CN109975561A - A method of the super sensitivity detection dopamine based on aptamer - Google Patents
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- G—PHYSICS
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- 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
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- G—PHYSICS
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- 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
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
- G01N33/9406—Neurotransmitters
- G01N33/9413—Dopamine
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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
- G01N2021/6417—Spectrofluorimetric devices
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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"
- G01N2021/6439—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 method of the present invention provides a kind of super sensitivity detection dopamine based on aptamer, forming tool in conjunction with first by ssDNA1 and dopamine aptamers, ssDNA2 by base pair complementarity principle, there are two the double-stranded DNAs 1 of 3 ' protruding terminus, wherein, the base number of ssDNA2 less than ssDNA1 and with ssDNA1 complementary pairing, dopamine aptamers are matched with ssDNA1 partial complementarity since the 3 ' ends of ssDNA2；Sample to be tested, hair clip DNA, fluorescent dye SYBR Green I and exonucleaseⅲ are added later, wherein, the base number of the hair clip DNA less than ssDNA1 and can pass through the double-stranded DNA 2 of base pair complementarity principle formation 3 ' distal tip of hair clip DNA chain with free ssDNA1.The present invention identifies dopamine by dopamine aptamers, is detached from aptamers from double-stranded DNA 1, to constantly cause endonuclease reaction, fluorescence signal is reduced, and realizes the super sensitivity detection to dopamine using this circulation amplifying technique.
Description
Technical field
The present invention relates to fields of biomedicine, and in particular to a kind of super sensitivity detection dopamine based on aptamer
Method.
Background technique
Parkinson's disease is a kind of common neurological dysfunction disease, mainly influences the middle-aged and the old, is after tumour, heart and brain blood
" the third killer " of person in middle and old age after pipe disease., it is surprising that global about 4,500,000 Parkinsonians, nearly half is in
State.And the most important pathological change of Parkinson's disease is the denaturation death of substantia nigra of midbrain dopaminergic neuron, causes line therefrom
Shape body dopamine (DA) content conspicuousness is reduced and is caused a disease.Therefore, the neurotransmitters such as dopamine are detected for ensureing national health
And the treatment and prevention of disease are all of great significance.
However, existing detection method is not easy to realize Sensitive Detection since content is lower in blood for dopamine (DA).It is another
Usually there is many kinds of substance, such as ascorbic acid, adrenaline, norepinephrine etc. in aspect body fluid, these substances are all
It is likely to affect the measurement of dopamine.Therefore, it is necessary to study a kind of identification probe that selectivity is good.Secondly, sampling is asked
Topic is also an aspect urgently to be resolved.The acquisition multi-pass of dopamine, which is crossed, takes blood, also there is cerebrospinal fluid sampling.The sampling at these positions
There is the features such as big to body wound, patient dependence is poor.With ultramicron detection, it is noninvasive or it is minimally invasive detection in medical field
In development, develop it is a kind of it is highly sensitive, highly selective, minimally invasively detect dopamine (DA) new method have important meaning
Justice.
Currently, the main method of measurement dopamine (DA) has spectroscopic methodology, mass spectrography, fluorescence method, chemoluminescence method, capillary
Electrophoresis, chromatography and electrochemical method.For example, Edyta N etc. is using luminol-ferricyanide chemical luminous system to more
Bar amine is determined.Andrej K etc. is using method associated with liquid chromatography mass to the neurotransmitter in mouse cerebrospinal fluid
It is determined research.The gold electrode of the graphene modified such as Ai Yongqing has been carried out while having been measured to dopamine and uric acid.Optics
Although method detection is convenient, fast, the sensitivity of detection is poor.Though chromatography is higher relative to optical means sensitivity,
Pre-treatment is cumbersome, expensive equipment, and reagent consumption is big, and operating cost is higher.Although electrochemical method equipment is simple, detection spirit
Sensitivity is not high, and the service life of electrode is shorter, and the reproducibility of testing result is poor.
Summary of the invention
The method of it is an object of the invention to provide a kind of super sensitivity detection dopamine based on aptamer, to solve
Existing detection method poor sensitivity, it is complicated for operation, using the big problem of wound.
The purpose of the present invention is what is be achieved through the following technical solutions: a kind of super sensitivity detection based on aptamer is more
The method of bar amine, first by ssDNA1 and dopamine aptamers, ssDNA2 by base pair complementarity principle in conjunction with formed and have
The double-stranded DNA 1 of two 3 ' protruding terminus, wherein the base number of ssDNA2 less than ssDNA1 and with ssDNA1 complementary pairing, DOPA
Amine aptamers are matched with ssDNA1 partial complementarity since the 3 ' ends of ssDNA2；Sample to be tested, hair clip DNA, glimmering is added later
Photoinitiator dye SYBR Green I and exonucleaseⅲ, wherein the base number of the hair clip DNA is less than ssDNA1 and can be with trip
From ssDNA1 by base pair complementarity principle formed 3 ' distal tip of hair clip DNA chain double-stranded DNA 2；
Dopamine in sample to be tested is competed from double-stranded DNA 1 obtains dopamine aptamers, so that remaining double-stranded DNA 1 is formed
The structure of 3 ' distal tips, so that the endonuclease reaction for causing exonucleaseⅲ forms free ssDNA1, hair clip DNA
(hDNA) double-stranded DNA 2 for forming 3 ' distal tip of hair clip DNA chain by base pair complementarity principle with free ssDNA1, into
And cause the endonuclease reaction of exonucleaseⅲ again, so circulation is so that be located at the fluorescent dye SYBR in double-stranded DNA structure
Green I is constantly released, and fluorescence signal is caused to reduce, and passes through the linear of the reduction amount of fluorescence signal and dopamine concentration
Relationship obtains the dopamine concentration of sample to be tested.
The method of the above-mentioned super sensitivity detection dopamine based on aptamer specifically includes the following steps:
A, the dopamine aptamers of equimolar amounts, ssDNA1, ssDNA2 are added in Tris-HCl solution, under room temperature into
Row is incubated for, and sample to be tested is then added, and reacted at room temperature；Wherein, dopamine aptamers sequence such as sequence table
In sequence 1 shown in, ssDNA1 sequence is as shown in the sequence 2 in sequence table, ssDNA2 sequence such as 3 institute of sequence in sequence table
Show；
B, hair clip DNA and I solution of fluorescent dye SYBR Green are added in the reaction solution obtained to step a, after fully reacting,
Exonucleaseⅲ is added and carries out endonuclease reaction, is cooled to room temperature after the reaction was completed；Wherein, in hair clip DNA sequence dna such as sequence table
Sequence 4 shown in；
C, the obtained reaction solution of step b is detected with sepectrophotofluorometer, obtains the corresponding fluorescence signal of sample to be tested
Reduction amount, then sample to be tested is obtained by the standard curve of the reduction amount of fluorescence signal and dopamine concentration that measure in advance
Dopamine concentration.
In step a, dopamine aptamers, ssDNA1, ssDNA2 incubation time be 5 hours.
In step b, endonuclease reaction condition are as follows: 70U exonucleaseⅲ is added, is incubated for 1 hour in 37 DEG C.
In step c, the excitation wavelength of sepectrophotofluorometer is 497nm, and fluorescence emission spectrum is detected in 510 ~ 600nm,
Excitation and transmite slit are 10.0 nm, and temperature is 25 DEG C.
The present invention identifies DA by specific aptamer, constructs the fluorescence detection platform of DA, meanwhile, it utilizes for the first time
Circulation amplifying technique realizes the super sensitivity detection to DA, mainly has the advantage that
(1) high sensitivity, conventional method detection sensitivity are mostly hundreds to thousands nanomolar concentration, and use this method, sensitive
Degree can reach 0.08 nanomole rank, and sensitivity improves hundreds of thousands of times, thus can be to the very low blood of DOPAMINE CONTENT IN RABBIT, urine
Sample is measured；
(2) sample is few with sample amount, is mostly several hectolambdas to several milliliters with the methods of electrochemistry, liquid chromatogram with sample amount, sampling amount
Greatly, it often causes to patient compared with major trauma, and then needs sample amount few using the method, it is only necessary to which several microlitres can meet measurement and want
It asks, not only alleviates the burden of patient, also create possibility for the household service in future；
(3) easy to operate, at low cost, clinically in order to obtain higher accuracy, it is often used the higher cerebrospinal fluid conduct of content
Test sample.But cerebrospinal fluid sampling is difficult, more demanding to sampling people's operation, and the present invention can be using blood, urine as sample
Product are measured；In addition, the present invention forms double-stranded DNA using unmarked single stranded DNA, insertion fluorescent dye carries out fluorescence inspection
It surveys, greatly reduces cost, and there is outstanding sensitivity.
Detailed description of the invention
Fig. 1 is the schematic diagram of the method for the present invention.
Fig. 2 is the standard curve that the embodiment of the present invention 1 measures.
Specific embodiment
Technical solution of the present invention is described in detail combined with specific embodiments below.DNA sequence involved in the present invention
Column are all made of conventional method synthesis, and the experimental condition that do not mention in the embodiment of the present invention and operation are by conventional method in that art
It carries out.
The drafting of 1 standard curve of embodiment
(a) configuration concentration is respectively the more of 0nM, 0.1nM, 0.3nM, 0.6nM, 0.9nM, 3.0nM, 4.0nM, 5.0nM, 10.0nM
Bar amine standard solution.
(b) according to the concentration gradient of dopamine solution, grouping carries out detection test, and every group of test operation is identical, specifically:
By 6 μ L ssDNA1(5 μM), 6 μ L ssDNA2(5 μM), 6 μ L dopamine aptamers (5 μM) are added to 230 μ L
Incubation at room temperature 5 hours in Tris-HCl (10 mM)；The dopamine solution that 20 μ L corresponding concentrations are added reacts at room temperature 1 hour,
6 DNA(15 μM of μ L hair clips are added) and 19 μ L SYBR Green I (purchased from the raw limited public affairs of work bioengineering (Shanghai) share
Department is diluted with 1000 times of water) room temperature reaction 45 minutes；Then 70U exonucleaseⅲ (Exo- Ш) is added, 37 DEG C of incubations 1 are small
When (demonstrate the progress of this endonuclease reaction using polyacrylamide gel electrophoresis), be cooled to room temperature and use sepectrophotofluorometer
It is detected, excitation wavelength 497nm, fluorescence emission spectrum is detected in 510 ~ 600nm, and excitation and transmite slit are 10.0
Nm, temperature are 25 DEG C.
Dopamine aptamers sequence is as shown in the sequence 1 in sequence table, ssDNA1 sequence such as 2 institute of sequence in sequence table
Show, ssDNA2 sequence is as shown in the sequence 3 in sequence table；Hair clip DNA sequence dna is as shown in the sequence 4 in sequence table；The mistake of reaction
Journey is as shown in Figure 1.The base number of above-mentioned ssDNA2 less than ssDNA1 and with ssDNA1 complementary pairing, dopamine aptamers from
3 ' the ends of ssDNA2 start to match with ssDNA1 partial complementarity, and so as to form tool, there are two the double-strands of 3 ' protruding terminus
DNA1.The base number of hair clip DNA is less than ssDNA1 and can form hair clip by base pair complementarity principle with free ssDNA1
The double-stranded DNA 2 of 3 ' distal tip of DNA chain.
Dopamine in sample is competed from double-stranded DNA 1 obtains dopamine aptamers, so that remaining double-stranded DNA 1 is formed
The structure of 3 ' distal tips, so that the endonuclease reaction for causing exonucleaseⅲ forms free ssDNA1, hair clip DNA and trip
From ssDNA1 form by base pair complementarity principle the double-stranded DNA 2 of 3 ' distal tip of hair clip DNA chain, and then cause again
The endonuclease reaction of exonucleaseⅲ, so circulation so that be located at double-stranded DNA structure in fluorescent dye SYBR Green I not
It is disconnected to be released, cause fluorescence signal to reduce, is obtained by the reduction amount of fluorescence signal and the linear relationship of dopamine concentration
The dopamine concentration of sample to be tested.
(c) the fluorescence signal reduction amount for measuring every group draws standard curve with corresponding dopamine concentration, such as Fig. 2 institute
Show.
DOPAMINE CONTENT IN RABBIT detects in 2 Mice brain tissues of embodiment
Intraperitoneal injection of anesthesia, anaesthesia dosage 0.1ml/10g, to holonarcosis are carried out to mouse using 1% yellow Jackets
Afterwards, its brain tissue is taken under cryogenic.Be added Tissue lysates in brain tissue according to 1g:7.5mL, be homogenized 30s, low temperature from
The heart (14000r/min, 4 DEG C) takes its supernatant to be centrifuged again by the same terms.By supernatant according to the detecting step of embodiment 1
Fluorescence detection is carried out, while conventionally carrying out enzyme linked immunosorbent assay (ELISA).Testing result is as shown in table 1, through statistics
P=0.235 > 0.1, the no significant difference of two methods are analyzed, it is thus regarded that two methods have preferable consistency.
Table 1:
Detection method is compared with the sensitivity of other methods in the prior art, as shown in table 2.It can be with by table 2
Find out, the present invention realizes super sensitivity detection, has marked improvement compared with the existing technology.
Table 2:
Bibliography in table 2:
[1] Raj D R, Prasanth S, Vineeshkumar T V, et al. Surface plasmon
resonance based fiber optic dopamine sensor using green synthesized silver
nanoparticles[J]. Sensors and Actuators B: Chemical, 2016, 224: 600-606.
[2] Liu S, Shi F, Zhao X, et al. 3-Aminophenyl boronic acid-
functionalized CuInS2 quantum dots as a near-infrared fluorescence probe for
the determination of dopamine[J]. Biosensors and Bioelectronics, 2013, 47:
379-384.
[3] Liu J M, Wang X X, Cui M L, et al. A promising non-aggregation
colorimetric sensor of AuNRs–Ag+ for determination of dopamine[J]. Sensors &
Actuators B Chemical, 2013, 176(6): 97-102.
[4] Yan Y, Liu Q, Du X, et al. Visible light photoelectrochemical sensor
for ultrasensitive determination of dopamine based on synergistic effect of
graphene quantum dots and TiO2 nanoparticles[J]. Analytica chimica acta,
2015, 853: 258-264.
[5] Mir T A, Akhtar M H, Gurudatt N G, et al. An amperometric
nanobiosensor for the selective detection of K+-induced dopamine released
from living cells[J]. Biosensors and Bioelectronics, 2015, 68: 421-428.
[6] Azadbakht A, Roushani M, Abbasi A R, et al. Design and
characterization of electrochemical dopamine–aptamer as convenient and
integrated sensing platform[J]. Analytical biochemistry, 2016, 507: 47-57.
[7] Zhou X, Ma P, Wang A, et al. Dopamine fluorescent sensors based on
polypyrrole/graphene quantum dots core/shell hybrids[J]. Biosensors &
Bioelectronics, 2015, 64: 404-410.
SEQUENCE LISTING
<110>Hebei Medical University
<120>a kind of method of the super sensitivity detection dopamine based on aptamer
<130> 4.23
<160> 4
<170> PatentIn version 3.3
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<211> 58
<212> DNA
<213>artificial synthesized
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<213>artificial synthesized
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Claims (5)
1. a kind of method of the super sensitivity detection dopamine based on aptamer, characterized in that first by ssDNA1 and DOPA
Amine aptamers, ssDNA2 combine the double-stranded DNA 1 for being formed and being had there are two 3 ' protruding terminus by base pair complementarity principle, wherein
The base number of ssDNA2 less than ssDNA1 and with ssDNA1 complementary pairing, dopamine aptamers since the 3 ' ends of ssDNA2 with
The pairing of ssDNA1 partial complementarity；Sample to be tested, hair clip DNA, fluorescent dye SYBR Green I and Exonucleolytic are added later
Enzyme III, wherein the base number of the hair clip DNA is less than ssDNA1 and can pass through base pair complementarity principle with free ssDNA1
Form the double-stranded DNA 2 of 3 ' distal tip of hair clip DNA chain；
Dopamine in sample to be tested is competed from double-stranded DNA 1 obtains dopamine aptamers, so that remaining double-stranded DNA 1 is formed
The structure of 3 ' distal tips, so that the endonuclease reaction for causing exonucleaseⅲ forms free ssDNA1, hair clip DNA and trip
From ssDNA1 form by base pair complementarity principle the double-stranded DNA 2 of 3 ' distal tip of hair clip DNA chain, and then cause again
The endonuclease reaction of exonucleaseⅲ, so circulation so that be located at double-stranded DNA structure in fluorescent dye SYBR Green I not
It is disconnected to be released, cause fluorescence signal to reduce, is obtained by the reduction amount of fluorescence signal and the linear relationship of dopamine concentration
The dopamine concentration of sample to be tested.
2. the method for the super sensitivity detection dopamine according to claim 1 based on aptamer, characterized in that including
Following steps:
A, the dopamine aptamers of equimolar amounts, ssDNA1, ssDNA2 are added in Tris-HCl solution, under room temperature into
Row is incubated for, and sample to be tested is then added, and reacted at room temperature；Wherein, dopamine aptamers sequence such as sequence table
In sequence 1 shown in, ssDNA1 sequence is as shown in the sequence 2 in sequence table, ssDNA2 sequence such as 3 institute of sequence in sequence table
Show；
B, hair clip DNA and I solution of fluorescent dye SYBR Green are added in the reaction solution obtained to step a, after fully reacting,
Exonucleaseⅲ is added and carries out endonuclease reaction, is cooled to room temperature after the reaction was completed；Wherein, in hair clip DNA sequence dna such as sequence table
Sequence 4 shown in；
C, the obtained reaction solution of step b is detected with sepectrophotofluorometer, obtains the corresponding fluorescence signal of sample to be tested
Reduction amount, then sample to be tested is obtained by the standard curve of the reduction amount of fluorescence signal and dopamine concentration that measure in advance
Dopamine concentration.
3. the method for the super sensitivity detection dopamine according to claim 2 based on aptamer, characterized in that step
In a, dopamine aptamers, ssDNA1, ssDNA2 incubation time be 5 hours.
4. the method for the super sensitivity detection dopamine according to claim 2 based on aptamer, characterized in that step
In b, endonuclease reaction condition are as follows: 70U exonucleaseⅲ is added, is incubated for 1 hour in 37 DEG C.
5. the method for the super sensitivity detection dopamine according to claim 2 based on aptamer, characterized in that step
In c, the excitation wavelength of sepectrophotofluorometer is 497nm, and fluorescence emission spectrum detects in 510 ~ 600nm, excites and emit narrow
Seam is 10.0 nm, and temperature is 25 DEG C.
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| CN111879738A (en) * | 2020-06-15 | 2020-11-03 | 山东师范大学 | Non-labeled aptamer probe system and detection method and application thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111879738A (en) * | 2020-06-15 | 2020-11-03 | 山东师范大学 | Non-labeled aptamer probe system and detection method and application thereof |
| CN111879738B (en) * | 2020-06-15 | 2023-03-14 | 山东师范大学 | Non-labeled aptamer probe system and detection method and application thereof |
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