CN107703112A - The proportionality fluorescent method of fluorescent label DNA based on carbon quantum dot quenching detects liquaemin - Google Patents
The proportionality fluorescent method of fluorescent label DNA based on carbon quantum dot quenching detects liquaemin Download PDFInfo
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- CN107703112A CN107703112A CN201710925956.6A CN201710925956A CN107703112A CN 107703112 A CN107703112 A CN 107703112A CN 201710925956 A CN201710925956 A CN 201710925956A CN 107703112 A CN107703112 A CN 107703112A
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- liquaemin
- quantum dot
- carbon quantum
- fam
- cqds
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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
-
- 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"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
Abstract
The present invention discloses a kind of proportional-type fluorescent method of the fluorescent label DNA based on carbon quantum dot quenching and detects liquaemin.It is characterized in that liquaemin can recover by the fluorescence of the FAM marker DNAs of carbon quantum dot quenching, and as the rise of concentration of heparin, FAM recovery increase therewith, while the fluorescence intensity holding stabilization that carbon quantum dot is original, accordingly, the Ratio-type fluorescence new method of detection liquaemin is established.Liquaemin detection method step of the present invention is simple, and liquaemin to DNA fluorescence recovers that good linear correlation is presented, and test limit is relatively low, reaches 9.04 ng/mL.The liquaemin recovery of standard addition of actual rabbit anteserum sample is in the range of 95.47 111.95%.It is close with standard curve to simulate the matched curve of the heparin sodium determination of serum, therefore, result of the blood serum sample without interference with detection.Compared with other liquaemin detection methods, this method has the characteristics of detection performance is excellent, easy to operate, and new method choice is provided for the detection of clinical liquaemin.
Description
Technical field
The present invention relates to the application field of carbon nanomaterial, in particular it relates to a kind of based on carbon quantum dot quenching FAM marks
SsDNA proportionality fluorescent method detection liquaemin.
Background technology
（1）Heparin (Heparin, referred to as Hep) is by gucosamine, L- idose aldehyde glycosides, N-Acetyl-D-glucosamine and D-
The glutinous Alginic Sodium Diester that glucuronic acid etc. alternately forms, Relative average molecular weight is 15 KD, there is strong acidity, simultaneous with
Very strong negative charge.Liquaemin can make blood coagulation become slow or mitigate, and be a kind of highly effective anticoagulation medicine.
In hospital, heparin can be used for preventing and treating thrombus, and be a kind of conventional anti-freezing for the patient for needing to carry out haemodialysis
Blood medicine.But can all have different optimum dosage for different diseases, heparin, this is due to the main secondary work of heparin
With being that can cause hematostaxis and thrombopenia, these side effects are serious complication during heparin therapy, are given
Patient brings great pain, it is also possible to cause medical dispute, therefore clinically, it is necessary to usage amount and patient to heparin
Internal heparin content is monitored, and can so prevent the adverse events such as bleeding or thrombus, and can is effectively instructed and adjusted
Clinical rational drug use.
（2）The method of measure heparin has a lot, mainly includes biological method and some chemical methodes.Examined in clinical treatment
Detected in disconnected frequently with the method for biology, to preventing various complication caused by the improper use of heparin from playing an important role.
But because bion is larger, monitoring restricted application of the biological method to heparin, certain limitation be present.Heparin monitors
Chemical method have photometry, high performance liquid chromatography, Resonance scattering, capillary electrophoresis, electrochemical sensor, modification electricity
Pole method etc..Wherein XRF is widely used in various lifes due to its higher degree of accuracy and sensitivity and operability
The monitoring of compound matter and medicine.It is reported that golden nanometer particle can be used for the measure of heparin, but still have not seen on ratio
Type fluorescence analysis method determines the report of heparin.
（3）Carbon quantum dot（CQDs）It is widely studied by scientist with special physics and chemical property.Normal conditions
Under, the existing big pi bond of carbon quantum dot, and possess a series of new optical properties.The biofacies showed by carbon quantum dot
The performances such as capacitive, hypotoxicity, chemical inertness, stability, luminescence generated by light so that carbon quantum dot is in bio-sensing imaging, phototube
Part etc. has broad application prospects.Find that the carbon quantum dot of specific preparation can be quenched according to the newest experiment in this laboratory
The characteristics of ssDNA of the fluorochrome labels such as FAM fluorescence signal, the present invention utilize liquaemin and the FAM of carbon quantum dot quenching
The ssDNA systems effect of mark, it is found that liquaemin can recover the FAM fluorescence being originally quenched, and carbon quantum dot itself is glimmering
Luminous intensity keeps stable.Both emission wavelength significant differences, signal ratio sensitive change with the change of concentration of heparin,
Ratio-type fluorescence analysis new method measure liquaemin is established based on this.
The content of the invention
（1）In view of this, the purpose of the present invention is the ssDNA for being quenched FAM by adding liquaemin and carbon quantum dot and marking
System acts on, and establishes proportionality fluorescent method detection liquaemin, this method is simple to operate, high sensitivity.
（2）To achieve these goals, the invention provides a kind of ratio of the fluorescent label DNA based on carbon quantum dot quenching
The method of rate type fluoroscopic examination liquaemin, it is characterised in that comprise the following steps：The liquaemin buffering for configuring various concentrations is molten
Liquid, carbon quantum dot and FAM marker DNAs are added, detect the fluorescence of carbon quantum dot and FAM marker DNAs respectively.Wherein, reagent configures
Simple to operate, reaction condition is steerable.
（3）The reagent room temperature lucifuge of preparation is reacted 10 minutes, sepectrophotofluorometer is moved into, respectively in 345 nm and 488
Nm wavelength excites, and reads 420 nm and 520 nm fluorescence intensity respectively.Reaction, condition determination are simple.
（4）The reaction system of platform liquaemin maximum fluorescence recovery efficiency is quenched to obtain measure based on CQDs, as excellent
Choosing, FAM marker DNAs are to the fluorescence change before and after adding liquaemin under the conditions of detecting various concentrations CQDs.
（5）The detection method of time dynamics is：Sample is prepared rapidly, starts timing after mixing, examines at regular intervals
First order fluorescence intensity is surveyed, in early days every detection in one minute once, the later stage can accordingly extend the time, until detection 1 hour, investigate inspection
Survey the time needed for liquaemin.
（6）To embody the generality of reaction system, the different length of FAM marks and the probe A of sequence are selected10（5’-FAM-
AAAAAAAAAA-3’）, A20, A40Tested instead of the DNA of original base sequence.
（7）For above-mentioned prior art, it is an object of the invention to provide a kind of easy to operate, high sensitivity, can effectively survey
Obtain the new application field of the carbon quantum dot of liquaemin.
（8）To achieve these goals, the invention provides a kind of quantitative detecting method of liquaemin, fluorescence spectrophotometer is passed through
Photometry carries out the detection of liquaemin, it is characterised in that solvent in the fluorescence spectrophotometry by cushioning liquid,
CQDs, FAM-ssDNA and liquaemin mix.
（9）The invention has the advantages that the present invention is reacted graphene quantum dot and the FAM ssDNA marked, carbon
Quantum dot can be quenched DNA fluorescence, and quenching efficiency reaches 85%, and DNA fluorescence can be recovered by being subsequently added into liquaemin, and carbon quantum
The fluorescence intensity that point keeps relative stability in whole process.By adding heparin sodium determination FAM marker DNA fluorescence intensities
Intensity of variation, establish the Ratio-type fluoroscopic examination side of the carbon quantum dot of the change of FAM marker DNAs fluorescence signal and stable fluorescence intensity
Method detects liquaemin, obtains good effect, and easy to operate, high sensitivity, the minimum detectability of liquaemin can reach
9.04 ng/mL。
（10）Other features and advantages of the present invention will be described in detail in subsequent specific embodiment part.
Brief description of the drawings
Accompanying drawing is for providing a further understanding of the present invention, and a part for constitution instruction, with following tool
Body embodiment is used to explain the present invention together, but is not construed as limiting the invention.In the accompanying drawings：
Fig. 1 is the synchronous scanning that CQDs recovers to FAM-ssDNA Quenching of fluorescences effect and liquaemin to FAM-ssDNA fluorescence
Fluorescence spectra.
Fig. 2 is CQDs transmission electron microscope photo.
Recovery relationship between efficiency figure of the liquaemin to ssDNA fluorescence when Fig. 3 is various concentrations CQDs.
Fig. 4 is that CQDs quenching FAM mark ssDNA and liquaemin recover CQDs quenchings FAM and mark ssDNA fluorescence strong
Spend change kinetics graph of a relation.
Fig. 5 is the comparison diagram of the recovery efficiency for the fluorescence that CQDs is quenched FAM-ssDNA systems under different interfering materials.
Fig. 6 is the comparison diagram of quenching and recoveries of the CQDs to the FAM different probes marked.
Fig. 7 is CQDss and ssDNA of the CQDs-ssDNA after the liquaemin of various concentrations is added fluorescence intensity collection of illustrative plates.
Fig. 8 is the graph of a relation between the fluorescence intensity ratio of ssDNA and CQDs in PBS solution and concentration of heparin.
Fig. 9 is the linear relationship chart between the fluorescence intensity ratio of ssDNA and CQDs in PBS solution and concentration of heparin.
Figure 10 is the linear pass between ssDNA and CQDs fluorescence intensities ratio and concentration of heparin in PBS and simulation serum
System compares figure.
Embodiment
（1）With reference to embodiment and accompanying drawing, the present invention is described in detail.It should be appreciated that this place
The embodiment of description is merely to illustrate and explain the present invention, and is not intended to limit the invention.
（2）Embodiment（Feasibility analysis）
The sample of the feasibility Experiment of the present embodiment is made by following steps：
Configure following solution：Solution 1 is CQDs；Solution 2 is FAM-ssDNA；Solution 3 is CQDs+FAM-ssDNA；Solution 4 is
CQDs, FAM-ssDNA and liquaemin.The wherein nM of 6.25 μ g/mL, FAM-ssDNA concentration of CQDs concentration 50, concentration of heparin
2.0 μ g/mL, three kinds of component solutions are all matched somebody with somebody with phosphate buffer, the cushioning liquid of the embodiment of the present invention（Buffer molten
Liquid is by Na2HPO4﹒ 12H2O、NaH2PO4﹒ 2H2O, NaCl is formed）Concentration be 10 mM, and the pH of cushioning liquid is 7.4.It is described
Carbon quantum dot（CQDs）, it is anti-by adding 10.0 mL polyethylene polyamines step by step after 2.0 g citric acids and the melting of 0.6 g glutathione
Answer 1 hour and be made.FAM-ssDNA refers to 6- Fluoresceincarboxylic acid labeled ssdnas, 5 '-FAM-AAAAAAAAAA-3 '.
（3）Fluoroscopic examination is synchronized to solution made from the present embodiment, it is 50 to set detection parameters Δ λ, scanning 200
Nm to 600 nm fluorescence spectrum, CQDs (420 nm), FAM-ssDNA are read respectively（520 nm）.Fig. 1 shows independent CQDs
It will not be interfered with each other with FAM-ssDNA fluorescence spectrum, when both mix and added before and after liquaemin, CQDs fluorescence is all the time not
Become, and DNA present quenching and recover two kinds of results, and recover efficiency can be with change in concentration.Illustrate that proportional-type can be established glimmering
Light detection method.
（4）Fig. 2 is the transmission electron microscope picture of CQDs in the present embodiment, as shown in Fig. 2 CQDs even particle distributions, size are
2.6 nm。
（5）Recovery relationship between efficiency figure of the liquaemin to ssDNA fluorescence when Fig. 3 is various concentrations CQDs, as shown in figure 3,
6.25 μ g/mL are optium concentration.
（6）Fig. 4 is the result for the kinetics that CQDs-ssDNA and CQDs-ssDNA adds liquaemin, such as Fig. 4 institutes
Show, the fluorescence intensity of mixture after 10 minutes keeps stable.
（7）Fig. 5 is the result of the recovery efficiency of FAM-ssDNA fluorescence under different interfering materials, as shown in figure 5, heparin
The recovery effects of sodium are best, illustrate that the detection method specificity of the present invention is good.
（8）Fig. 6 is that quenchings and liquaemin of the CQDs to the FAM different probes marked recover fluorescence intensity collection of illustrative plates, such as Fig. 6 institutes
Show, A10, A20, A40, and the present embodiment probe have quenching and recover result.
（9）The invention provides a kind of detection method of liquaemin, and the inspection of liquaemin is carried out by fluorescence spectrophotometry
Survey, wherein, the solution in the fluorescence spectrophotometry passes through cushioning liquid, FAM-ssDNA, graphene quantum dot and liquaemin
Mix.
1. 10 μ L CQDs solution, 10 μ L FAM-ssDNA and a series of various concentrations liquaemins of 10 μ L are accurately measured,
Holding cumulative volume is 200 μ L, and resulting solution is placed in sepectrophotofluorometer by lucifuge after reacting 10 min.Wherein, it is all
Component solution is all matched somebody with somebody with phosphate buffer, the nM of 6.25 μ g/mL, FAM-ssDNA concentration of CQDs concentration 50.
2. under the conditions of the nm of excitation wavelength 488, fluorescence intensity level at the nm of launch wavelength 520 is read, according to fluorescence intensity
Data can draw known FAM-ssDNA fluorescence intensity level, draw standard curve.
3. in a kind of preferred embodiment provided by the invention, the content of each component is specially in the solution：
CQDs concentration is 6.25 μ g/mL, cushioning liquid（Cushioning liquid is by Na2HPO4﹒ 12H2O、NaH2PO4﹒ 2H2O, NaCl is formed）
Concentration be 10 mM, and the pH of the cushioning liquid is 7.4.Corresponding concentration of heparin is 0 to curve in Fig. 7 from bottom to top
µg/mL、0.01 µg/mL、0.05 µg/mL、0.1 µg/mL、0.5 µg/mL、1.0 µg/mL、1.5 µg/mL、2.0 µg/
ML, 2.5 μ g/mL, 3.0 μ g/mL, 5.0 μ g/mL, 10 μ g/mL, it can thus be seen that with the increase of concentration of heparin,
DNA fluorescence intensity gradually increases, stable more than holding after 3.0 μ g/mL.Ordinate is DNA fluorescence and CQDs fluorescence in Fig. 8
Ratio value, with the increase of concentration of heparin, DNA fluorescence intensity gradually increases, more than keeping stable after 3.0 μ g/mL,
As seen in Figure 9, in 0-2 μ g/mL, its concentration and ssDNA and CQDs fluorescence ratio have concentration of heparin to be measured
Good linear relationship, linear equation are：F/FCQDs=0.20791+0.52224X, and R2For 0.99796, F and FCQDsRespectively
SsDNA and the fluorescent value of carbon quantum dot after addition liquaemin；X is concentration of heparin minimum detection limit（LOD）=9.04 ng/mL
（S/N=3）.Figure 10 is PBS and simulates the linear ratio in serum between ssDNA and CQDs fluorescence intensities ratio and concentration of heparin
Compared with figure, two lines similar temperament, illustrate to simulate detection of the serum without interference with liquaemin.
4. the present invention investigates the reappearance of the DNA measure different activities concentration heparin sodium based on CQDs quenching fluorescence labelings,
Under the condition determination of step 3., repeat, independently determine 0.01 μ g/mL, 0.1 μ g/mL respectively six times, the RSD difference of measure
For 4.23% and 4.76%.
5. above example is only the preferred embodiments of the present invention, and not all.Based on the embodiment in embodiment,
Those skilled in the art obtain other embodiments on the premise of creative work is not made, and belong to the protection of the present invention
Scope.
Claims (10)
1. a kind of method of the Ratio-type fluoroscopic examination liquaemin of the fluorescent label DNA based on carbon quantum dot quenching, its feature exist
In comprising the following steps：The cushioning liquid containing various concentrations liquaemin is configured, adds carbon quantum dot and fluorescent label DNA, point
Not Jian Ce carbon quantum dot and DNA fluorescence.
2. the method as described in claim 1, it is characterised in that the DNA is 5 ' end mark FAM single stranded DNA（FAM-
ssDNA）,-FAM-TCA ACA TCA GTC TGA TAA GCT the A-3 ' of sequence 5 '.
3. method as claimed in claim 1 or 2, it is characterised in that the carbon quantum dot（CQDs）, by 2.0 g citric acids and
10.0 mL polyethylene polyamine reactions are added after the melting of 0.6 g glutathione step by step to be made within 1 hour.
4. method as claimed in claim 3, it is characterised in that the described nm of CQDs maximum excitations wavelength 345.
5. the method as described in claim 1-4 is any, it is characterised in that to CQDs concentration in 3.125 ~ 25 μ g/mL and instead
Optimized between seasonable in 0 ~ 60 minute scope.
6. method as claimed in claim 5, it is characterised in that select optimal CQDs concentration as 6.25 μ g/mL.
7. the method as described in claim 1-5, it is characterised in that after adding liquaemin and carbon quantum dot and fluorescent label DNA
Solution mixes room temperature avoid light place and reacts 10 min, then carries out fluoroscopic examination.
8. the method as described in claim 1-7, it is characterised in that cushioning liquid PBS, buffer concentration are 10 mM, and institute
PH of buffer is stated as 7.4.
A kind of 9. detection method of liquaemin, by fluorescence spectrophotometry to FAM- before and after addition various concentrations liquaemin
SsDNA fluorescence is detected, it is characterised in that the solution used in the fluorescence spectrophotometry by cushioning liquid,
CQDs, FAM-ssDNA and liquaemin mix.
10. detection method as claimed in claim 9, it is characterised in that the absorption drawn by the fluorescence spectrophotometry
Curve of spectrum equation is：Y=0.20791+0.52224X, R2=0.99796, Y F/FCQD, F and FCQDsRespectively add liquaemin
SsDNA and the fluorescent value of carbon quantum dot afterwards；X is concentration of heparin, minimum detection limit (LOD)=9.04 ng/mL；In the solution
The content of each component is：The concentration that CQDs concentration is 6.25 μ g/mL, FAM-ssDNA is 50 nM.
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| CN109813666A (en) * | 2019-02-12 | 2019-05-28 | 福建医科大学 | Trypsase detection kit based on pro- platinum nanocell simulations oxidizing ferment |
| CN110155992A (en) * | 2019-06-14 | 2019-08-23 | 福州大学 | A kind of preparation method of the sulfur and nitrogen co-doped graphene quantum dot electrolyte suitable for supercapacitor |
| CN111321205A (en) * | 2020-03-11 | 2020-06-23 | 昆明理工大学 | MiRNA detection method |
| CN111705112A (en) * | 2020-05-08 | 2020-09-25 | 江苏大学 | Mercury ion fluorescence detection method based on silicon quantum dots, fluorescein labeled DNA and shear enzyme |
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| CN111705112A (en) * | 2020-05-08 | 2020-09-25 | 江苏大学 | Mercury ion fluorescence detection method based on silicon quantum dots, fluorescein labeled DNA and shear enzyme |
| CN111705112B (en) * | 2020-05-08 | 2023-07-18 | 江苏大学 | Fluorescent detection method for mercury ions based on DNA marked by silicon quantum dots and fluorescein and shearing enzyme |
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