CN105717081A - Detection solution containing DNA fragments and organic dyestuff and application thereof - Google Patents
Detection solution containing DNA fragments and organic dyestuff and application thereof Download PDFInfo
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- ZCFFYALKHPIRKJ-UHFFFAOYSA-N 3-[18-(2-carboxylatoethyl)-8,13-bis(ethenyl)-3,7,12,17-tetramethyl-22,23-dihydroporphyrin-21,24-diium-2-yl]propanoate Chemical compound N1C(C=C2C(=C(C)C(=CC=3C(C)=C(CCC(O)=O)C(N=3)=C3)N2)C=C)=C(C)C(C=C)=C1C=C1C(C)=C(CCC(O)=O)C3=N1 ZCFFYALKHPIRKJ-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- 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
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
Abstract
The invention discloses a detection solution containing DNA fragments and organic dyestuff. The detection solution comprises the single-stranded DNA rich in guanine, sodions and the small organic molecule dyestuff adopted as output molecules. The invention further discloses a detection method for K+ inside a biological body on the basis of the DNA technology, fluorescent signals output through the DNA technology have the good linear relation for the content of K+ in a system to be tested within the certain concentration range, and therefore K+ high-sensitivity detection is achieved. The detection system is hardly disturbed by other common physiological activity small molecules and other metal ions inside a biological system, and therefore K+ detection has the high selectivity. According to the method, an instrument is simple, operation is easy and convenient, the cost is low, the sample needed amount is small, continuous and rapid detection of K+ inside the biological body can be achieved, and therefore the high practical application value is achieved.
Description
Technical field
The invention belongs to field of bioanalysis, be specifically related to a kind of detection solution containing DNA fragmentation and organic dyestuff and application thereof.
Background technology
K+It is the metal ion that in organism, abundance second is high, participates in a series of biochemical reaction in organism, especially in signal conduction, K+Participation activates/regulates substantial amounts of signal transduction pathway.K in human body+Concentration is unbalance, and meeting causes a series of disease, including cardiovascular disease, renal dysfunction, nervous dysfunction and Other diseases.Additionally, the K of tumor cell surface+Passage is also the target spot of some antitumor drug.Therefore, it is achieved K in organism+Simplicity, high-selectivity analysis real-time, highly sensitive have particularly important meaning for the pathogenesis exploring some pathological processes, signal conductive process and some disease from molecular level.Traditional detection K+Method mainly have ion-selective electrode (ion-selectiveelectrode, ISE), coupled plasma atomic absorption spectrography (AAS) (inductivecoupledplasmaatomicabsorptionspectrophotometry, ICP-AAS), the chromatography of ions etc..But these classical ways K in detection organism+There is many weak points.First, these methods need the instrument of costliness and complexity mostly, it is necessary to specialized instrumentation person is operated, therefore, it is difficult to realize field assay.Secondly, biological sample needs the steps such as complicated pre-treatment, preenrichment and separation, and analysis time is long, it is impossible to realize K in biological sample+Quick analysis, temporal resolution is also poor.Compared with additive method, K+Although ion-selective electrode is by a relatively simple, however it is necessary that sample size is relatively big, therefore have significant limitation equally in field of biological sample analysis.
Therefore, a kind of novel detection technique of highly desirable development is for K in organism+Selective enumeration method highly sensitive, high.
Summary of the invention
Goal of the invention: the present invention is directed to K in current organism+The present situations such as the complicated operation that detection method exists, length consuming time, temporal resolution are poor, by Development of Novel DNA technique to K+Carry out the detection of high selectivity, high sensitivity.This technology is used for K+Detection has the advantage that 1) this detection method is to K+There are good sensitivity and monitoring limit, fully meet K in organism+Testing requirement；2) selectivity is good, and in organism, other materials do not interfere with；3) with low cost, it is not necessary to specially design and synthesis are to K+The fluorescent probe molecule of selective response；4) need sample amount only small, detect easy and simple to handle, analyze speed, it is possible to achieve K in organism in pathological processes+Dynamic observation.
Technical scheme: in order to solve above-mentioned technical problem, the invention provides a kind of DNA technique and quickly detects K in organism for highly sensitive high selectivity+.The method comprises one rich in the single stranded DNA of guanine (G base) and the fluorescent dye that can interact with this DNA.This DNA is at Na+There are antiparallel four stranded structure of lower formation, after this dyestuff interacts therewith, photoluminescent property does not change substantially.Once add K+, this DNA is preferential and K+Interacting and form four serobilas of positive parallel construction, itself and selected dyestuff can be obviously enhanced the fluorescence of organic dyestuff after interacting.
The invention provides a kind of detection solution containing DNA fragmentation and organic dyestuff, this detection solution includes the Single-stranded DNA fragments rich in guanine, sodium ion and the organic molecule dyestuff as signal output molecule.
This Single-stranded DNA fragments is individually containing Na+Solution in form antiparallel DNA tetra-stranded structure.
Na at this Single-stranded DNA fragments+Solution adds K+, then it is become just parallel DNA tetra-stranded structure by antiparallel DNA tetra-stranded structure.Owing to selected DNA is rich in G base, it can at Na+There are antiparallel four stranded structure of lower formation；Add K+After this DNA is preferential and K+Interact, thus be transformed into four serobilas of positive parallel construction from four serobilas of antiparallel configuration.Selected organic fluorescent dye interacts with antiparallel DNA tetra-serobila, and fluorescence is not changed in substantially；Interacting with just parallel DNA tetra-serobila, its fluorescence significantly changes.
The base sequence of described Single-stranded DNA fragments is 5 '-GTGGGTAGGGCGGGTTGG-3 '.
A kind of based on K in the organism of DNA technique+Detection method, comprise the following steps:
1) prepared by specific sequence DNA: utilizes the Single-stranded DNA fragments that solid phase synthesis technique preparation is designed, obtains the Single-stranded DNA fragments of described particular sequence after separated purification；
2) preparation DNA storing solution: buffer solution and TE buffer that preparation is suitable for pH value and chemical constituent are stand-by；Take a certain amount of step 1) in Single-stranded DNA fragments be dissolved in this buffer and obtain DNA storing solution；
3) preparation antiparallel ssdna detection liquid: take a certain amount of DNA storing solution, it is added thereto to a certain amount of NaCl and organic dyestuff (such as porphyrins, riboflavin compounds, compounds etc. is attained in fen, sale is all had in Ge great Reagent Company) and use TE buffer, dilution, obtains antiparallel ssdna detection liquid after heated annealing；(TE buffer: T:Tris-HCl；E:EDTA；50mMTris-HCl (pH7.0), 2.5mMEDTA)；
4) K+ detection: by above-mentioned detection liquid respectively with the blank sample without K+ and containing K+Sample with applicable volume ratio (such as 1:1) the mixing reaction that is placed in 37 DEG C of environment lucifuge, then the fluorescence signal in detection gained sample, can obtain K according to the ratio of both fluorescence intensity+Concentration value.
Specifically include following steps:
1) prepared by DNA: utilizes the DNA that PCR reaction preparation is designed, separates through gel electrophoresis, obtain the DNA (ProbeG) of particular sequence after purification；
2) preparation DNA storing solution: preparation pH is 7.0, and 50mMTris-HCl buffer solution and TE buffer solution containing 2.5mMEDTA are stand-by；Taking a certain amount of Single-stranded DNA fragments and be dissolved in this TE buffer, compound concentration is the DNA storing solution of 400 μMs；
3) preparation antiparallel ssdna detection liquid: take a certain amount of DNA storing solution, is added thereto to a certain amount of NaCl and organic dyestuff and dilutes with TE buffer, and containing DNA concentration in final solution is 20 μMs, and organic dyestuff concentration is 20 μMs, Na+Concentration is 200mM, is placed at 90 DEG C by this solution after heating in water bath 10min slow cooling and, to room temperature, then keeps in Dark Place stand-by；
4)K+Detection: by above-mentioned detection liquid respectively with the blank sample without K+ and containing K+Sample mix with the volume ratio of 1:1 and be placed in 37 DEG C of environment lucifuge reaction, then the fluorescence signal in detection gained sample, can obtain K according to the ratio of both fluorescence intensity+Concentration value.
The present invention is to K+Detection by quantitative principle: this DNA probe is at Na+There are antiparallel four stranded structure of lower formation.This structure is substantially suitable with organic dyestuff body fluorescence with the fluorescence of the complex that organic dyestuff interaction is formed.When detection system adds K+Afterwards, this DNA probe is preferential and K+Interacting, four serobilas of its formation are transformed into positive parallel construction by antiparallel configuration.And there is notable change in the organic dyestuff in solution and this just parallel DNA tetra-serobila its fluorescence after forming complex that interacts, and the degree of change in fluorescence and added K+Concentration has certain dependence.
Beneficial effect: the fluorescence signal that the DNA technique of the present invention exports is to K in system to be measured+Content within the scope of finite concentration, have good linear relationship, thus realize to K+Highly sensitive detection.This detection system is little affected by the interference of living things system other little molecules of common physiologically active interior and other metal ions, therefore to K+Detection has high selectivity.In addition the method instrument is simple, easy and simple to handle, with low cost, need sample amount few, it is possible to achieve K in organism+Continuous, quickly detect, be therefore of very high actual application value.
The present invention is directed to K in current organism+The present situations such as the complicated operation that detection method exists, length consuming time, time resolution rate variance, by utilizing DNA technique to realize K+High selectivity, highly sensitive detection.This technology is for K in organism+Detection has the advantage that 1) selectivity is good, and detection signal is neither by the interference of other compositions in living things system, also by the interference of biotic environment change in pathological processes；2) highly sensitive；3) need sample amount few, analyze speed, be effectively improved in organism K+The temporal resolution of detection；4) easy and simple to handle, can be used for clinical K+Quick analysis.
Accompanying drawing explanation
Fig. 1 DNA/PPIX is to K+The working curve of detection；
Fig. 2 DNA/PPIX is to K+The selectivity of detection；(1, artificial cerebrospinal fluid (aCSF)；2, aCSF+500 μM of glucose；3, aCSF+500 μM of lactic acid；4, aCSF+10 μM of ascorbic acid (AA)；5, aCSF+10 μM of 5-hydroxy tryptamine (5-HT)；6, aCSF+10 μM of UA (uric acid)；7, aCSF+10 μM of DA (dopamine)；8, aCSF+10 μM of DOPAC (3,4-dihydroxyphenyl acetic acid))；
Fig. 3 DNA technique is for K in Mus brain dialysis solution+Detection；
Fig. 4 DNA technique is for hippocampus K in Mus brain in ischemia-reperfusion process+Dynamic monitoring；
Fig. 5 DNA technique is for K in tumor+Dynamic monitoring；
Fig. 6 DNA technique is for K in organism+The principle of detection.
Detailed description of the invention
Below technical solution of the present invention is described in detail, but protection scope of the present invention is not limited to described embodiment.
The example being described DNA with single stranded DNA (ProbeG), its sequence is 5 '-GTGGGTAGGGCGGGTTGG-3 ' (being synthesized by Nanjing Jin Sirui Bioisystech Co., Ltd).
The example being described organic dyestuff with protoporphyrin (protoporphyrinIX, PPIX).
Embodiment 1DNA technology is at K+Application in detection
1) prepared by DNA:
Utilize the DNA that solid phase synthesis technique preparation is designed, after separated purification, obtain the DNA (ProbeG) of particular sequence.
2) preparation DNA storing solution:
Preparation pH is 7.0, and the 50mMTris-HCl buffer solution (TE buffer) containing 2.5mMEDTA is stand-by；Taking a certain amount of DNA and be dissolved in this TE buffer, compound concentration is the DNA storing solution of 400 μMs；
3) preparation antiparallel ssdna/PPIX solution:
Taking a certain amount of DNA storing solution, be added thereto to a certain amount of NaCl and PPIX and dilute constant volume with TE buffer, in final solution, contained DNA concentration is 20 μMs, and PPIX concentration is 20 μMs, Na+Concentration is 200mM.Being placed at 90 DEG C by this solution after heating in water bath 10min slow cooling, to room temperature, then keeps in Dark Place stand-by.
4) positive parallel ssdna/PPIX solution is prepared:
By the antiparallel ssdna/PPIX solution of preparation respectively with the blank sample without K+ and containing K+Sample mix with the volume ratio of 1:1 and be placed in 37 DEG C of environment lucifuge reaction 2h, then detect the fluorescence signal of reacted solution.
We have initially set up K+The working curve of detection.As seen from Figure 1, the fluorescence intensity of the just parallel DNA/PPIX formed and the ratio of blank solution and affiliated K+Concentration presents extraordinary linear relationship within the scope of finite concentration, and linearly dependent coefficient has reached 0.997, and the range of linearity is 500 μMs of 10mM, and its lowest detection is limited to 100 μMs.This range of linearity completely covers K in organism+Concentration range, therefore may be used for K in organism+Mensuration.
Embodiment 2DNA technology is to K+High selective enumeration method
The method is to K+Show good selectivity.As in figure 2 it is shown, the little molecule of physiologically active common in organism is to this K+Detection system, almost without interference, illustrates that this DNA technique can be used for K in organism+High selective enumeration method.
Embodiment 3DNA technology is to K in Mus brain+High selective enumeration method
Take 10 μ L antiparallel DNA/PPIX solution, respectively with 10 μ L without K+Artificial cerebrospinal fluid and Mus brain dialysis solution mixing after the static 2h of lucifuge.Afterwards sample is taken out, detect its fluorescence intensity, the ratio according to two fluorescent intensity, bring working curve into, calculate and obtain K in Mus brain dialysis solution+Content be 1.71 ± 0.22mM (Fig. 3).These data and traditional plasma atomic absorption spectrographic method (ICP-AAS) coincide (table 4) substantially, it was demonstrated that our method may be used for K in mobiles+Simplicity, reliable determination.
Table 4 traditional method (ICP-AAS) and K in our method detection Mus brain+Comparison；
| K+/mM | Our method | ICP-AAS |
| Rat 1 | 1.95 | 1.83 |
| Rat 2 | 1.51 | 1.40 |
| Rat 2 | 1.67 | 1.56 |
Embodiment 4DNA technology is to K in mobiles in physiology/pathological process+High selective enumeration method
With reference to embodiment 3, K in mobiles in detection physiology/pathological process+Dynamic change.Fig. 4 show in ischemia-reperfusion process K in rat brain+The dynamic change of concentration.After we have found that generation global brain ischemia, the K of hippocampus in rat brain+Concentration significantly rises and slowly recovers to basic value in refilling process.Fig. 5 show after tumor-bearing mice internal injection 5-ALA in tumor K+Dynamic change.It was found that the K in tumor tissues+Concentration is apparently higher than normal structure.K in its tumor after mouse peritoneal injection 5-ALA+There is not notable change in concentration, and K after the mice internal injection 5-ALA of normal group+Then present of short duration rising, it was demonstrated that our method may be used in optical dynamic therapy process K in tumor+Dynamic monitoring.
Claims (6)
1. the detection solution containing DNA fragmentation and organic dyestuff, it is characterised in that include the Single-stranded DNA fragments rich in guanine, sodium ion and the organic molecule dyestuff as signal output molecule in this detection solution.
2. the detection solution containing DNA fragmentation and organic dyestuff according to claim 1, it is characterised in that this Single-stranded DNA fragments is individually containing Na+Solution in form antiparallel DNA tetra-stranded structure.
3. the detection solution containing DNA fragmentation and organic dyestuff according to claim 1, it is characterised in that at the Na of this Single-stranded DNA fragments+Solution adds K+, then it is become just parallel DNA tetra-stranded structure by antiparallel DNA tetra-stranded structure.
4. the detection solution containing DNA fragmentation and organic dyestuff according to claim 1, it is characterised in that the base sequence of described Single-stranded DNA fragments is 5 '-GTGGGTAGGGCGGGTTGG-3 '.
5. one kind based on K in the organism of DNA technique+Detection method, it is characterised in that comprise the following steps:
1) prepared by specific sequence DNA: utilize the Single-stranded DNA fragments that solid phase synthesis technique preparation is designed, obtains the Single-stranded DNA fragments of particular sequence described in claim 1 after separated purification；
2) preparation DNA storing solution: buffer solution and TE buffer that preparation is suitable for pH value and chemical constituent are stand-by；The Single-stranded DNA fragments taking a certain amount of step 1) is dissolved in this buffer and obtains DNA storing solution；
3) preparation antiparallel ssdna detection liquid: take a certain amount of DNA storing solution, is added thereto to a certain amount of NaCl and organic dyestuff and dilutes with TE buffer, obtains antiparallel ssdna detection liquid after heated annealing.
4) K+Detection: the antiparallel ssdna in step 3) is detected liquid respectively with the blank sample without K+ and containing K+Sample with the applicable volume ratio mixing reaction that is placed in 37 DEG C of environment lucifuge, then the fluorescence signal in detection gained sample, can obtain K according to the ratio of both fluorescence intensity+Concentration value.
6. according to claim 5 a kind of based on K in the organism of DNA technique+Detection method, it is characterised in that specifically include following steps:
1) prepared by DNA: utilizes the DNA that PCR reaction preparation is designed, separates through gel electrophoresis, obtain the DNA of particular sequence after purification；
2) preparation DNA storing solution: preparation pH is 7.0, and 50mMTris-HCl buffer solution and TE buffer solution containing 2.5mMEDTA are stand-by；Taking a certain amount of single stranded DNA and be dissolved in this TE buffer, compound concentration is the DNA storing solution of 400 μMs；
3) preparation antiparallel ssdna detection liquid: take a certain amount of DNA storing solution, is added thereto to a certain amount of NaCl and organic dyestuff and dilutes with TE buffer, and containing DNA concentration in final solution is 20 μMs, and organic dyestuff concentration is 20 μMs, Na+Concentration is 200mM, is placed at 90 DEG C by this solution after heating in water bath 10min slow cooling and, to room temperature, then keeps in Dark Place stand-by；
4) K+Detection: the antiparallel ssdna in step 3) is detected liquid respectively with the blank sample without K+ and containing K+Sample mix with the volume ratio of 1:1 and be placed in 37 DEG C of environment lucifuge reaction, then the fluorescence signal in detection gained sample, can obtain K according to the ratio of both fluorescence intensity+Concentration value.
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| CN201610064718.6A CN105717081A (en) | 2016-01-29 | 2016-01-29 | Detection solution containing DNA fragments and organic dyestuff and application thereof |
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