, specific embodiment
Embodiment 1
The technical scheme of the invention to solve the technical problem is: a kind of fluorescence sense method for detecting Exo I,
Specific step is as follows:
(1) be 3.28 μM of CCP by 90 μ L concentration with 1 μ L concentration it is that 1mM complex of iridium mixes, respective volume is then added
10 × Exo I buffer, obtain 100 μ L for detecting the fluorescence sense based on CCP Yu complex of iridium FRET effect of Exo I
Body system.Fluorescence intensity calculates the fluorescence intensity of CCP (415nm) and the fluorescence intensity ratio of complex of iridium (530nm).
(2) ssDNA to the system in step (1), the variation of fluorescence intensity, calculating CCP of 10 μM of 2 μ L is added
The fluorescence intensity of (415nm) and the fluorescence intensity ratio of complex of iridium (530nm).
(3) the Exo I of various concentration is added in step (2), then 10 × Exo I buffer of respective volume, and 37 DEG C
Lower incubation 30min, the variation of fluorescence intensity calculate the glimmering of fluorescence intensity and the complex of iridium (530nm) of CCP (415nm)
Intensity ratio obtains a series of corresponding fluorescence intensity ratio of Exo I of various concentrations, establishes fluorescence intensity ratio and Exo
Quantitative relationship between I concentration；It can detecte the activity of Exo I in sample according to this quantitative relationship.
Embodiment 2
The technical scheme of the invention to solve the technical problem is: a kind of fluorescence sense method for detecting Exo I,
Specific step is as follows:
(1) be 4 μM of CCP by 85 μ L concentration with 2 μ L concentration it is that 1mM complex of iridium mixes, is then added the 10 of respective volume
× Exo I buffer obtains 100 μ L for detecting the fluorescence sense body based on CCP Yu complex of iridium FRET effect of Exo I
System.Fluorescence intensity calculates the fluorescence intensity of CCP (415nm) and the fluorescence intensity ratio of complex of iridium (530nm).
(2) ssDNA to the system in step (1), the variation of fluorescence intensity, calculating CCP of 15 μM of 1 μ L is added
The fluorescence intensity of (415nm) and the fluorescence intensity ratio of complex of iridium (530nm).
(3) the Exo I of various concentration is added in step (2), then 10 × Exo I buffer of respective volume, and 34 DEG C
Lower incubation 30min, the variation of fluorescence intensity calculate the glimmering of fluorescence intensity and the complex of iridium (530nm) of CCP (415nm)
Intensity ratio obtains a series of corresponding fluorescence intensity ratio of Exo I of various concentrations, establishes fluorescence intensity ratio and Exo
Quantitative relationship between I concentration；It can detecte the activity of Exo I in sample according to this quantitative relationship.
Embodiment 3
The technical scheme of the invention to solve the technical problem is: a kind of fluorescence sense method for detecting Exo I,
Specific step is as follows:
(1) be 3 μM of CCP by 95 μ L concentration with 0.5 μ L concentration it is that 2mM complex of iridium mixes, respective volume is then added
10 × Exo I buffer obtains 100 μ L for detecting the fluorescent optical sensor based on CCP Yu complex of iridium FRET effect of Exo I
System.Fluorescence intensity calculates the fluorescence intensity of CCP (415nm) and the fluorescence intensity ratio of complex of iridium (530nm).
(2) ssDNA to the system in step (1), the variation of fluorescence intensity, calculating CCP of 5 μM of 3 μ L is added
The fluorescence intensity of (415nm) and the fluorescence intensity ratio of complex of iridium (530nm).
(3) the Exo I of various concentration is added in step (2), then 10 × Exo I buffer of respective volume, and 32 DEG C
Lower incubation 30min, the variation of fluorescence intensity calculate the glimmering of fluorescence intensity and the complex of iridium (530nm) of CCP (415nm)
Intensity ratio obtains a series of corresponding fluorescence intensity ratio of Exo I of various concentrations, establishes fluorescence intensity ratio and Exo
Quantitative relationship between I concentration；It can detecte the activity of Exo I in sample according to this quantitative relationship.
Two, feasibility Experiment
Measure the fluorescence intensity of CCP/ complex of iridium system；The ssDNA composition CCP/ssDNA/ iridium that 66 bases are added is matched
Objects system is closed, its fluorescence intensity is measured；It is added Exo I (10U/mL, 1 μ L), cultivates 30min at 37 DEG C, measure the fluorescence of system
Intensity.In addition Exo I (10U/mL, 1 μ L) is added in the only system of CCP/ complex of iridium, as control experiment.Together
Shi Liyong chemiluminescence fluorescence imaging system obtains the fluorescent image (specific implementation example 1) of each solution.When in system there are CCP and
Efficient FRET occurs when complex of iridium；After ssDNA is added, FRET efficiency is strongly reduced；Due to being hydrolyzed in the presence of Exo I
Fluorescence intensity at ssDNA, 530nm enhances, and FRET (such as Fig. 1) occurs again for the CCP and complex of iridium in system.So we
The fluorescent optical sensor based on CCP and complex of iridium FRET effect of design can detecte the activity of Exo I, and can quantitative detection
The concentration of Exo I has certain application potential.
Three, Exo I detection application
1, the method for the biological sensor detection Exo I of above-mentioned specific embodiment 1 preparation is utilized
Using fluorescence analysis, slit width is set are as follows: 10nm, excitation wavelength are 380nm, and scanning voltage is 700V, detection
The fluorescence intensity ratio of the fluorescence intensity of CCP (415nm) and complex of iridium (530nm) in CCP/ssDNA/ complex of iridium system
I530/I415To the quantitative relationship of various concentration Exo I, according to quantitative relationship between the two, Exo I in sample to be tested is determined
Content (specific implementation example 1).Fluorescence intensity of the Fig. 2 at 530nm enhances with the increase of Exo I concentration, this means that
Efficient FRET occurs again for CCP and complex of iridium, therefore FRET efficiency increases.
2, sensitivity test
Using fluorescence analysis, slit width is set are as follows: 10nm, excitation wavelength are 380nm, and scanning voltage is 700V, above-mentioned
The fluorescence intensity ratio I of CCP/ssDNA/ complex of iridium system prepared by specific embodiment 1530/I415, the range of Exo I concentration
For 0~2U/mL (specific implementation example 1).Test result explanation, as shown in figure 3, increase of the explanation with Exo I concentration, I530/
I415It is more obvious；Sensor I530/I415Linearly related equation to the logarithm of Exo I concentration is I530/I415=1.29+0.45 ×
IgC (U/mL), R2=0.9926, the range of linearity is 0.01~2.0U/mL, learns that detection is limited to 0.01U/ according to S/N calculating
mL.Illustrate that sensor can realize highly sensitive detection to Exo I.
3, specificity experiments
Selectivity experiment is as shown in Figures 4 and 5 with interference--free experiments, and wherein the concentration of Exo I and other enzymes is 0.1U/
ML, other used enzymes are as follows: exonuclease (Exo III), limitation restriction endonuclease (Hind III), exonuclease (λ-
Exo), restriction endonuclease (EcoR I) is limited.
(1) selectivity experiment
Using fluorescence analysis, slit width is set are as follows: 10nm, excitation wavelength are 380nm, and scanning voltage is 700V, above-mentioned
Exonuclease (the Exo that CCP/ssDNA/ complex of iridium system difference detectable concentration prepared by specific embodiment 1 is 0.1U/mL
III), restriction endonuclease (Hind III), exonuclease (λ-Exo), limitation restriction endonuclease (EcoR I) are limited.Such as Fig. 4, with Exo I
Comparison, sensor is very small to the response of other enzymes, substantially close to blank signal, illustrates that sensor has the detection of Exo I
Selectivity well.
(2) interference--free experiments,
Using fluorescence analysis, slit width is set are as follows: 10nm, excitation wavelength are 380nm, and scanning voltage is 700V, above-mentioned
CCP/ssDNA/ complex of iridium system prepared by specific embodiment 1, it is 0.1U/mL that detectable concentration is distinguished in the presence of 0.1U/mL
Exonuclease (Exo III), limitation restriction endonuclease (Hind III), exonuclease (λ-Exo), limitation restriction endonuclease (EcoR
I), comparing sensor, to four systems and only the fluorescence response in the presence of Exo I, such as Fig. 5 observe I530/I415Size with
I in the presence of only Exo I530/I415Substantially there is no difference, illustrate that sensor realizes the specific detection to Exo I.
Certainly, above description is not limitation of the present invention, and the present invention is also not limited to the example above.The art
The variations, modifications, additions or substitutions that those of ordinary skill makes within the essential scope of the present invention also should belong to protection of the present invention
Range.
Claims (2)
1. a kind of fluorescence sense method for detecting exonuclease I-Exo I, the specific steps are as follows:
It (1) is 3~4 μM of cation conjugated polymer CCP by 85~95 μ L concentration and 0.5~2 μ L concentration is that 0.5~2mM iridium is matched
Close object mixing, then be added respective volume 10 × Exo I buffer, obtain 100 μ L for detect Exo I based on cation
The fluorescent optical sensor system of conjugated polymer CCP and complex of iridium fluorescence resonance energy transfer FRET effect；Fluorescence intensity,
Calculate CCP 415nm fluorescence intensity and complex of iridium 530nm fluorescence intensity ratio；
(2) ssDNA to the system in step (1), the variation of fluorescence intensity, calculating CCP of 5~15 μM of 1~3 μ L is added
415nm fluorescence intensity and complex of iridium 530nm fluorescence intensity ratio；
(3) the Exo I of various concentration is added in step (2), then 10 × Exo I buffer of respective volume, and 30~40 DEG C
25~35min of lower incubation, the variation of fluorescence intensity calculate CCP in the fluorescence intensity and complex of iridium of 415nm in 530nm
Fluorescence intensity ratio, obtain a series of corresponding fluorescence intensity ratio I of Exo I of various concentrations530/I415, it is strong to establish fluorescence
Spend ratio I530/I415With the quantitative relationship between Exo I concentration；It can detecte Exo I in sample according to this quantitative relationship
Activity.
2. fluorescence sense method according to claim 1, it is characterised in that: slit width are as follows: 10nm, excitation wavelength are
380nm, scanning voltage are 700V, fluorescence intensity I of the CCP in 415nm in detection CCP/ssDNA/ complex of iridium system415With iridium
Fluorescence intensity ratio I of the complex in 530nm530/I415To the quantitative relationship of various concentration Exo I.
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