CN108588180A - A kind of plasma nano platform of Visual retrieval and its application - Google Patents
A kind of plasma nano platform of Visual retrieval and its application Download PDFInfo
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Abstract
The present invention provides plasma nano platform and its application of a kind of Visual retrieval, belongs to base mutation detection field.Plasma nano platform, including following components：The nano fibrous membrane of hair clip type nucleic acid probe H2 functionalization；Hair clip type nucleic acid probe H1 solution；H2O2Concentration is not less than the MES buffer solutions of 120 μm of ol/L；The hemin solution of 1~6 μm of ol/L；Contain 0.2~0.4mmol/L HAuCl4MES buffer solutions；The glutathione solution of 100~120 μm of ol/L.The plasma nano platform has the characteristics that simple, unmarked, hypersensitive and reusable.The plasma nano platform can visually detect the target DNA of super low concentration in human serum sample, and can improve single base and distinguish ratio 0.001% with fully-complementary sequence concentration, ensure that the reliability of single base mismatch identification.
Description
Technical field
The invention belongs to base mutation detection fields, and in particular to a kind of plasma nano platform of Visual retrieval and
It is applied.
Background technology
Single base mutation is the principal element of human diseases neurological susceptibility and drug response difference in genome.Base in blood
Mutant DNA concentration relatively low (10-16~10-12Mol/L) and compared with normal expression difference is small, and there is an urgent need to develop highly sensitive list
Base mutation diagnostic method.
Currently used single base mutation detection method mainly has DNA sequencing method, quantitative PCR method, electrochemical process, is based on rolling
Circle amplification technology (RCA) colorimetric method etc..But there is instruments complicated for operation, time-consuming and that needs are valuable etc. to lack for these methods
Point, it is difficult to be widely used in medical diagnosis.Existing report distinguishes the concentration ratio of single base mismatch and complete complementary, i.e. concentration area
Indexing, also rests on 0.01% ratio.There is an urgent need for Development of Novel, simplicity, overdelicate single base mutation detection methods, further
Concentration discrimination is improved, ensures the reliability of single base mismatch.
Invention content
In view of this, the purpose of the present invention is to provide a kind of plasma nano platform of Visual retrieval and its answering
With with higher sensitivity and resolution in terms of Visual retrieval list base mismatch and double base mismatch mutation.
In order to achieve the above-mentioned object of the invention, the present invention provides following technical scheme：
The present invention provides a kind of plasma nano platforms of Visual retrieval, including following components：
1) nano fibrous membrane of hair clip type nucleic acid probe H2 functionalization；Hair clip type nucleic acid probe H2 is affine by biotin-
Element is immobilized on nano fibrous membrane；
2) hair clip type nucleic acid probe H1 solution；
3) concentration is not less than the H of 120 μm of ol/L2O2MES buffer solutions；
4) the hemin solution of 1~6 μm of ol/L；
5) contain 0.2~0.4mmol/L HAuCl4MES buffer solutions；
6) glutathione solution of 100~120 μm of ol/L.
Preferably, immobilized a concentration of 0.7~1 μm ol/Ls of the hair clip type nucleic acid probe H2 on nano fibrous membrane.
Preferably, the nano fibrous membrane is that average fibre diameter is 100~1000nm and smooth pipe/polyhenylethylene nano
Fiber accumulates the 3D structural membranes of formation at random.
Preferably, the hair clip type nucleic acid probe H1 solution is the hair clip type nucleic acid probe H1 containing 0.7~1.0 μm of ol/L
Reaction solution；
The reaction solution includes following content component：10~20mmol/L Tris-HCl, 5~10mmol/L MgCl2With
The KCl of 10~30mmol/L；The pH of the reaction solution is 7~8.
Preferably, the construction method of the nano fibrous membrane of hair clip type nucleic acid probe H2 functionalization includes the following steps：
1) prepared polymer solution stirs 24~26h, the electrostatic spinning solution 1~3h of spinning that will be obtained, obtained nanometer
Tunica fibrosa；
The polymer solution be comprising mass fraction be 10~30% polystyrene, mass-volume concentration be 0.1~
The DMF solution of 0.3% TBAB；
2) nano fibrous membrane in the step 1) in argon plasma atmosphere is handled into 1~6min, obtains surface band
There is the tunica fibrosa of a large amount of oxygen-content active group and negative electrical charge；
The condition of the processing is as follows：Voltage is 30~60V, and electric current is 1.8~3.6A；
3) by nano fibrous membrane of the surface in the step 2) with a large amount of oxygen-content active group and negative electrical charge wait it is electric
1~2h is impregnated in the avidin solution that point pI is 10, it is primary to clean, it is then molten in the hair clip type nucleic acid probe H2 of biotin labeling
1~2h is impregnated in liquid, secondary cleaning obtains the nano fibrous membrane of hair clip type nucleic acid probe H2 functionalization.
Preferably, the condition of the step 1) spinning is as follows：12~18kV voltages, 0.2~0.5mL/h sample introduction speed, connect
It is 7~12cm to receive distance.
Preferably, a concentration of 2.0~4.0 μm of ol/L of the avidin solution；The hair clip type core of the biotin labeling
A concentration of 0.7~1 μm of ol/L of acid probe H2 solution.
The present invention provides the plasma nano platforms in the mono- base mismatch of Visual retrieval HIV DNA and double mistakes
With the application in base.
Preferably, the method for the Visual retrieval list base mismatch includes the following steps：
A. hair clip type nucleic acid probe H1 solution and sample to be tested solution are mixed, by hair clip type nucleic acid probe H2 functionalization
Nano fibrous membrane impregnates 15~17h in obtained mixed liquor, cleaning, then impregnates 1.5 in the hemin solution of 1~6 μm of ol/L
~2.5h, cleaning, obtains DNAzyme nano fibrous membranes；
B. by the DNAzyme nano fibrous membranes in H2O2Concentration not less than reaction 30 in 120 μM of MES buffer solutions~
40min is added containing 0.2~0.4mmol/L HAuCl4MES buffer solutions react 20~25min after, add 100~
The glutathione solution of 120 μm of ol/L, ultraviolet-uisible spectrophotometer detect the absorbance value of target DNA；According to absorbance
Value calculates the concentration of HIV DNA in testing sample solution.
Preferably, the hair clip type nucleic acid probe H2 has the nucleotide sequence as shown in SEQ ID NO.1 in sequence table；
The hair clip type nucleic acid probe H1 has the nucleotide sequence as shown in SEQ ID NO.2 in sequence table.
The present invention provides a kind of plasma nano platform of Visual retrieval, the plasma nano platform has
Simply, unmarked, hypersensitive and reusable feature.The plasma nano platform can visually detect human serum sample
The target DNA of super low concentration in product, and single base can be improved and distinguish ratio 0.001% with fully-complementary sequence concentration, it ensure that list
The reliability of base mispairing identification.
The present invention provides the plasma nano platforms in the mono- base mismatch of Visual retrieval HIV DNA and double mistakes
With the application in base.When detecting single base mismatch and double base mismatch respectively based on plasma nano platform, have relatively low
Detection sensitivity, detection sensitivity respectively reached 10-11Mol/L and 10-8The concentration of mol/L, and to the area of single base mismatch
Indexing reaches 0.001%.
Description of the drawings
Fig. 1 is the principle that plasma nano platform is used to detect target DNA；
Fig. 2 is that the SEM of Electrospun nano-fibers film schemes；
Fig. 3 is that the TEM of reaction solution schemes, and wherein Fig. 3-A scheme for the TEM of the AuNPs of no target DNA, and Fig. 3-B are to have target
The TEM of the AuNPs of DNA schemes；
Fig. 4 is the target DNA results in plasma nano platform detection human serum sample；
Fig. 5 is the detection base mismatch result of plasma nano platform.
Specific implementation mode
The present invention provides a kind of plasma nano platforms of Visual retrieval, including following components：
1) nano fibrous membrane of hair clip type nucleic acid probe H2 functionalization；Hair clip type nucleic acid probe H2 is affine by biotin-
Element is immobilized on nano fibrous membrane；
2) hair clip type nucleic acid probe H1 solution；
3) concentration is not less than 120 μM of H2O2MES buffer solutions；
4) the hemin solution of 1~6 μm of ol/L；
5) contain 0.2~0.4mmol/L HAuCl4MES buffer solutions；
6) glutathione solution of 100~120 μm of ol/L.
Plasma nano platform provided by the invention includes the nano fibrous membrane of hair clip type nucleic acid probe H2 functionalization.
The concentration of immobilized hair clip type nucleic acid probe H2 is preferably 0.7~1.0 μm of ol/L on nano fibrous membrane, more preferably 1.0 μm of ol/
L.The nano fibrous membrane is 100~1000nm by styroflex average diameter and smooth nanofiber accumulates shape at random
At 3D structural membranes, be specifically shown in Fig. 2.The styroflex average diameter is more preferably 400nm.
In the present invention, the construction method of the nano fibrous membrane of the hair clip type nucleic acid probe H2 functionalization, preferably includes
Following steps：
1) prepared polymer solution stirs 24~26h, the electrostatic spinning solution spinning 1-3h that will be obtained, obtained nanometer
Tunica fibrosa；
The polymer solution be comprising mass fraction be 10~30% polystyrene, mass-volume concentration be 0.1~
The DMF solution of 0.3% TBAB；
2) nano fibrous membrane in the step 1) in argon plasma atmosphere is handled into 1~6min, obtains surface band
There is the nano fibrous membrane of a large amount of oxygen-content active group and negative electrical charge；
The condition of the processing is as follows：Voltage is 30~60V, and electric current is 1.8~3.6A；
3) by nano fibrous membrane of the surface in the step 2) with a large amount of oxygen-content active group and negative electrical charge wait it is electric
1~2h is impregnated in the avidin solution that point pI is 10, it is primary to clean, then in the biotin mark of 100 μ L, 0.7~1.0 μm of ol/L
1~2h is impregnated in the hair clip type nucleic acid probe H2 solution of note, secondary cleaning obtains the nanometer of hair clip type nucleic acid probe H2 functionalization
Tunica fibrosa.
Prepared polymer solution of the present invention, stirs 24~26h, and the electrostatic spinning solution 1~3h of spinning that will be obtained is obtained
Fiber membrane；The polymer solution be comprising mass fraction be 10~30% polystyrene, mass-volume concentration be 0.1~
The DMF solution of 0.3% TBAB.The time of the stirring is preferably 24-28h.The condition of the spinning is as follows：12~18kV electricity
Pressure, 0.2~0.5mL/h sample introduction speed, it is 7~12cm to receive distance.The instrument of spinning is not particularly limited, using this field
Known spinning instrument.Preferably obtained nano fibrous membrane is dried after spinning.The temperature of the drying is excellent
60~80 DEG C are selected as, the time of the drying is preferably 3~6h.Nano fibrous membrane after drying is cut into a diameter of 4mm's
Circular membrane.
After obtaining nano fibrous membrane, the nano fibrous membrane handles in argon plasma atmosphere 1 by the present invention~
6min obtains the tunica fibrosa that surface carries a large amount of oxygen-content active group and negative electrical charge；The condition of the processing is as follows：Voltage is
30~60V, electric current are 1.8~3.6A.The type of the oxygen activity group is including-OH ,-COOH ,-C=O etc..The processing
Time is preferably 2~5min, more preferably 3~4min.The a large amount of active group and negative electrical charge that nanofiber film surface generates
Be conducive to it is subsequently through electrostatic interaction that Avidin is uniform, be effectively immobilized on nanofiber film surface.
After obtaining nano fibrous membrane of the surface with a large amount of oxygen-content active group and negative electrical charge, the present invention is by the surface
Nano fibrous membrane with a large amount of oxygen-content active group and negative electrical charge isoelectric point pI be 10 avidin solution in impregnate 1~
2h, it is primary to clean, 1~2h is then impregnated in the hair clip type nucleic acid probe H2 solution of biotin labeling, secondary cleaning is sent out
The nano fibrous membrane of clip-type nucleic acid probe H2 functionalization.
In the present invention, Avidin is positively charged in the avidin solution that the isoelectric point pI is 10, is conducive to and fiber
The surface of film is fixed by electrostatic interaction.The concentration of the avidin solution is preferably 2.0~4.0 μm of ol/L, and more preferably 2.0
μmol/L.The volume of the avidin solution is preferably 100 μ L.Cleaning is preferably secondary water with solution.The number of the cleaning
Preferably 3 times.The purpose once cleaned is the extra Avidin removal that will be not associated on tunica fibrosa.The biotin labeling
The concentration of hair clip type nucleic acid probe H2 solution is preferably 0.7~1.0 μm of ol/L, more preferably 1.0 μm of ol/L.The biotin mark
The concentration of the hair clip type nucleic acid probe H2 solution of note not only contributes to improve supported quantity and then improves detection sensitivity, but also protects
The accuracy of testing result will not be influenced because of space steric effect by demonstrate,proving in detection process.The method of the secondary cleaning is the same as primary clear
It washes.Secondary cleaning removes the hair clip type nucleic acid probe H2 for the biotin labeling not combined with Avidin.
Plasma nano platform provided by the invention includes hair clip type nucleic acid probe H1 solution.The hair clip type nucleic acid is visited
Needle H1 solution is preferably the reaction solution of the hair clip type nucleic acid probe H1 containing 0.7~1.0 μm of ol/L, more preferably 1.0 μm of ol/L；
The reaction solution preferably further includes following content component：10~20mmol/L Tris-HCl, 5~10mmol/L MgCl2With 10
The KCl of~30mmol/L；The pH of the reaction solution is 7~8.
In the present invention, the sequence design methodology of hair clip type the nucleic acid probe H2 and H1 are as follows：
The sequence of target DNA is divided into 3 segments, is followed successively by CBA by 5 ' to 3 ', the base number of wherein C is 6, the alkali of B
Radix is 9, and A base numbers are 8；The sequence of H1 is designed as A successively by 5 ' to 3 '-B-C-ADCB；Wherein A-Indicate the complementation of A
Sequence；B-Indicate the complementary series of B；C-Indicate that the complementary series of C, D are the sequence of poly G and poly T.The sequence of H2 by 5 ' to
3 ' are designed as C successively-D-A-CBADE；Wherein A-Indicate the complementary series of A；B-Indicate the complementary series of B；C-Indicate the complementary sequence of C
Row；D-Indicate the complementary series of D.
Plasma nano platform provided by the invention includes H2O2Concentration is not less than the MES buffer solutions of 120 μm of ol/L.Institute
State H2O2Concentration is preferably 120~240 μm of ol/L, more preferably 120 μm of ol/L.The present invention is not special to the MES buffer solutions
Limitation, using MES buffer solutions known in the art.
Plasma nano platform provided by the invention includes hemin solution.The concentration of the hemin solution is preferably 1
~6 μm of ol/L, more preferably 5 μm of ol/L.
Plasma nano platform provided by the invention includes containing 0.2~0.4mmol/L HAuCl4MES buffer solutions.
The HAuCl4Concentration be preferably 0.2mmol/L.
Plasma nano platform provided by the invention includes the glutathione solution of 100~120 μm of ol/L.Glutathione
The concentration of solution is preferably 100 μm of ol/L.The effect of the glutathione solution is to prevent the growth of gold nanoparticle.
Said components are preferably assembled into a kind of for detecting single mispairing alkali in plasma nano platform provided by the invention
The detection kit of base and double base mismatch.
The present invention provides the plasma nano platforms in the mono- base mismatch of Visual retrieval HIV DNA and double mistakes
With the application in base.
In the present invention, the method for the mono- base mismatch of the Visual retrieval HIV DNA preferably includes following steps：
A. hair clip type nucleic acid probe H1 solution and sample to be tested solution are mixed, by hair clip type nucleic acid probe H2 functionalization
Nano fibrous membrane impregnates 15~17h in obtained mixed liquor, cleaning, then impregnates 1.5 in the hemin solution of 1~6 μm of ol/L
~2.5h, cleaning, obtains DNAzyme nano fibrous membranes；
B. by the DNAzyme nano fibrous membranes in H2O2Concentration not less than reaction 30 in 120 μM of MES buffer solutions~
40min is added containing 0.2~0.4mmol/L HAuCl4MES buffer solutions react 20~25min after, add 100~
The glutathione solution of 120 μm of ol/L, ultraviolet-uisible spectrophotometer detect the absorbance value of target DNA；According to the target
Mark the HIV DNA concentrations in the absorbance value calculating sample to be tested solution of DNA.
The present invention mixes hair clip type nucleic acid probe H1 solution and sample to be tested solution, by hair clip type nucleic acid probe H2 functions
The nano fibrous membrane of change impregnates 15~17h in obtained mixed liquor, cleaning, then is soaked in the hemin solution of 1~6 μm of ol/L
1.5~2.5h is steeped, cleaning obtains DNAzyme nano fibrous membranes.The hair clip type nucleic acid probe H1 solution and sample to be tested solution
Volume ratio be preferably 110:1~50:1, more preferably 100:1.In the present invention, the time of immersion is preferably 16h.The hair
Clip-type nucleic acid probe H1 preferably has the nucleotide sequence as shown in SEQ ID NO.1 in sequence table；The hair clip type nucleic acid is visited
Needle H2 preferably has the nucleotide sequence as shown in SEQ ID NO.2 in sequence table.Hair clip type nucleic acid probe H2 functionalization is received
When rice tunica fibrosa is the circular membrane of diameter 4mm, the mixed liquor of hair clip type nucleic acid probe H1 solution and the formation of sample to be tested solution
Volume is preferably 100 μ L.The volume of the hemin solution is 100 μ L.
After obtaining DNAzyme nano fibrous membranes, the present invention is by the DNAzyme nano fibrous membranes in H2O2Concentration is not less than
The H of 120 μm of ol/L2O2MES buffer solutions in react 30~40min, add containing 0.2~0.4mmol/L HAuCl4MES
After buffer solution reacts 20~25min, the glutathione solution of 100~120 μm of ol/L is added, ultraviolet-uisible spectrophotometer comes
Detect the absorbance value of target DNA；It is dense that the HIV DNA in sample to be tested solution are calculated according to the absorbance value of the target DNA
Degree.The computational methods are obtained according to the regression equation that standard curve obtains.The standard curve is using known in the art
Conventional method is prepared.
In the present invention, the DNAzyme nano fibrous membranes and contain H2O2MES buffer solutions reaction before preferably will described in
DNAzyme nano fibrous membranes are blotted is placed in 96- orifice plates bottom again.The method blotted is carried out with filter paper.
In the present invention, the DNAzyme nano fibrous membranes are in H2O2Concentration is reacted in the MES buffer solutions not less than 120 μM
Time be preferably 30min.The HAuCl4MES buffer solutions reaction time be preferably 20min.When nano fibrous membrane is straight
When the circular membrane of diameter 4mm, the H2O2Concentration is not less than 120 μM of MES buffer solutions and HAuCl4MES buffer solutions volume it is only
It stands as 100 μ L, the volume of glutathione solution stands alone as 50 μ L.
The detection principle diagram of application provided by the invention as shown in Figure 1.When there are target detections in sample solution to be detected
When the DNA fragmentation of HIV, the DNA fragmentation of the DNA fragmentation of hair clip type nucleic acid probe H1 and target HIV, target HIV first will be in solution
The loop-stem structure of H1 is opened, and the parts the H1 chain of opening makes the H2 loop-stem structures of the biotin labeling on fiber membrane open, simultaneously
The DNA fragmentation of target HIV is replaced, and carries out the CHA reactions of next step, is formed in fiber film surface after repeatedly recycling
A large amount of H1-H2 double helix compounds；The rich G segments for being hidden in biotin-H2 at this time are exposed to form tetra- serobilas of G-, and
Tetra- serobilas of G-/hemin DNAzyme with peroxidase activity are formed in the presence of hemin.Functional fiber film over-assemble
DNAzyme can efficient catalytic H2O2It decomposes, the growth of AuNPs is regulated and controled with this, develop plasma visual signals, specially
DNAzyme, efficient catalytic H can be quickly assembled on tunica fibrosa2O2It decomposes, makes the reduction of its concentration.Low concentration H2O2It can make AuNPs
The speed of growth is slow, generates the AuNPs of aspherical aggregation, and solution colour is blue.When in sample to be detected do not contain target
When DNA fragmentation, H2O2Up to 120 μM of concentration, can quickly restore HAuCl4Spherical and good dispersion AuNPs is generated, solution is in red
Color.When therefore, by observing red, can determine whether can when observing blue without containing detection target DNA in sample to be detected
Judge in sample to be detected containing detection target DNA.
The plasma nano platform to a kind of Visual retrieval provided by the invention and its application with reference to embodiment
It is described in detail, but they cannot be interpreted as limiting the scope of the present invention.
Embodiment 1
One, the application in the structure and detection of plasma nano platform
As shown in Figure 1, the plasma nano platform in the present invention is caused by PS nano fibrous membranes sensing interface, HIV DNA
CHA iodines and film surface DNAzyme regulation and control AuNPs plasma signal three parts compositions, specific steps occur for film surface
It is as follows：
(1) preparation of PS nano fibrous membranes.Using DMF as solvent, the PS/TBAB/DMF that mass fraction is 10~30% is prepared
Solution, wherein TBAB are 0.1~0.3% (w/v), and 24~26h is stirred at room temperature and obtains electrostatic spinning solution；By electrostatic spinning
Solution receives the fiber membrane that 1~3h of spinning is obtained under conditions of distance is 10cm in 15kV voltages, 0.3mL/h sample introduction speed；
It is spare that tunica fibrosa is put in 3~6h of drying, then size that nano fibrous membrane is cut into diameter 5mm in 60~80 DEG C of baking ovens.Such as
Shown in Fig. 2, tunica fibrosa is the 3D structural membranes that the nanofiber uniform and smooth by diameter accumulates formation at random, and nanofiber is average
A diameter of 100~1000nm.
(2) tunica fibrosa of biotin-H2 functionalization.The tunica fibrosa prepared argon plasma is handled into 1~6min,
Voltage is 30~60V, and electric current is 1.8~3.6A.Then plasma treated PS nano fibrous membranes are soaked in 100 immediately
In the avidin solution of L2.0~4.0 μm μ ol/L, after 1~2h taking-up cleaned three times with secondary water, then in 100 μ L, 1.0 μ
Biotin-H2 (the 5 '-Biotin-CTG ACT AAA ACC CAA AAC CCG CTA GAG AAG TCA GTG of mol/L
TGG AAA ATC TCT AGC GGG TTT TGG GTT TTG GGT TTT GGG-3 ' SEQ ID No.1) 1 is impregnated in solution
The tunica fibrosa for obtaining biotin-H2 functionalization three times is cleaned in~2h, taking-up with secondary water.
(3) tunica fibrosa of biotin-H2 functionalization is immersed in 100 μ L and contains H1 (5- ' GCT AGA GAT TTT CCA
CAC TGA CTT CTC TAG CGG GTT TTG GGT TTT AGT CAG TGT GGA AAA-3 ' SEQ ID No.2) and
(DNA reaction solutions (pH=7~8 of (5- ' AGT CAG TGT GGA AAA TCT CTA GC-3 ' SEQ ID No.3) HIV DNA
10~20mmol/L Tris-HCl, 5~10mmol/LMgCl2With the KCl of 10~30mmol/L) in 15~17h, take out with two
Secondary water cleans tunica fibrosa, impregnates 1.5-2.5h in 100 μ L hemin solution later, taking-up is cleaned three times with secondary water.In this way
DNAzyme/PS nano fibrous membranes have just been obtained, and have been stored in 4 DEG C of refrigerators.
(4) it is blotted DNAzyme/PS nano fibrous membranes are once purged with filter paper and is positioned over 96- orifice plates bottom, into hole
100 μ L are added and contain not less than 240 μm ol/L H2O2MES buffer solutions.After reacting 30min, be added 100 μ L contain 0.2~
0.4mmol/L HAuCl4MES buffer solutions reaction 20min after, be added 50 100~120 μm of ol/L of μ L glutathione solution.
It uses digital camera to shoot photo later, and is detected by ultraviolet-uisible spectrophotometer, wavelength measurement ranging from 400-
800nm, and record the absorbance value at 550nm.Absorbance reduced value is named as-Δ A=A-A0, wherein A0Indicate chromophoric solution
In there is no an absorbance value of target DNA, and A indicates the absorbance value for having target DNA in chromophoric solution.
Two, the testing result of plasma nano platform
The plasma nano platform detects HIV DNA markers, and the results are shown in Figure 4, can detect low in human serum
To 10-17The HIV DNA of mol/L.And for nothing and there is the AuNPs of HIV DNA markers to be characterized to (A) and (B), such as Fig. 3
It is shown.Testing result to base mismatch is as shown in figure 5, detect respectively down to 10-11Mol/L and 10-8Single mispairing alkali of mol/L
Base and double base mismatch, reach 0.001% to the discrimination of single base mismatch.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Sequence table
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Claims (10)
1. a kind of plasma nano platform of Visual retrieval, which is characterized in that including following components：
1) nano fibrous membrane of hair clip type nucleic acid probe H2 functionalization；Hair clip type nucleic acid probe H2 is solid by biotin-avidin
It is loaded on nano fibrous membrane；
2) hair clip type nucleic acid probe H1 solution；
3) concentration is not less than 120 μM of H2O2MES buffer solutions；
4) the hemin solution of 1~6 μm of ol/L；
5) contain 0.2~0.4mmol/L HAuCl4MES buffer solutions；
6) glutathione solution of 100~120 μm of ol/L.
2. plasma nano platform according to claim 1, which is characterized in that the hair clip type nucleic acid probe H2 is receiving
Immobilized a concentration of 0.7~1 μm of ol/L on rice tunica fibrosa.
3. plasma nano platform according to claim 1 or 2, which is characterized in that the nano fibrous membrane is average
A diameter of 100~1000nm and smooth polystyrene nano fiber accumulate the 3D structural membranes of formation at random.
4. plasma nano platform according to claim 1, which is characterized in that the hair clip type nucleic acid probe H1 solution
For the reaction solution of the hair clip type nucleic acid probe H1 containing 0.7~1 μm of ol/L；
The reaction solution further includes following content component：10~20mmol/L Tris-HCl, 5~10mmol/L MgCl2With 10~
The KCl of 30mmol/L；The pH of the reaction solution is 7~8.
5. plasma nano platform according to claim 1 or 2, which is characterized in that hair clip type nucleic acid probe H2 functions
The construction method of the nano fibrous membrane of change includes the following steps：
1) prepared polymer solution, stirs 24~26h, and the electrostatic spinning solution 1~3h of spinning that will be obtained obtains nanofiber
Film；
The polymer solution be comprising mass fraction be 10~30% polystyrene, mass-volume concentration be 0.1~0.3%
TBAB DMF solution；
2) nano fibrous membrane in the step 1) in argon plasma atmosphere is handled into 1~6min, obtains surface with big
The oxygen-content active group of amount and the nano fibrous membrane of negative electrical charge；
The condition of the processing is as follows：Voltage is 30~60V, and electric current is 1.8~3.6A；
3) by nano fibrous membrane of the surface in the step 2) with a large amount of oxygen-content active group and negative electrical charge in isoelectric point pI
It is primary to clean to impregnate 1~2h in 10 avidin solution, then in the hair clip type nucleic acid probe H2 solution of biotin labeling
1~2h is impregnated, secondary cleaning obtains the tunica fibrosa of hair clip type nucleic acid probe H2 functionalization.
6. plasma nano platform according to claim 5, which is characterized in that the condition of the step 1) spinning is such as
Under：12~18kV voltages, 0.2~0.5mL/h sample introduction speed, it is 7~12cm to receive distance.
7. plasma nano platform according to claim 5 or 6, which is characterized in that the concentration of the avidin solution
For 2.0~4.0 μm of ol/L；A concentration of 0.7~1.0 μm of ol/L of the hair clip type nucleic acid probe H2 solution of the biotin labeling.
8. the plasma nano platform described in claim 1~7 any one is in the mono- base mismatch of Visual retrieval HIV DNA
With the application in double base mismatch.
9. application according to claim 8, which is characterized in that the side of the mono- base mismatch of the Visual retrieval HIV DNA
Method includes the following steps：
A. hair clip type nucleic acid probe H1 solution and sample to be tested solution are mixed, by the nanometer of hair clip type nucleic acid probe H2 functionalization
Tunica fibrosa impregnates 15~17h in obtained mixed liquor, cleaning, then in the hemin solution of 1~6 μm of ol/L impregnate 1.5~
2.5h, cleaning, obtains DNAzyme nano fibrous membranes；
B. by the DNAzyme nano fibrous membranes in H2O2Concentration reacts 30~40min in the MES buffer solutions not less than 120 μM,
It adds containing 0.2~0.4mmol/L HAuCl4MES buffer solutions react 20~25min after, add 100~120 μm of ol/
The glutathione solution of L, ultraviolet-uisible spectrophotometer detect the absorbance value of target DNA；It is waited for according to absorbance value calculating
The concentration of HIV DNA in sample solution.
10. application according to claim 1, which is characterized in that the hair clip type nucleic acid probe H2 has as in sequence table
Nucleotide sequence shown in SEQ IDNO.1；The hair clip type nucleic acid probe H1 has as shown in SEQ ID NO.2 in sequence table
Nucleotide sequence.
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| CN109884299A (en) * | 2019-03-27 | 2019-06-14 | 四川大学 | One-step method fluorescent detection system and blood coagulation enzyme assay method |
| CN109884304A (en) * | 2019-03-27 | 2019-06-14 | 四川大学 | A kind of the CHA iodine system and hypersensitive visible detection method of HCV Core Protein |
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| CN103293298A (en) * | 2013-07-05 | 2013-09-11 | 常熟理工学院 | Toxin detection method based on G-quadruplex nuclease |
| CN104726560B (en) * | 2015-02-11 | 2015-12-02 | 青岛大学 | A kind of colorimetry detecting zymoplasm |
| CN107828861B (en) * | 2017-11-21 | 2021-06-04 | 湖南工程学院 | Kit for detecting circulating nucleic acid based on microfluidic chip and G-quadruplex-heme DNA enzyme, and preparation method and application thereof |
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| CN109884299A (en) * | 2019-03-27 | 2019-06-14 | 四川大学 | One-step method fluorescent detection system and blood coagulation enzyme assay method |
| CN109884304A (en) * | 2019-03-27 | 2019-06-14 | 四川大学 | A kind of the CHA iodine system and hypersensitive visible detection method of HCV Core Protein |
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