CN107179305A - Detection method based on embedded dyestuff and the DNA saxitoxin interacted - Google Patents
Detection method based on embedded dyestuff and the DNA saxitoxin interacted Download PDFInfo
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- CN107179305A CN107179305A CN201710365017.0A CN201710365017A CN107179305A CN 107179305 A CN107179305 A CN 107179305A CN 201710365017 A CN201710365017 A CN 201710365017A CN 107179305 A CN107179305 A CN 107179305A
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- dna
- saxitoxin
- interacted
- detection method
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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
- G01N21/6402—Atomic fluorescence; Laser induced fluorescence
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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
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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- General Health & Medical Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
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- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
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- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Optics & Photonics (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a kind of detection method based on embedded dyestuff and the DNA saxitoxin interacted, including step in detail below：DNA is dissolved in buffer solution first, then saxitoxin is added into buffer solution to be interacted with DNA, the time of interaction is 25min~35min, it is to be interacted that time is up, embedded dyestuff is added into buffer solution, is waited after 25min~35min, fluorescence spectrometry is carried out, the peak value composed by institute's light-metering, contrast standard curve calculates the toxin concentration of saxitoxin.The present invention can be achieved with the quantitative determination of the toxin concentration for saxitoxin by using the measure to fluorescence, instrument and detection platform that need not be complicated be built, detection mode is simple, quick, accurate, the technical bottleneck for determining the instrument for needing to rely on complicated, high cost is passed so as to break away from, and overcomes the complexity for needing to build to being fixed of DNA and detection platform.
Description
Technical field
The present invention relates to the detection of saxitoxin research, and in particular to one kind is based on embedded dyestuff and DNA interactions
Saxitoxin detection method.
Background technology
Paralytic shellfish poison's element is a class neurotoxin, and toxicity is extremely strong, can hinder the conduction of nerve impulse and make nervous centralis
It is dead that system is suppressed generation respiratory system paralysis.Its Poisoning most strong saxitoxin can make one dead in 15min
Die, destroyed by it can not be digested enzyme, colorless and odorless, therefore the detection research to it is particularly important.Traditional mouse inspection
Fluorescence analysis detection technique causes practical application wide due to being limited to zoopery and large-scale instrument after survey method and HPLC
It is general be applied to it is daily.All kit is the protein reagent box using antibody as base on the market at present.It is limited to it stable
Property and the limitation for preparing on a large scale, the identification technology for the saxitoxin that DNA is base is have developed at present.
It is high by SELEX (systematic evolution ofligands by exponential enrichment)
Throughtput screening technologies, Handy, Sara M. etc. report the DNA sequence dna APTSTX1, (First that first case is directed to saxitoxin
report of the use of a saxitoxin-protein conjugate to develop a DNA aptamer
To a small molecule toxin, toxin, 2013,61,30-37), then Zheng Xin etc. reports changing for first case
DNA sequence dna M-30f (the A saxitoxin-binding aptamer with higher affinity and of good version
inhibitory activity optimizedby rational site-directed mutagenesis
andtruncation,toxin,2015,101,41-47)。
DNA has the advantages that to be easy to extensive synthesis, modified and design for the bio-sensing recognition component of base, therefore wide
The general preparation and application for sensor.Can be with molecule and instrument with signals such as light, electricity, magnetic by DNA conformation changes
It is combined, reaches the purpose detected using DNA conformation changes to detected material.
But found from 2013 after the DNA sequence dna for saxitoxin, for the biography of the saxitoxin of its sequence
The design report of sensor is no rapidly to be increased such as other detectable substance sequences.Used in during being reported in itself except article
BLI detecting instruments, the sensor reported currently with above-mentioned two sequence only has electrochemical method (Amperometric
aptasensor for saxitoxin using a gold electrode modified with carbon
nanotubes on a self-assembled monolayer,and methylene blue as an
Electrochemical indicator probe, 2016,183,6,1971-1980).To find out its cause, mainly two kinds sequences
Length is longer, and secondary structure is more complicated, causes to be difficult to using base pairing and the replacement eliminated, amplification, fixation, and sharp
Complexity effect of the optical signal produced with conformation change again due to sequence in itself be not obvious.
Using DNA only BLI and electrochemical analysis method, these methods are built at present for the sensor of saxitoxin
It is that detection process is monitored in real time after DNA probe is fixed using surface and interface analytical instrument, strong depend-ence instrument
And the cycle is longer, testing cost is high, and instrument is more difficult obtains, it is therefore desirable to which finding a kind of pervasive method can be to the sequence of research and development
Row are designed to reach the purpose of application.
The content of the invention
Goal of the invention：Need to rely on to overcome the deficiencies in the prior art to break away from biography measure there is provided one kind
The instrument of complicated, high cost and overcoming is needed to being fixed of DNA and the complexity of detection platform structure based on embedded
The detection method of dyestuff and the saxitoxin of DNA interactions.
Technical scheme：To achieve the above object, the present invention provides a kind of stone room interacted based on embedded dyestuff and DNA
The detection method of clam toxin, including step in detail below：
Step 1)：DNA is dissolved in buffer solution first and Quenching Treatment is carried out；
Step 2)：Then add saxitoxin into buffer solution to be interacted with DNA, the time of interaction is
25min~35min；
Step 3)：To be interacted time is up, to step 2) in buffer solution in add embedded dyestuff, wait
After 25min~35min, fluorescence spectrometry, the peak value composed by institute's light-metering are carried out, contrast standard curve calculates Saxidomus poison
The toxin concentration of element.
The step 3) in embedded dyestuff have and can recognize and with reference to the feature of the serobilas of DNA secondary structures G- tetra-, by
The serobila secondary con features of G- tetra- are respectively provided with the DNA sequence dna found, and research finds that saxitoxin has intercalation of DNA sequence
The characteristics of arranging generated tetra- serobila conformations of G, therefore the combination of saxitoxin certainly will influence the packing interaction of embedded dyestuff, because
This can carry out Fluorescence Identification by this effect to whetheing there is toxin and toxin concentration.
Further, the step 1) in buffer solution be Tris-HCl buffer solutions, and the pH=7 of buffer solution.
Further, the step 3) in embedded dyestuff for fluorescence is purple or peacock green.
Further, the step 3) it is middle using sepectrophotofluorometer progress fluorescence spectrometry.
Further, the content ratio of the DNA and embedded dyestuff is 1:1.
Further, the step 3) in peak value at the 652nm that is composed by institute's light-metering, contrast standard curve calculates stone
The toxin concentration of STX.
Beneficial effect：The present invention compared with prior art, can be achieved with for Saxidomus by using the measure to fluorescence
The quantitative determination of the toxin concentration of toxin, it is not necessary to which complicated instrument and detection platform are built, detection mode is simple, quick, accurate
Really, so as to break away from the technical bottleneck for passing and determining the instrument for needing to rely on complicated, high cost, and overcome and need to enter DNA
The complexity that row immobilization and detection platform are built.
Brief description of the drawings
Fig. 1 is the peak value of spectrum and the curve relation figure of fluorescence intensity；
Fig. 2 is the graph of a relation between fluorescence intensity and the toxin concentration of saxitoxin.
Embodiment
The present invention is further described with reference to embodiment.
Embodiment 1：
1 μM of DNA is dissolved in pH=7 10mM Tris-HCl buffer solutions first and Quenching Treatment is carried out, then
Add saxitoxin into Tris-HCl buffer solutions to be interacted with DNA, the time of interaction is 25min, treats phase
Interaction is after time is up, and the fluorescence that 1 μM is added into Tris-HCl buffer solutions is purple, waits after 25min, utilizes fluorescence spectrophotometer
Photometer carries out fluorescence spectrometry, the peak value at 652nm composed by institute's light-metering, and contrast standard curve calculates Saxidomus poison
The toxin concentration of element.
Embodiment 2：
1 μM of DNA is dissolved in pH=7 10mM Tris-HCl buffer solutions first and Quenching Treatment is carried out, then
Add saxitoxin into Tris-HCl buffer solutions to be interacted with DNA, the time of interaction is 35min, treats phase
Interaction is after time is up, and the fluorescence that 1 μM is added into Tris-HCl buffer solutions is purple, waits after 35min, utilizes fluorescence spectrophotometer
Photometer carries out fluorescence spectrometry, the peak value at 652nm composed by institute's light-metering, and contrast standard curve calculates Saxidomus poison
The toxin concentration of element.
Embodiment 3：
1 μM of DNA is dissolved in pH=7 10mM Tris-HCl buffer solutions first and Quenching Treatment is carried out, then
Add saxitoxin into Tris-HCl buffer solutions to be interacted with DNA, the time of interaction is 30min, treats phase
Interaction is after time is up, and the fluorescence that 1 μM is added into Tris-HCl buffer solutions is purple, waits after 30min, utilizes fluorescence spectrophotometer
Photometer carries out fluorescence spectrometry, the peak value at 652nm composed by institute's light-metering, and contrast standard curve calculates Saxidomus poison
The toxin concentration of element.
Embodiment 4：
If Fig. 1 is to carry out fluorescence spectrometry, the peak value and fluorescence intensity of obtained spectrum using sepectrophotofluorometer
Curve relation figure, the content ratio that the curve in Fig. 1 is respectively from top to bottom DNA and embedded dyestuff is 0,0.3,0.5,1,1.5,
1.8、2；
Fig. 2 be institute light-metering spectrum in the case of the peak value at 656nm, the toxin concentration of fluorescence intensity and saxitoxin it
Between graph of a relation；
By peak values of the Fig. 1 in institute light-metering spectrum 656nm at respectively obtain when the content ratio of DNA and embedded dyestuff be 0,
0.3rd, 0.5,1,1.5,1.8,2 when corresponding fluorescence intensity, then contrasted according to Fig. 2 and obtain the poison of corresponding saxitoxin
Plain concentration.
Described above is only the preferred embodiment of the present invention, it should be pointed out that：Come for those skilled in the art
Say, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should be regarded as
Protection scope of the present invention.
Claims (6)
1. the detection method based on embedded dyestuff and the DNA saxitoxin interacted, it is characterised in that：Including in detail below
Step：
Step 1)：DNA is dissolved in buffer solution first and Quenching Treatment is carried out；
Step 2)：Then add saxitoxin into buffer solution to be interacted with DNA, the time of interaction is
25min~35min；
Step 3)：To be interacted time is up, to step 2) in buffer solution in add embedded dyestuff, wait 25min~
After 35min, fluorescence spectrometry is carried out, the peak value composed by institute's light-metering, contrast standard curve calculates the poison of saxitoxin
Plain concentration.
2. the detection method according to claim 1 based on embedded dyestuff and the DNA saxitoxin interacted, it is special
Levy and be：The step 1) in buffer solution be Tris-HCl buffer solutions, and the pH=7 of buffer solution.
3. the detection method according to claim 1 based on embedded dyestuff and the DNA saxitoxin interacted, it is special
Levy and be：The step 3) in embedded dyestuff for fluorescence is purple or peacock green.
4. the detection method according to claim 1 based on embedded dyestuff and the DNA saxitoxin interacted, it is special
Levy and be：The step 3) it is middle using sepectrophotofluorometer progress fluorescence spectrometry.
5. the detection method according to claim 1 based on embedded dyestuff and the DNA saxitoxin interacted, it is special
Levy and be：The content ratio of the DNA and embedded dyestuff is 1:1.
6. the detection method according to claim 1 based on embedded dyestuff and the DNA saxitoxin interacted, it is special
Levy and be：The step 3) in peak value at the 652nm that is composed by institute's light-metering, contrast standard curve calculates saxitoxin
Toxin concentration.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710365017.0A CN107179305A (en) | 2017-05-22 | 2017-05-22 | Detection method based on embedded dyestuff and the DNA saxitoxin interacted |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710365017.0A CN107179305A (en) | 2017-05-22 | 2017-05-22 | Detection method based on embedded dyestuff and the DNA saxitoxin interacted |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1636340A2 (en) * | 2003-06-18 | 2006-03-22 | The Scripps Research Institute | Unnatural reactive amino acid genetic code additions |
| CN104630230A (en) * | 2015-01-06 | 2015-05-20 | 江南大学 | Group of nucleic acid aptamers for specifically recognizing okadaic acid |
| CN104894135A (en) * | 2015-04-28 | 2015-09-09 | 中国人民解放军第二军医大学 | High affinity adapter body capable of specifically binding with saxitoxin acetate and application thereof |
-
2017
- 2017-05-22 CN CN201710365017.0A patent/CN107179305A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1636340A2 (en) * | 2003-06-18 | 2006-03-22 | The Scripps Research Institute | Unnatural reactive amino acid genetic code additions |
| CN104630230A (en) * | 2015-01-06 | 2015-05-20 | 江南大学 | Group of nucleic acid aptamers for specifically recognizing okadaic acid |
| CN104894135A (en) * | 2015-04-28 | 2015-09-09 | 中国人民解放军第二军医大学 | High affinity adapter body capable of specifically binding with saxitoxin acetate and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| JUN-HONG GUO 等: "Triphenylmethane dyes as fluorescent probes for G-quadruplex recognition", 《TALANTA》 * |
| X.ZHENG 等: "A saxitoxin-binding aptamer with higher affinity and inhibitory activity optimized by rational site-directed mutagenesis and truncation", 《TOXICON》 * |
| 郭亚辉 等: "Ｇ四链体在生物传感器中的应用", 《武汉大学学报（理学版）》 * |
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Application publication date: 20170919 |