CN106957908A - MiRNA and/or target molecules with aptamer detection method and detection probe - Google Patents
MiRNA and/or target molecules with aptamer detection method and detection probe Download PDFInfo
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Abstract
The invention discloses a kind of miRNA and/or the target molecules with aptamer detection method and detection probe, including：Golden nanometer particle H1 compounds, golden nanometer particle H2 compounds and object recognition complex；Golden nanometer particle H1 compounds and golden nanometer particle the H2 compound is combined by neck ring DNA H1 and H2 and formed with golden nanometer particle respectively；The object recognition complex includes：At least one in carboxyl magnetic microsphere DNA1/2 compounds for recognizing miRNA, and DNA3 for recognizing target molecules.The detection method of the present invention is without chemical labeling, and color change naked eyes are visible, and method is simple, applied widely, available for miRNA, the detection of the target molecules with aptamer.
Description
Technical field
The present invention relates to nucleic acid hybridisationdetection technology field, and in particular to a kind of miRNA and/or with aptamer
The detection method and detection probe of target molecules.
Background technology
MiRNA is a class endogenous non-coding small molecule (18-22nt), is played in gene expression or reverse transcription important
Adjustment effect.It is related to extensive bioprocess, such as cell propagation, Apoptosis and death.MiRNA unconventionality expression is related to
To various diseases, particularly human cancer, the nervous system disease, virus infection and diabetes.MiRNA is in physiology and pathologic process
In obvious regulating and controlling effect miRNA is used for the diagnosis, gene therapy and the discovery of new anti-cancer drug thing of clinical disease.For example,
MiRNA-203 is in the overexpression of various malignant tumours, including breast cancer, cervical carcinoma, leukaemia and laryngocarcinoma etc..Therefore, for
The diagnosis and prognosis of morbid state, miRNA-203 expression is promising biomarker, to losing in gene therapy
Hereditary diseases and potential drug targets provide an attractive approach.MiRNA-21 is in kinds of tumors sample and cell
System all detects the rise extremely of its expression, and miRNA-21 is a generally acknowledged carcinogenicity tiny RNA.
However, the features such as due to having short, the higher sequence homology of low abundance, size between miRNA members of the same clan, because
This, detection miRNA is a challenge, and the detection method for miRNA needs to have good specificity, sensitivity and stably
Property.MiRNA traditional detection method has：The gene core of Northern blottings, real-time polymerase chain reaction (PCR) and miRNA
Piece detection etc..Wherein, Northern blottings are considered as the gold standard method of miRNA detections, but are due to that its operation is numerous
It is trivial, waste time and energy, sensitivity is relatively low and make it that it is not suitable for routine the shortcomings of sample requirement big (10 μ g samples)
Clinical diagnosis；PCR and genechip detection method have that efficiency is low, test limit is low, time-consuming, economy is low and complex operation lacked
Point, limits their biology and biomedical applications, therefore, and it is significant to develop new miRNA inspection policies
's.
Aptamer is the single stranded oligonucleotide that a class developed in recent years is filtered out through external artificial synthesized,
Can efficiently, specifically combine various biological target molecules, aptamer appears as chemical-biological educational circles and biomedicine
Boundary provides a kind of new research platform.Aptamer have self stability it is good, prepare synthesis it is relatively easy, quick, easy
The advantages of acquisition, easy functional modification are with mark, therefore, using flexibly extensive in biosensor design.
At present, the biology sensor based on ATP aptamers has been utilized to detect ATP report, but in ATP detection
During, it is necessary to carry out chemical labeling to aptamer, further according to label before and after identification reaction in itself or its catalytic substrate
Change detected that therefore complex operation, cost is higher.
Golden nanometer particle unique chemical stability, catalytic activity, machinability and metalline, become one and draw
The nano material that people gazes at, and make extensively with the related optics of unique size and characteristic electron, therefore, golden nanometer particle
In the technical fields such as catalysis, nanoelectronics and biomedicine (sensing, diagnosis, image and mark).However, at present also not
See to have and be combined golden nanometer particle with DNA hybridization chain reaction to detect miRNA and/or target with aptamer point
The report of son.
The content of the invention
For above-mentioned the deficiencies in the prior art, it is an object of the invention to provide a kind of miRNA and/or with nucleic acid
The detection method and detection probe of the target molecules of aptamers.The detection method is without chemical labeling, and color change naked eyes are visible,
Method is simple, applied widely, available for miRNA, the target molecules with aptamer (such as ATP, fibrin ferment, bacteriolyze
Enzyme, EGF-R ELISA etc.) detection.
To achieve the above object, the present invention uses following technical proposals：
The first aspect of the present invention includes there is provided a kind of probe, the probe：Golden nanometer particle-H1 compounds, Jenner's grain of rice
Son-H2 compounds and object recognition complex；
Golden nanometer particle-H1 compounds and golden nanometer particle-the H2 compound is respectively by neck ring DNA H1 and H2 and gold
Nano-particle is combined and formed；
The object recognition complex includes：Carboxyl magnetic microsphere-DNA1/2 compounds for recognizing miRNA, and
At least one in DNA3 for recognizing target molecules.
In above-mentioned probe, the sequence of the neck ring DNA H1 is as shown in SEQ ID NO.1, H2 sequence such as SEQ ID
It is specific as follows shown in NO.2：
H1：5’-SH-GCG ATT CCT AGG TTG AGC CCA GGG TTT TTT CAC AGT CCC TGG GCT
CAA CCT AGG-3’；(SEQ ID NO.1)
H2：5’-CCC TGG GCT CAA CCT AGG AAT CGC TTT TTT CCT AGG TTG AGC CCA GGG
ACT GTG-SH-3’；(SEQ ID NO.2).
In above-mentioned probe, the carboxyl magnetic microsphere-DNA1/2 compounds by two partial complementarities DNA1 and DNA2 chains
It is connected on the magnetic microsphere of carboxyl modified and forms.
The sequence with miRNA to be detected complementation and/or partial complementarity is included in the DNA1.
The nucleic acid aptamer sequence specifically bound with target molecules to be detected is included in the DNA3.
It is preferred that, the particle diameter of the golden nanometer particle is 5nm.
It is preferred that, the carboxyl magnetic microsphere particle diameter is 1 μm.
It is preferred that, the miRNA is miR-203 or miR-21.
When miRNA to be detected is miR-203, in the probe, DNA1 includes the sequence with miR-203 partial complementarities
Row, its sequence is as shown in SEQ ID NO.3, and DNA2 sequence is specific as follows as shown in SEQ ID NO.4：
DNA1：5’-GGG C TAG TGG TCC TAA ACA TTT CAC-NH2-3’；(SEQ ID NO.3)
DNA2：5’-GGA CCA CTA G CCC TGG GCT CAA CCT AGG AAT CGC-3’；(SEQ ID
NO.4)。
When miRNA to be detected is miR-21, in the probe, DNA1 ' includes the sequence with miR-21 partial complementarities,
Its sequence is as shown in SEQ ID NO.5, and DNA2 ' sequence is specific as follows as shown in SEQ ID NO.6：
DNA1’：5’-GGG T CAA CAT CAG TCT GAT AAG CTA-NH2-3’；(SEQ ID NO.5)
DNA2’：5’-CTG ATG TTG A CCC TGG GCT CAA CCT AGG AAT CGC-3’；(SEQ ID
NO.6)。
It is preferred that, the target molecules are ATP.
When the target molecules of detection are ATP, in the probe, DNA3 sequence is as shown in SEQ ID NO.7, specifically such as
Under：
5’-CCC AGG T CCC TGG GCT CAA CCT AGG AAT CGC GGG ACC TGG GGG AGT ATT
GCG GAG GAA GGT-3’；(SEQ ID NO.7)
The present invention also provides purposes of the above-mentioned probe in detection miRNA and/or target molecules with aptamer.
Purposes of the above-mentioned probe in the kit of detection miRNA and/or target molecules with aptamer is prepared
It is also protection scope of the present invention.
The second aspect of the present invention comprises the following steps there is provided the preparation method of above-mentioned probe：
(1) preparation of golden nanometer particle-H1 compounds and golden nanometer particle-H2 compounds：Respectively by neck ring DNA H1 and
H2 is incubated with golden nanometer particle in buffer solution, reacts 5-7h；Separated using agarose gel electrophoresis, by separation
Band is put into dialysis membrane and dialysed, and produces；
(2) preparation of carboxyl magnetic microsphere-DNA1/2 compounds：The magnetic bead of carboxyl modified is subjected to activation process, to work
DNA1 is added in magnetic bead after change, 3-5h is reacted, Magnetic Isolation is carried out to gained compound, carboxyl magnetic microsphere-DNA1 is produced
Compound；DNA2 is added, 1-3h is reacted, then carries out Magnetic Isolation, carboxyl magnetic microsphere-DNA1/2 compounds are produced.
In step (1), the preparation method of the golden nanometer particle is：Added in gold chloride and trisodium citrate mixed liquor
The sodium borohydride of ice, stirring to solution becomes pink, reacts 2-5h, produces.
It is preferred that, in gold chloride and trisodium citrate mixed liquor, the concentration of gold chloride is 0.25mM, trisodium citrate it is dense
Spend for 0.25mM, the concentration of the sodium borohydride of ice is 0.1M.
In step (1), the mol ratio that H1 and H2 are added with golden nanometer particle is 1：2.
In step (1), the buffer solution is 0.5 × tbe buffer liquid, consisting of：89mMTris, 89mM boric acid, 2mM
EDTA, pH 8.0.
In step (2), the activation method of the magnetic bead of carboxyl modified is：N- hydroxysuccinimidyl acyls are added into carboxyl modified magnetic bead
Imines sodium salt (NHS) and 1- ethyls -3- (3- dimethylamino-propyls) carbodiimide hydrochloride (EDC), react 1h at room temperature, will be mixed
Compound carries out Magnetic Isolation, produces.
A kind of side of target molecules of the third aspect of the present invention there is provided detection miRNA and/or with aptamer
Method, step is as follows：
(1) object recognition complex is mixed with the object solution to be detected of series concentration gradient respectively, reacted, to
Golden nanometer particle-H1 compounds and golden nanometer particle-H2 compounds are added in reacted solution, 3-5h is incubated, determines and is incubated
The ultra-violet absorption spectrum of solution, builds the working curve between target concentration to be detected and UV absorption peak value afterwards；
(2) object in detected sample is extracted, using the method for step (1), the ultraviolet suction of solution after being incubated is measured
Spectrum is received, working curve is substituted into, produces the concentration of object in detected sample.
Specifically, detection miRNA method is：
(1) the miRNA solution to be detected by carboxyl magnetic microsphere-DNA1/2 compounds respectively with series concentration gradient is mixed
Close, 1-3h is reacted under the conditions of 20-30 DEG C, Magneto separate and Aspirate supernatant are carried out after reaction；Jenner's grain of rice is added into supernatant
Son-H1 compounds and golden nanometer particle-H2 compounds, are incubated 3-5h, determine the ultra-violet absorption spectrum of solution after being incubated, and build
Working curve between miRNA concentration and UV absorption peak value；
(2) RNA in detected sample is extracted, using the method for step (1), the UV absorption light of solution after being incubated is measured
Spectrum, substitutes into working curve, produces the concentration of miRNA in detected sample.
The method of target molecules (by taking ATP as an example) of the detection with aptamer be：
(1) DNA3 is mixed with the ATP solution of series concentration gradient, 1-3h is reacted under the conditions of 20-30 DEG C, to reacted
Golden nanometer particle-H1 compounds and golden nanometer particle-H2 compounds are added in solution, 3-5h is incubated, solution after being incubated is determined
Ultra-violet absorption spectrum, builds the working curve between ATP concentration and UV absorption peak value；
(2) ATP in detected sample is extracted, using the method for step (1), the UV absorption light of solution after being incubated is measured
Spectrum, substitutes into working curve, produces the concentration of ATP in detected sample.
The detection miRNA of the present invention and/or the principle of the target molecules with aptamer are：
For miRNA detection, the present invention constructs a kind of based on golden nanometer particle-DNA hybridization chain reaction first
Probe, the probe includes：Golden nanometer particle-H1 compounds, golden nanometer particle-H2 compounds and carboxyl magnetic microsphere-DNA1/2
Compound；Wherein, carboxyl magnetic microsphere-DNA1/2 compounds are magnetic microsphere two partial complementarities of link in carboxyl modified
Comprising the sequence with miRNA partial complementarities to be detected in DNA (i.e. DNA1 and DNA2), DNA1, when with the presence of object miRNA
When, object miRNA and DNA1 is acted on, so as to discharge DNA2.Add golden nanometer particle-H1 compounds and Jenner's grain of rice
Son-H2 compounds, under DNA2 induction, H1 and H2 neck ring structure are opened, so that golden nanometer particle-H1 and Jenner
Hybridization chain reaction occurs for rice corpuscles-H2 so that golden nanometer particle mutual distance is close, and color changes, and passes through colorimetric method
The identification and detection to miRNA can be achieved, concrete principle figure is as shown in Figure 1.
For the detection of the target molecules with aptamer, present invention structure first is included can be with target to be detected
The DNA3 for the nucleic acid aptamer sequence that molecular specificity is combined is marked, is neck ring structure under the DNA3 normal conditions, when there is object
In the presence of target molecules, DNA3 and the specific combination of target molecules, the neck ring structures of DNA3 in itself are opened, and add Jenner
Rice corpuscles-H1 compounds and golden nanometer particle-H2 compounds, the DNA3 after neck ring structure is opened can induce H1 and H2 neck ring
Structure is opened, so that hybridization chain reaction occurs for golden nanometer particle-H1 and golden nanometer particle-H2 so that golden nanometer particle
Mutual distance is close, and color changes, and is that the identification and detection to target molecules, concrete principle figure can be achieved by colorimetric method
As shown in Figure 2.
One of key of the present invention is to miRNA and/or the target molecules with aptamer have detectability
How the design of probe, i.e., be designed to H1 and H2, enables its one side stable existence, on the other hand, knows in object
Neck ring structure can be smoothly opened under the induction of other compound, it is further to occur hybridization chain reaction.Wherein, if H1 and H2
The base number of middle hybridization is excessive, then H1 and H2 neck ring structure can be made excessively to stablize, luring in object recognition complex is difficult
Lead lower opening；If the base number hybridized in H1 and H2 is very few, H1 and H2 neck ring structure can be made unstable, in no object
In the presence of be also easily opened, cause detect false positive situation occur.The present invention devises multigroup in process of the test
The H1 and H2 of different structure, different hybridization base numbers, compare through actually detected effect, using with SEQ ID NO.1 and SEQ
H1 and H2 shown in ID NO.2, it is the most excellent to miRNA and the Detection results of target molecules.
Beneficial effects of the present invention：
The present invention first by golden nanometer particle with DNA hybridization chain reaction is ingenious connects, pass through golden nanometer particle
Mutual distance close to and the color change that occurs detects miRNA and target molecules with aptamer, detection method
Simply, sensitivity is high, and the least concentration detected to miRNA is up to 1.0 × 10-11M；The least concentration detected to ATP is up to 1.0
×10-8 M。
Brief description of the drawings
Fig. 1：Present invention detection miRNA schematic diagram；
Fig. 2：Present invention detection ATP schematic diagram；
Fig. 3：The transmission electron microscope picture of golden nanometer particle in embodiment 1；
Fig. 4：Detect various concentrations miR-203 working curve (A) and detection solution thereon (B)；
Fig. 5：Selectivity of the present invention to detection miR-203；
Fig. 6：Detect various concentrations miR-21 working curve.
Fig. 7 A：Various concentrations ATP uv absorption spectra and photo is detected in embodiment 7；
Fig. 7 B：Detect various concentrations ATP working curve.
Embodiment
The present invention is further illustrated in conjunction with the embodiments, it should explanation, and the description below is merely to explain this
Invention, is not defined to its content.
Embodiment 1：Detect the preparation of miRNA probes：
The probe includes：Golden nanometer particle-H1 compounds, golden nanometer particle-H2 compounds and carboxyl magnetic microsphere-
DNA1/2 compounds；Wherein：H1, H2 sequence are as follows：
H1：5’-SH-GCG ATT CCT AGG TTG AGC CCA GGG TTT TTT CACAGT CCC TGG GCT
CAA CCT AGG-3’；(SEQ ID NO.1)
H2：5’-CCC TGG GCT CAA CCT AGG AAT CGC TTT TTT CCT AGG TTGAGC CCA GGG
ACT GTG-SH-3’；(SEQ ID NO.2).
The sequence with miRNA to be detected complementation and/or partial complementarity is included in DNA1；DNA1 and DNA2 partial complementarities.
Wherein, golden nanometer particle-H1 compounds, the preparation method of golden nanometer particle-H2 compounds are：
(1) preparation of diameter 5nm golden nanometer particle：In 20mL gold chloride (0.25mM) and trisodium citrate (0.25
MM the sodium borohydride of 0.6mL0.1M ice) is added in mixed liquor, stirring to solution becomes pink, reacts 2-5h.Golden nanometer particle
Solution is placed at 4 DEG C and saved backup.The transmission electron microscope picture of golden nanometer particle is shown in Fig. 3, embodies preferably dispersiveness and particle diameter equal
One property.
(2) preparation of golden nanometer particle-H1 compounds and golden nanometer particle-H2 compounds：H1 and H2 respectively with gold nano
Particle is with 1:2 ratio is incubated in 0.5 × tbe buffer liquid (89mMTris, 89mM boric acid, 2mM EDTA, pH 8.0),
A small amount of repeatedly addition NaCl in 6h, course of reaction is reacted at room temperature, makes its concentration up to cause 50mM.Utilize 3% Ago-Gel electricity
Swimming is separated (running gel buffer solution：0.5×TBE,70V,1.5h).Band is put into dialysis membrane (molecular cut off 14000)
Middle dialysis, golden nanometer particle-DNA compounds are stored in 4 DEG C.
The preparation method of carboxyl magnetic microsphere-DNA1/2 compounds is：
Add in 200 μ L carboxyl modifieds magnetic beads (1 μm) 100 μ L 0.01M N-hydroxy-succinamides sodium salts (NHS) and
100 μ L 0.01M 1M1- ethyls -3- (3- dimethylamino-propyls) carbodiimide hydrochlorides (EDC), react 1h at room temperature, will
Mixture carries out Magnetic Isolation, and is washed with deionized water three times, and the magnetic bead after activation is resuspended in 200 μ L deionized waters, and adds
Enter 200 μ L1.0 × 10-5Mol/L DNA1, then, reacts 4h at room temperature, finally, and Magnetic Isolation is carried out to gained compound,
It is resuspended in 200 μ LmL PBS cushioning liquid, is just obtained again after being washed 3 times for 7.4 PBS cushioning liquid with 0.01M pH
Carboxyl magnetic microsphere-DNA1 compounds.200 μ L1.0 × 10 are added in above-mentioned carboxyl magnetic microsphere-DNA1 compounds-4mol/
L DNA2, then, reacts 2h at room temperature, and Magnetic Isolation is carried out to gained carboxyl magnetic microsphere-DNA1/2 compounds, uses
0.01M pH are resuspended in 100 μ LPBS cushioning liquid again after being washed 3 times for 7.4 PBS cushioning liquid.
Embodiment 2：Application of the probe of the present invention in detection miR-203
1. test material：
MiR-203 nucleotide sequence is as follows：
5’-GUG AAA UGU UUA GGA CCA CUA G-3’。
In the composition and preparation method be the same as Example 1 of probe, probe：
H1, H2 sequence are as follows：
H1：5’-SH-GCG ATT CCT AGG TTG AGC CCA GGG TTT TTT CACAGT CCC TGG GCT
CAA CCT AGG-3’；(SEQ ID NO.1)
H2：5’-CCC TGG GCT CAA CCT AGG AAT CGC TTT TTT CCT AGG TTGAGC CCA GGG
ACT GTG-SH-3’；(SEQ ID NO.2)；
DNA1, DNA2 sequence are as follows：
DNA1：5’-GGG C TAG TGG TCC TAA ACA TTT CAC-NH2-3’；(SEQ ID NO.3)
DNA2：5’-GGA CCA CTA G CCC TGG GCT CAA CCT AGG AAT CGC-3’；(SEQ ID
NO.4)。
2. detection method：
Specific method is as follows：
(1) preparation of miR-203 working solutions：The preparation of miR-203 working solutions：The DEPC water of 1 ‰ (volume ratios) will be contained
PBS cushioning liquid (pH 7.4) sterilized, and with its prepare various concentrations miR-203 solution.
(2) drafting of working curve：By 100 μ L carboxyl magnetic microsphere-DNA1/2 compounds and 100 μ L various concentrations
MiR-203 solution (0,1.0 × 10-11M, 5.0 × 10-11M, 1.0 × 10-10M, 3.0 × 10-10M) it is well mixed, it is anti-at 25 DEG C
Answer and Magneto separate and Aspirate supernatant carried out after 2h, then, added in supernatant 100 μ L golden nanometer particle-H1 compounds and
100 μ L golden nanometer particle-H2 compounds, are incubated record solution colour change after 4h, and survey ultra-violet absorption spectrum.With miR-203
Concentration is abscissa, using the UV absorption peak value ratio at 620nm and 520nm as ordinate drawing curve map.Can be with from Fig. 4
Find out, along with the raising of miR-203 concentration, respective change occurs for UV absorption peak value ratio at 620nm and 520nm.This method
Detect that (Y represents UV absorption peak value at 620nm and 520nm to miR-203 working curve equation for Y=0.022X+0.437
Than X represents miR-203 concentration, unit 10-10M, R=0.99), miR-203 concentration range is 1.0 × 10-11M-3.0×
10-10M.With the raising of miR-203 concentration, while it was observed that system color is gradually changed into purple from shallow claret is changed into shallow again
Blue (Fig. 4), illustrates the increase of miR-203 concentration, improves the aggregation extent of golden nanometer particle, and the color of system is just not yet
Together.
(3) miR-203 detection：The RNA in detected sample is extracted, using the method for step (1), is measured molten after incubation
The ultra-violet absorption spectrum of liquid, substitutes into working curve, produces the concentration of miR-203 in detected sample.
Using the probe colorimetric determination miR-203 least concentrations of golden nanometer particle-DNA hybridization chain reaction of the present invention
For 1.0 × 10-11M。
Embodiment 3：The selectivity of miR-203 detection methods is investigated
MiR-203, miR-21 (nucleotide sequence：5 '-UAG CUU AUC AGA CUG AUG UUG A-3 ') and miR-16
(nucleotide sequence：5 '-UAG CAG CAC GUA AAU AUU GGC G-3 ') solution preparation：1 ‰ (volume ratios) will be contained
The PBS cushioning liquid (pH 7.4) of DEPC water is sterilized, and prepares 1.0 × 10 with it-10M miR-203、2.0×10-9M
MiR-21 and 2.0 × 10-9M miR-16 solution.
By 100 μ L carboxyl magnetic microsphere-DNA1/2 compounds and 100 μ LmiR-203 solution (1.0 × 10-10M) or
MiR-21 solution (1.0 × 10-9) or miR-16 solution (1.0 × 10 M-9M), it is well mixed, reacts and carried out after 2h at 25 DEG C
Magneto separate and Aspirate supernatant, then, add 100 μ L golden nanometer particle-H1 compounds and 100 μ L Jenner's grain of rices in supernatant
Son-H2 compounds, are incubated after 4h, the UV absorption peak value ratio at record 620nm and 520nm.As shown in figure 5, only miR-
203 solution cause obvious change to the ultraviolet spectra of golden nanometer particle, and solution colour also generates change.Although miR-21
High 1 order of magnitude of concentration ratio miR-203 solution concentrations of solution and miR-16 solution, but do not cause ultraviolet absorption peak still
Value than significant change, therefore this method has good selectivity to miR-203.
Embodiment 4：Intracellular miR-203 Concentration Testings
RNA extraction in cell：37 DEG C, contain 5%CO2Moist environment in, with containing 10% hyclone and
1% dual anti-DMEM nutrient solution culture MCF-7 cells, 1.0 × 10 are taken with blood counting chamber meter4Individual MCF-7 cells simultaneously use PBS
(10mM, pH 7.4) is rinsed twice, the circulation under 37 DEG C and two environment of liquid nitrogen of MCF-7 cells is placed in 3min 3 times,
300 μ L cell pyrolysis liquids are added, then, 40 μ L chloroform and 200 μ L PBS (10mM, pH 7.4) are added in cell,
And 15s is stirred vigorously, 3min is then placed at room temperature, mixed solution is centrifuged 15min under 12000 rotating speeds, collects supernatant,
Then, 100 μ L isopropanol is added in supernatant, 10min is placed at room temperature after being well mixed, by mixture 13000
Centrifuge 10min under rotating speed to precipitate RNA, finally, obtained RNA is redispersed in the PBS cushioning liquid (pH 7.4) of DEPC processing
In.
MiR-203 in colorimetric probe method detection cell：By 100 μ L carboxyl magnetic microsphere-DNA1/2 compounds and carefully
The 100 μ LRNA solution extracted in born of the same parents are well mixed, and reacted at 25 DEG C and Magneto separate and Aspirate supernatant are carried out after 2h, then,
100 μ L golden nanometer particle-H1 compounds and 100 μ L golden nanometer particle-H2 compounds are added in supernatant, record molten after being incubated 4h
Liquid color change, colorimetric determination miR-203.Ultra-violet absorption spectrum is surveyed, the peakedness ratio at 620nm and 520nm is calculated, and according to
Calculated according to the working curve diagram drawn in Fig. 4 and obtain the concentration of miR-203 in cell for 460copys/cell.
Embodiment 5：Application of the probe based on golden nanometer particle-DNA hybridization chain reaction in detection miR-21
1. test material：
MiR-21 nucleotide sequence is as follows：
5’-GUG AAA UGU UUA GGA CCA CUA G-3’。
In the composition and preparation method be the same as Example 1 of probe, probe：
H1, H2 sequence are as follows：
H1：5’-SH-GCG ATT CCT AGG TTG AGC CCA GGG TTT TTT CACAGT CCC TGG GCT
CAA CCT AGG-3’；(SEQ ID NO.1)
H2：5’-CCC TGG GCT CAA CCT AGG AAT CGC TTT TTT CCT AGG TTGAGC CCA GGG
ACT GTG-SH-3’；(SEQ ID NO.2)；
DNA1 ', DNA2 ' sequence it is as follows：
DNA1’：5’-GGG T CAA CAT CAG TCT GAT AAG CTA-NH2-3’；(SEQ ID NO.5)
DNA2’：5’-CTG ATG TTG A CCC TGG GCT CAA CCT AGG AAT CGC-3’；(SEQ ID
NO.6)。
2. detection method：
Specific method is as follows：
(1) preparation of miR-21 working solutions：The preparation of miR-21 working solutions：The DEPC water of 1 ‰ (volume ratios) will be contained
PBS cushioning liquid (pH 7.4) is sterilized, and with its prepare various concentrations miR-21 solution.
(2) drafting of working curve：By 100 μ L carboxyl magnetic microsphere-DNA1/2 compounds and 100 μ L various concentrations
MiR-21 solution (0,1.0 × 10-11M, 5.0 × 10-11M, 1.0 × 10-10M, 3.0 × 10-10M) it is well mixed, it is anti-at 25 DEG C
Answer and Magneto separate and Aspirate supernatant carried out after 2h, then, added in supernatant 100 μ L golden nanometer particle-H1 compounds and
100 μ L golden nanometer particle-H2 compounds, are incubated record solution colour change after 4h, and survey ultra-violet absorption spectrum.It is dense with miR-21
Spend for abscissa, using the UV absorption peak value ratio at 620nm and 520nm as ordinate drawing curve map.Can from Fig. 6
Go out, along with the raising of miR-21 concentration, respective change occurs for UV absorption peak value ratio at 620nm and 520nm.This method is detected
MiR-21 working curve equation is that (Y represents UV absorption peak value ratio at 620nm and 520nm to Y=0.025X+0.359, and X is represented
MiR-21 concentration, unit 10-10M, R=0.99), miR-21 concentration range is 1.0 × 10-11 M-3.0×10-10M.With
The raising of miR-21 concentration, while it was observed that system color is gradually changed into purple from shallow claret is changed into light blue, explanation again
The increase of miR-21 concentration, improves the aggregation extent of golden nanometer particle, and the color of system is also just different.
(3) miR-21 detection：The RNA in detected sample is extracted, using the method for step (1), is measured molten after incubation
The ultra-violet absorption spectrum of liquid, substitutes into working curve, produces the concentration of miR-21 in detected sample.
Embodiment 6：The detection of intracellular miR-21 concentration
RNA extraction in cell：37 DEG C, contain 5%CO2Moist environment in, with containing 10% hyclone and
1% dual anti-DMEM nutrient solution culture MCF-7 cells, 1.0 × 10 are taken with blood counting chamber meter4Individual MCF-7 cells simultaneously use PBS
(10mM, pH 7.4) is rinsed twice, the circulation under 37 DEG C and two environment of liquid nitrogen of MCF-7 cells is placed in 3min 3 times,
300 μ L cell pyrolysis liquids are added, then, 40 μ L chloroform and 200 μ L PBS (10mM, pH 7.4) are added in cell,
And 15s is stirred vigorously, 3min is then placed at room temperature, mixed solution is centrifuged 15min under 12000 rotating speeds, collects supernatant,
Then, 100 μ L isopropanol is added in supernatant, 10min is placed at room temperature after being well mixed, by mixture 13000
Centrifuge 10min under rotating speed to precipitate RNA, finally, obtained RNA is redispersed in the PBS cushioning liquid (pH 7.4) of DEPC processing
In.
MiR-21 in colorimetric probe method detection cell：By 100 μ L carboxyl magnetic microsphere-DNA1/2 compounds and cell
100 μ LRNA solution of middle extraction are well mixed, and reacted at 25 DEG C and Magneto separate and Aspirate supernatant are carried out after 2h, then, upper
100 μ L golden nanometer particle-H1 compounds and 100 μ L golden nanometer particle-H2 compounds are added in clear liquid, recording solution after 4h is incubated
Color change, colorimetric determination miR-21.Ultra-violet absorption spectrum is surveyed, the peakedness ratio at 620nm and 520nm, and foundation is calculated
The working curve diagram drawn in Fig. 6, which is calculated, obtains the concentration of miR-21 in cell for 7600copys/cell.
Embodiment 7：Application of the probe of the present invention in detection ATP
(1) by 100 μ L DNA3 and the ATP solution (0,1.0 × 10 of 100 μ L various concentrations-8M, 5.0 × 10-8M, 1.0 ×
10-7M, 5.0 × 10-7M) it is well mixed, is reacted at 25 DEG C after 1h, adds 100 μ L golden nanometer particle-H1 compounds and 100 μ L
Golden nanometer particle-H2 compounds, are incubated record solution colour change after 4h.As can be seen from Figure 7A, carrying along with ATP concentration
Height, system color is gradually changed into purple from shallow claret and is changed into light blue (Fig. 7 A) again, illustrates the increase of ATP concentration, improves
The aggregation extent of golden nanometer particle, the color of system is also just different.Using ATP concentration as abscissa, at 620nm and 520nm
UV absorption peak value ratio is ordinate drawing curve map.As can be seen from Figure 7B, along with the raising of ATP concentration,
Respective change occurs for UV absorption peak value ratio at 620nm and 520nm.This method detection ATP working curve equation is Y=
(Y represents UV absorption peak value ratio at 620nm and 520nm to 1.62X+0.17, and X represents ATP concentration, unit 10-6M, R=0.99),
ATP concentration range is 1.0 × 10-8M-5.0×10-7M。
(2) ATP detection：The ATP in detected sample is extracted, using the method for step (1), solution after being incubated is measured
Ultra-violet absorption spectrum, substitutes into working curve, produces the concentration of ATP in detected sample.
DNA3 sequence is as follows：
5’-CCC AGG T CCC TGG GCT CAA CCT AGG AAT CGC GGG ACC TGG GGG AGT ATT
GCG GAG GAA GGT-3’。
Embodiment 8：The detection of ATP concentration in clasmatosis liquid
37 DEG C, contain 5%CO2Moist environment in, cultivated with the dual anti-DMEM of 10% hyclone and 1% is contained
Liquid culture MCF-7 cells.Rinsed twice by MCF-7 cells and with PBS (10mM, pH 7.4), ice-bath ultrasonic obtains clasmatosis
Liquid.100 μ L DNA3 are well mixed with 100 μ L clasmatosis liquid, reacted at 25 DEG C after 1h, 100 μ L Jenner's grain of rices are added
Son-H1 compounds and 100 μ L golden nanometer particle-H2 compounds, be incubated 4h after, survey ultra-violet absorption spectrum, calculate 620nm with
Peakedness ratio at 520nm, and be according to the concentration that the working curve diagram calculating drawn in Fig. 7 B obtains ATP in clasmatosis liquid
3.2×10-6M。
Above-described embodiment is preferably embodiment, but embodiments of the present invention are not by above-described embodiment of the invention
Limitation, other any Spirit Essences without departing from the present invention and the change made under principle, modification, replacement, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
SEQUENCE LISTING
<110>Linyi University
<120>MiRNA and/or target molecules with aptamer detection method and detection probe
<130> 2017
<160> 7
<170> PatentIn version 3.5
<210> 1
<211> 54
<212> DNA
<213>Artificial sequence
<400> 1
gcgattccta ggttgagccc agggtttttt cacagtccct gggctcaacc tagg 54
<210> 2
<211> 54
<212> DNA
<213>Artificial sequence
<400> 2
ccctgggctc aacctaggaa tcgctttttt cctaggttga gcccagggac tgtg 54
<210> 3
<211> 25
<212> DNA
<213>Artificial sequence
<400> 3
gggctagtgg tcctaaacat ttcac 25
<210> 4
<211> 34
<212> DNA
<213>Artificial sequence
<400> 4
ggaccactag ccctgggctc aacctaggaa tcgc 34
<210> 5
<211> 25
<212> DNA
<213>Artificial sequence
<400> 5
gggtcaacat cagtctgata agcta 25
<210> 6
<211> 34
<212> DNA
<213>Artificial sequence
<400> 6
ctgatgttga ccctgggctc aacctaggaa tcgc 34
<210> 7
<211> 61
<212> DNA
<213>Artificial sequence
<400> 7
cccaggtccc tgggctcaac ctaggaatcg cgggacctgg gggagtattg cggaggaagg 60
t 61
Claims (10)
1. a kind of probe, it is characterised in that including：Golden nanometer particle-H1 compounds, golden nanometer particle-H2 compounds and target
Thing recognition complex；
Golden nanometer particle-H1 compounds and golden nanometer particle-the H2 compound is respectively by neck ring DNA H1 and H2 and gold nano
Particle is combined and formed；
The object recognition complex includes：For recognizing miRNA carboxyl magnetic microsphere-DNA1/2 compounds, and it is used for
Recognize at least one in the DNA3 of target molecules.
2. probe as claimed in claim 1, it is characterised in that the sequence of the neck ring DNA H1 as shown in SEQ ID NO.1,
H2 sequence is as shown in SEQ ID NO.2.
3. probe as claimed in claim 1 or 2, it is characterised in that the miRNA is miR-203 or miR-21；The target
Molecule is ATP, fibrin ferment, lysozyme or EGF-R ELISA.
4. probe as claimed in claim 3, it is characterised in that when miRNA is miR-203, in the probe, DNA1 sequence
As shown in SEQ ID NO.3, DNA2 sequence is as shown in SEQ ID NO.4.
5. probe as claimed in claim 3, it is characterised in that when miRNA is miR-21, in the probe, DNA1 sequence
As shown in SEQ ID NO.5, DNA2 sequence is as shown in SEQ ID NO.6.
6. probe as claimed in claim 3, it is characterised in that when target molecules are ATP, DNA3 sequence such as SEQ ID
Shown in NO.7.
7. probe described in claim any one of 1-6 is in detection miRNA and/or target molecules with aptamer
Purposes；Or the purposes in the kit of detection miRNA and/or target molecules with aptamer is prepared.
8. the preparation method of the probe described in claim any one of 1-6, it is characterised in that comprise the following steps：
(1) preparation of golden nanometer particle-H1 compounds and golden nanometer particle-H2 compounds：Respectively by H1 and H2 and Jenner's grain of rice
Son is incubated in buffer solution, reacts 5-7h；Separated using agarose gel electrophoresis, the band of separation is put into dialysis
Dialyse, produce in film；
(2) preparation of carboxyl magnetic microsphere-DNA1/2 compounds：The magnetic bead of carboxyl modified is subjected to activation process, to after activation
Magnetic bead in add DNA1, react 3-5h, Magnetic Isolation carried out to gained compound, produce carboxyl magnetic microsphere-DNA1 and be combined
Thing；DNA2 is added, 1-3h is reacted, then carries out Magnetic Isolation, carboxyl magnetic microsphere-DNA1/2 compounds are produced.
9. preparation method as claimed in claim 8, it is characterised in that in step (1), the preparation method of the golden nanometer particle
For：The sodium borohydride of ice is added in gold chloride and trisodium citrate mixed liquor, stirring to solution becomes pink, reacts 2-
5h, is produced；
It is preferred that, in gold chloride and trisodium citrate mixed liquor, the concentration of gold chloride is 0.25mM, and the concentration of trisodium citrate is
0.25mM, the concentration of the sodium borohydride of ice is 0.1M.
10. the probe in detecting miRNA described in a kind of utilization claim any one of 1-6 and/or the target with aptamer
The method of molecule, it is characterised in that step is as follows：
(1) object recognition complex is mixed with the object solution to be detected of series concentration gradient respectively, reacted, to reaction
Golden nanometer particle-H1 compounds and golden nanometer particle-H2 compounds are added in solution afterwards, 3-5h is incubated, determines molten after being incubated
The ultra-violet absorption spectrum of liquid, builds the working curve between target concentration to be detected and UV absorption peak value；
(2) object in detected sample is extracted, using the method for step (1), the UV absorption light of solution after being incubated is measured
Spectrum, substitutes into working curve, produces the concentration of object in detected sample.
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