CN109991421A - The method of DNA gel detection miRNA-21 based on blood glucose meter - Google Patents
The method of DNA gel detection miRNA-21 based on blood glucose meter Download PDFInfo
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Abstract
The method for the DNA gel detection miRNA-21 that the invention discloses a kind of based on blood glucose meter, specifically follows the steps below: the step S1, synthesis of DNA polymeric chain PS- I and DNA polymeric chain PS- II；Step S2, the preparation that DNA polymeric chain PS- I and DNA polymeric chain PS- II is packaged the DNA gel of amylase is prepared with step S1；Step S3, miRNA-21 is measured with blood glucose meter.The present invention solve the problems, such as easily operated blood glucose meter can the real time measure miRNA-21 content, realize the public for the detection demand of determinand miRNA-21.
Description
Technical field
The invention belongs to technical field of molecular biology, detect more particularly to a kind of DNA gel based on blood glucose meter
The method of miRNA-21.
Background technique
MicroRNA (miRNA) is a kind of endogenous tiny RNA with 20-24 base, and this tiny RNA is sent out in early stage
It educates, play important regulatory function in the life processes such as cell Proliferation, apoptosis and death.In the recent period, studies have shown that the table of miRNA
Closely related with the generation, diffusion and post-operative recovery of tumour up to level, therefore, it can be used as the potential life of ideal of corresponding cancer
Object marker.Particularly, miRNA is not only expressed in cancer cell and tumor tissues, is equally expressed in peripheral blood.Moreover,
Expression of the miRNA in peripheral blood has obvious correlation with the expression in cancer cell and tumor tissues.In addition,
Peripheral blood detection has many advantages, such as noninvasive, therefore, realizes identification of the miRNA in peripheral blood and quantifies, for cancer
Early diagnosis, treatment and postoperative evaluation are very important.
The Sensitive Detection of miRNA is constantly subjected to it with less base number, similar nucleic acid sequence and low content
Etc. features limitation.Although as RNA marking method, microarray analysis, RT-polymerase chain reaction, flow cytometry and enzyme are urged
Change the methods of amplifying technique and have been applied to medical center and Research-oriented Lab, but these methods nevertheless suffer from some limits
System, such as large-scale instrument and equipment is needed, accurate temperature control needs longer detection time, further needs exist for professional people
Member is operated.So far, the public can detect in nonspecific place to miRNA for few detection techniques.On the contrary,
The public needs to take a substantial amount of time sample is sent to professional institution with money to detect, this has not only delayed the early stage of cancer
Diagnosis, and prevent the post-operative recovery of cancer from obtaining effective monitoring.Therefore, it is badly in need of a kind of letter that the public can be used directly
Single, quick, sensitive miRNA detection method.
MiRNA-21 as miRNA detect in more part and parcel, be often used in measurement, but identification miRNA-21
Existing method is all complex.
In past many years, blood glucose meter is to realize the most successful example of patient's self-test.Blood glucose meter have small size,
Easy to operate, the advantages that price is low, testing result is reliable, still, traditional blood glucose meter only can be used to monitor the content of glucose, this pole
Big limits its extensive use.
Summary of the invention
The method for the DNA gel detection miRNA-21 that the purpose of the present invention is to provide a kind of based on blood glucose meter, solves
The public existing in the prior art, which needs to take a substantial amount of time, to be sent to professional institution for sample with money and detects and blood
Sugared instrument is only capable of the problem of detection glucose content.
The technical scheme adopted by the invention is that the method for the DNA gel detection miRNA-21 based on blood glucose meter, is specifically pressed
It is carried out according to following steps:
Step S1, the synthesis of DNA polymeric chain PS- I and DNA polymeric chain PS- II；
Step S2, with step S1 prepare DNA polymeric chain PS- I and DNA polymeric chain PS- II be packaged amylase DNA it is solidifying
The preparation of glue；
Step S3, miRNA-21 is measured with blood glucose meter.
Further, the step S1 mixes the acrylamide aqueous solution that mass fraction is 25% with buffer solution, so
It is added in I aqueous solution of Strands- of acrylamide label of 3mM afterwards, mixed solution is deaerated in vacuum desiccator
The ammonium persulfate aqueous solution that mass fraction is 5% and N, N, N' that mass fraction is 5%, N'- tetramethyl second two is added in 10min
Amine aqueous solution continues the 18min that deaerates, obtains DNA polymeric chain PS- I, be placed in 4 DEG C of preservations；
The nucleotide sequence of Strands- I is as shown in SEQ ID No.1；
The acrylamide aqueous solution that mass fraction is 25% is mixed with buffer solution, is then added to the acryloyl of 3mM
In II aqueous solution of Strands- of amine label, mixed solution is deaerated 10min in vacuum desiccator, mass fraction, which is added, is
5% ammonium persulfate aqueous solution, the N, N, N' that mass fraction is 5%, N'- tetramethylethylenediamine aqueous solution continue to deaerate
18min obtains DNA polymeric chain PS- II, is placed in 4 DEG C of preservations；
The nucleotide sequence of Strands- II is as shown in SEQ ID No.2.
Further, the step S2 is specifically followed the steps below:
By with DNAzyme cleavage site Substrate-link and EA-21 and EB-21 be mixed to form mixed liquor, 37
DEG C 30min is incubated, mixed liquor is taken to be added to containing DNA polymeric chain PS- I, DNA polymeric chain PS- II and 0.4 μ g/ μ L amylase
Hydrogel is formed in centrifuge tube, so that Strands- I, Strands- II and the Substrate- with DNAzyme cleavage site
The mass ratio of the material of link is 1:1:1；
The nucleotide sequence of DNAzyme is as shown in SEQ ID No.5；
The nucleotide sequence of Substrate-link with DNAzyme cleavage site is as shown in SEQ ID No.7.
Further, the step S3 will contain 4-6 μM of Pb2+Step is added to the solution of various concentration miRNA-21
In the centrifuge tube containing hydrogel of S2 preparation, 4h is reacted under the conditions of 37 DEG C, the amidin of 14 μ g/ μ L is added, 37
DEG C the reaction was continued 2h, is taken supernatant, is detected using blood glucose meter to the glucose content in supernatant, obtain various concentration
The corresponding glucose content of miRNA-21, to determine the concentration of miRNA-21 according to glucose content.
Further, in the step S3 nucleotide sequence of EA-21 as shown in SEQ ID No.3；
The nucleotide sequence of EB-21 is as shown in SEQ ID No.4.
Further, the nucleotide sequence of the miRNA-21 is as shown in SEQ ID No.6.
Further, the acrylamide aqueous solution that the mass fraction is 25%: buffer solution: the acrylamide mark of 3mM
The ammonium persulfate aqueous solution that the Strands- I: mass fraction of note is 5%: the N that mass fraction is 5%, N, N', N'- tetramethyl second
The volume ratio of two amine aqueous solutions is 4:12:8:1:1；
The acrylamide aqueous solution that mass fraction is 25%: buffer solution: the Strands- of the acrylamide label of 3mM
II: the ammonium persulfate aqueous solution that mass fraction is 5%: the N that mass fraction is 5%, N, N', N'- tetramethylethylenediamine aqueous solution
Volume ratio be 4:12:8:1:1；
Buffer solution pH is 7.3, Na in buffer solution+Concentration and Cl-Concentration be 100-150mM.
Further, it is with DNAzyme cleavage site that the amount of the substance of described EA-21, EB-21 is equal
/ 10th of the amount of the substance of Substrate-link；
Substrate-link and EA-21 and EB-21 with DNAzyme cleavage site are mixed to form the volume of mixed liquor
It is equal with the volume containing DNA polymeric chain PS- I, DNA polymeric chain PS- II and 0.4 μ g/ μ L amylase.
Further, the hydrogel solution of the step S2 preparation: contain 4-6 μM of Pb2+With the solution of miRNA-21: forming sediment
The volume ratio of amidin is 6:1:4.
The invention has the advantages that the DNA gel used in the present invention has the advantages that easily to prepare, easily store, solution
Easily operated blood glucose meter of having determined can the real time measure miRNA-21 content the problem of, therefore the present invention realize the public for
The detection demand of determinand miRNA-21.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
Some embodiments of invention for those of ordinary skill in the art without creative efforts, can be with
It obtains other drawings based on these drawings.
Figure 1A is working principle diagram of the present invention for miRNA-21 detection；
Figure 1B is the working principle for the hydrogel of miRNA-21 detection and used nucleic acid hybridization sequences signal
Figure；
Fig. 2A is the proof diagram for being formed and being hydrolyzed to DNA gel based on starch/iodine solution；
Fig. 2 B is the proof diagram responded to DNA gel to determinand miRNA-21 based on starch/iodine solution；
Fig. 3 A is investigation figure of the DNA gel to amylase encapsulation ability；
Fig. 3 B is that blood glucose meter measured value changes with time figure under the conditions of 0.4 μ g/ μ L amylase and 4 μ g/ μ L starch；
Fig. 3 C is variation diagram of the blood glucose meter measured value with starch concentration；
Fig. 3 D is EA-21 and the influence diagram that EB-21 concentration responds blood glucose meter to miRNA-21 signal；
Fig. 4 A is the dependency graph of blood glucose meter measured value Yu miRNA-21 concentration；
Fig. 4 B is the specific investigation figure to the DNA gel of miRNA-21 response.
Specific embodiment
Below in conjunction with the embodiment of the present invention, technical scheme in the embodiment of the invention is clearly and completely described,
Obviously, described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Based in the present invention
Embodiment, every other embodiment obtained by those of ordinary skill in the art without making creative efforts, all
Belong to the scope of protection of the invention.
DNA gel is skeleton using polymer, and functionalization DNA is cross linked chain, is widely used in small molecule, ion or egg
In white detection.DNAzyme is a kind of DNA molecular with catalytic activity, has been applied to DNA, protein, metal ion
Detection, complete DNAzyme are divided into multi-part nuclease i.e. MNAzyme, and more flexible application is provided in biological detection for it.
Accordingly, it is considered to the ease for operation of blood glucose meter is arrived, and the extraordinary performance of the encapsulation of gel and releasability and DNAzyme, the present invention
Amylase is encapsulated into DNA gel, with the starch separation in solution, starch cannot be hydrolyzed and generate glucose.It is to be measured when being added
After object miRNA-21, MNAzyme is reset, and forms the DNAzyme with catalytic capability, and being catalyzed has DNAzyme cleavage site
Substrate-link is disconnected, and hydrogel disintegrates, and for amylase release into solution, hydrolysis starch generates glucose.Use blood glucose
The concentration of the determinand miRNA-21 of instrument detection glucose content, glucose content and addition is positively correlated, and then realizes and treat
Survey the Sensitive Detection of object miRNA-21 concentration.
The method of DNA gel detection miRNA-21 based on blood glucose meter, specifically follows the steps below:
Step S1, the synthesis of DNA polymeric chain PS- I and DNA polymeric chain PS- II:
The acrylamide aqueous solution that mass fraction is 25% is mixed with buffer solution, is then added to the acryloyl of 3mM
In I aqueous solution of Strands- of amine label, mixed solution is deaerated 10min in vacuum desiccator, degassing time is to polymeric chain
Synthesis have an impact, then be added mass fraction be 5% ammonium persulfate aqueous solution and mass fraction be 5% N, N, N', N'-
Tetramethylethylenediamine aqueous solution continues the 18min that deaerates, obtains DNA polymeric chain PS- I, be placed in 4 DEG C of preservations；
The nucleotide sequence of Strands- I is as shown in SEQ ID No.1, specially 5 '-Acrydite-
ATGTGTGGTTGTAG-3'；
The acrylamide aqueous solution that mass fraction is 25% is mixed with buffer solution, is then added to the acryloyl of 3mM
In II aqueous solution of Strands- of amine label, mixed solution is deaerated 10min in vacuum desiccator, mass fraction is then added
For 5% ammonium persulfate aqueous solution, mass fraction be 5% N, N, N', N'- tetramethylethylenediamine aqueous solution continues to deaerate
18min obtains DNA polymeric chain PS- II, is placed in 4 DEG C of preservations；
The nucleotide sequence of Strands- II is as shown in SEQ ID No.2, specially 5 '-Acrydite-
CTGTTTGGTAGTTG-3'；
The acrylamide aqueous solution that mass fraction is 25%: buffer solution: the Strands- I of the acrylamide label of 3mM:
The ammonium persulfate aqueous solution that mass fraction is 5%: the N that mass fraction is 5%, N, N', the body of N'- tetramethylethylenediamine aqueous solution
Product is than being 4:12:8:1:1；
The acrylamide aqueous solution that mass fraction is 25%: buffer solution: the Strands- of the acrylamide label of 3mM
II: the ammonium persulfate aqueous solution that mass fraction is 5%: the N that mass fraction is 5%, N, N', N'- tetramethylethylenediamine aqueous solution
Volume ratio be 4:12:8:1:1；
Buffer solution pH is 7.3, Na in buffer solution+Concentration and Cl-Concentration be 100-150mM；
N, N, N', N'- tetramethylethylenediamine catalysis ammonium persulfate generate free radicals, and cause acrylamide monomer polymerization；
Step S2, the preparation of the DNA gel of amylase is encapsulated:
By with DNAzyme cleavage site Substrate-link and EA-21 and EB-21 be mixed to form mixed liquor, 37
DEG C 30min is incubated, mixed liquor is taken to be added to containing DNA polymeric chain PS- I, DNA polymeric chain PS- II and 0.4 μ g/ μ L amylase
Hydrogel is formed in centrifuge tube, so that Strands- I, Strands- II and the Substrate- with DNAzyme cleavage site
The mass ratio of the material of link is 1:1:1；
The nucleotide sequence of DNAzyme is as shown in SEQ ID No.5, specially 5 '-CATCTCTTCTCCGAGCCGGTCG
AAATAGTGAGT-3'；
The nucleotide sequence of Substrate-link with DNAzyme cleavage site is as shown in SEQ ID No.7, specifically
For 5 '-CTACAACCACACATAACTCACTATrAGGAAGAGATGAACAACTACCAAACA G-3 '；
The nucleotide sequence of EA-21 is as shown in SEQ ID No.3, specially 5 '-TAGCTTTCAACATCAGTCGGTCGA
AATAGTGAGT-3'；
The nucleotide sequence of EB-21 is as shown in SEQ ID No.4, and specially 5 '-
CATCTCTTCTCCGAGCCTGATAAGCTA-3'；
The nucleotide sequence of DNAzyme is as shown in SEQ ID No.5, specially 5 '-CATCTCTTCTCCGAGCCGGTCG
AAATAGTGAGT-3'；
Since PS- I, PS- II are polymeric chains, concentration difficulty is determined, so PS- I can only be controlled, on II polymeric chain of PS-
The concentration of Strands- I, Strands- II；
The amount of the substance of EA-21, EB-21 is equal be the Substrate-link with DNAzyme cleavage site object
/ 10th of the amount of matter；
Substrate-link and EA-21 and EB-21 with DNAzyme cleavage site are mixed to form the volume of mixed liquor
It is equal with the volume containing DNA polymeric chain PS- I, DNA polymeric chain PS- II and 0.4 μ g/ μ L amylase；
Step S3, miRNA-21 is measured with blood glucose meter:
4-6 μM of Pb will be contained2+With the solution of various concentration miRNA-21 be added to step S2 preparation contain hydrogel
Centrifuge tube in, 4h is reacted under the conditions of 37 DEG C, the reaction time has an impact to the glucose content in later period, by the shallow lake of 14 μ g/ μ L
Amidin is added, and 37 DEG C of the reaction was continued 2h are taken supernatant, examined using blood glucose meter to the glucose content in supernatant
It surveys, the corresponding glucose content of various concentration miRNA-21 is obtained, to determine miRNA-21's according to glucose content
Concentration；
The nucleotide sequence of miRNA-21 is as shown in SEQ ID No.6, and specially 5 '-
UAGCUUAUCAGACUGAUGUUGA-3'；
The hydrogel solution of step S2 preparation: contain 4-6 μM of Pb2+With the solution of miRNA-21: the body of amidin
Product is than being 6:1:4；
DNAzyme is a metal ion species enzyme, and catalytic action needs Pb2+Presence could activate；Pb is added2+Be for
Activation DNAzyme, DNAzyme, which are split into after MNAzyme, to be needed to be added determinand miRNA-21 and could form enzymatic structure, Pb2+
Excessively could DNAzyme be activated.
Figure 1A is working principle diagram of the present invention for miRNA-21 detection, as shown in Figure 1, firstly, passing through acrylamide
The combined polymerization of the Strands- II and acrylamide monomer of Strands- I or the acrylamide label of label, by Strands- I
It is connected on linear polyacrylamide polymer with Strands- II, forms DNA polymeric chain PS- I and DNA polymeric chain PS- II；
DNAzyme splits into multi-part nuclease i.e. MNAzyme, and MNAzyme is made of EA-21, EB-21 and miRNA-21, only EA-
21, when EB-21 and miRNA-21 are existed simultaneously, the enzymatic structure for being formed and there is cavity could be hybridized, DNAzyme catalytic activity is swashed
It is living；Secondly, EA-21 and EB-21 is complementary with having the Substrate-link of DNAzyme cleavage site, formed unactivated
DNAzyme structure, Strands- II in Strands- I and DNA polymeric chain PS- II in DNA polymeric chain PS- I with have
The two end regions Complementary hybridizations of the Substrate-link of DNAzyme cleavage site, so that DNA polymeric chain PS- I and DNA be gathered
It closes chain PS- II and is cross-linked into hydrogel, amylase is encapsulated in inside hydrogel；Initially, amylase is steadily encapsulated in water-setting
Inside glue, starch is located in the solution outside hydrogel, it is achieved that the separation of amylase and starch；Finally, when there is determinand
When miRNA-21 occurs, the conformation of MNAzyme activates the catalytic capability of its own by rearranging, therefore, in Pb2+In the presence of
The Substrate-link with DNAzyme cleavage site is cut, the decomposition of hydrogel and the release of amylase, amylase are caused
Catalytic starch hydrolysis generates a large amount of glucose and quantitative determines for blood glucose meter.In addition, the DNAzyme of activation will be automatically moved to
Neighbouring substrate carries out next step cutting, to generate the signal of enzymatic amplification.Therefore, by using blood glucose meter in supernatant
Glucose content is detected, and while the present invention realizes quantitative analysis miRNA-21, also maintains quick and easy operation.
Figure 1B is the working principle for the miRNA-21 hydrogel detected and used nucleic acid hybridization sequences signal
Figure.
Fig. 2A is the proof diagram for being formed and being hydrolyzed to DNA gel based on starch/iodine solution.Only encapsulating amylase
When in hydrogel including DNAzyme sequence, that is, Fig. 2A (c), starch/iodine solution navy blue becomes colorless, and blank hydrogel is
It Fig. 2A (a) and encapsulates amylase but is added without in the DNAzyme i.e. hydrogel sample of Fig. 2A (b), upper layer starch/iodine is kept
It is blue constant.The result shows that being successfully prepared the DNA hydrogel of encapsulation amylase, and amylase is encapsulated in hydrogel
Inside, starch are located in the outer solution of gel, realize the physical separation of amylase and starch.In addition, only existing in DNAzyme
Under, the Substrate-link with DNAzyme cleavage site can be catalyzed and disconnected, cause hydrogel to disintegrate and discharge starch
Enzyme, amylase is by Starch Hydrolysis at glucose.
Fig. 2 B is the proof diagram responded to DNA gel to determinand miRNA-21 based on starch/iodine solution.There is no amylase
It is encapsulated in gel with EA-21 and EB-21, and that miRNA-21 is not added, the clear gel layer of test tube bottom and upper layer are formed sediment
Powder/iodine mixture blue solution forms obvious comparison, i.e. hydrogel is highly stable under normal operation.Equally, for not having
EA-21, EB-21 and miRNA-21, that is, Fig. 2 B (b) have EA-21, EB-21 but do not have miRNA-21 i.e. Fig. 2 B (c) or do not have
EA-21, EB-21 but the hydrogel for encapsulating amylase for having the i.e. Fig. 2 B (d) of miRNA-21, all do not show color change,
It is essential part in enzyme-hydrogel reaction system that this, which shows amylase, EA-21, EB-21 and miRNA-21 all,.It will
MiRNA-21 is added to containing EA-21, EB-21 i.e. Fig. 2 B (e) and encapsulates in the hydrogel of amylase, and upper layer blue solution becomes
Be it is colourless, show when object chain miRNA-21 sequence occurs, gel section disintegrate, release amylase to starch/iodine solution
In, Starch Hydrolysis.All of above result further demonstrates the feasibility of experiment mechanism.
Fig. 3 A is investigation figure of the DNA gel to amylase encapsulation ability.On the one hand, the amount for increasing amylase will increase grape
The yield of sugar, to improve the sensitivity of sensing detection.On the other hand, it is limited for giving the amylase load capacity of hydrogel
, once exceeding its capacity, excessive amylase cannot be encapsulated in inside hydrogel well, even if in no miRNA- to be measured
Starch can be also hydrolyzed in the case where 21 appearance, blood glucose meter is caused false positive signal occur.It, will in order to optimize the load capacity of amylase
Starch/iodine solution is added to be packaged in a series of hydrogels of various concentration amylase (0-1.0 μ g/ μ L) in advance.From Fig. 3 A
In as can be seen that for encapsulation Acarbose concentrations be lower than 0.4 μ g/ μ L hydrogel, even if after 6 hours, being also not observed
Color change, on the contrary, observing that blue starch/iodine solution color is obvious when the concentration of amylase increases to 0.6 μ g/ μ L
It fades, which shows that amylase leakage will seriously affect testing result if using excessive amylase.Therefore, 0.4 is selected
The amylase of μ g/ μ L encapsulates the hydrogel of amylase to prepare.
Fig. 3 B is that blood glucose meter measured value changes with time figure under the conditions of 0.4 μ g/ μ L amylase and 4 μ g/ μ L starch, research
The influence that catalyzed by amylase starch reaction time generates blood glucose meter registration.The result shows that blood glucose meter signal is with hydrolysis time
Increase and increase, tend towards stability after 2 hours.Therefore, 2 hours Best Times as amylorrhexis starch are selected.
Fig. 3 C is variation diagram of the blood glucose meter measured value with starch concentration.Initially, blood glucose meter signal is with amylose concentration
Increase and increase, when Acarbose concentrations reach 14 μ g/ μ L, blood glucose meter signal tends to be steady, and shows dense in fixed starch
Under degree, Acarbose concentrations reach saturation.Therefore, best item of the 14 μ g/ μ L Acarbose concentrations as amylorrhexis starch is selected
Part.
Fig. 3 D is EA-21 and the influence diagram that EB-21 concentration responds blood glucose meter signal to miRNA-21.In view of activation
DNAzyme is moved to the adjacent Substrate-link with DNAzyme cleavage site automatically further to be cut
It cuts, and generates the signal of enzymatic amplification, therefore the dosage of EA-21, EB-21 will affect the degree of hydrogel hydrolysis.Pass through detection
The content of glucose optimizes the amount of EA-21, EB-21 in the starch solution of upper layer.It is being shown in Fig. 3 D the result shows that blood glucose meter believe
Number with EA-21 and EB-21/ have DNAzyme cleavage site Substrate-link the mass ratio of the material example increase and increase
Add.However, EA-21 and EB-21/ has the mass ratio of the material of the Substrate-link of DNAzyme cleavage site is excessively high will lead
Blood glucose meter signal is caused to gradually decrease.This phenomenon is to will limit the DNAzyme of activation since the density of EA-21 and EB-21 are too high
It is spontaneous be moved to adjacent substrate generated with further being cut signal amplification.Therefore, the object of EA-21 and EB-21 is selected
The amount of matter is the amount of the Substrate-link substance with DNA enzymatic cleavage site 1/10 for constructing subsequent hydrogel.
Fig. 4 A is the dependency graph of blood glucose meter measured value Yu miRNA-21 concentration.As miRNA-21 concentration rises, blood glucose
Instrument signal dramatically increases.In addition, apparent positive is presented in the logarithm of the miRNA-21 in blood glucose meter signal and 0.5-250fmol
It closes.In addition, being limited to 0.325fmol according to the estimation detection of 3 σ rules, show that the present invention being capable of effectively Sensitive Detection determinand
miRNA-21。
Fig. 4 B is specific investigation figure of the invention；Due to the high similitude of miRNA family's family sequence, the spy of miRNA detection
The opposite sex is to evaluate the important parameter of detection performance.Using the miRNA with miRNA-21 sequence with different number base mismatch, such as
MiRNA-21-1, miRNA-21-2, miRNA-21-3 and the corresponding segment of the miRNA of some other types, as miRNA-335,
MiRNA-155, miRNA-122 study the sequence-specific of the DNA gel of miRNA, if Fig. 4 B is shown, miRNA-21 are added and produces
Raw blood glucose meter signal is more much higher than the blood glucose meter signal that the target fragments of other more than one base mismatch generate is added.More than
The result shows that the specificity of prepared hydrogel is very high, illustrate that it is applied to the miRNA family with high sequence similarity
The potentiality of miRNA identification in member.
The nucleotide sequence of miRNA-335 is as shown in SEQ ID No.8, and specially 5 '-
UCAAGAGCAAUAACGAAAAAUGU-3'；
The nucleotide sequence of miRNA-155 is as shown in SEQ ID No.9, and specially 5 '-
UUAAUGCUAAUCGUGAUAGGGGU-3'；
The nucleotide sequence of miRNA-122 is as shown in SEQ ID No.10, and specially 5 '-
UGGAGUGUGACAAUGGUGUUUGU-3'；
The nucleotide sequence of miRNA-21-1 is as shown in SEQ ID No.11, and specially 5 '-
UAGCUUAUCAGACUGAAGUUGA-3'；
The nucleotide sequence of miRNA-21-2 is as shown in SEQ ID No.12, and specially 5 '-
UAGCUUAUCAGACUGAAGUUCA-3'；
The nucleotide sequence of miRNA-21-3 is as shown in SEQ ID No.13, and specially 5 '-
UAGCUUAUCAGACUGGAGUUCA-3’。
Each embodiment in this specification is all made of relevant mode and describes, same and similar portion between each embodiment
Dividing may refer to each other, and each embodiment focuses on the differences from other embodiments.Especially for system reality
For applying example, since it is substantially similar to the method embodiment, so being described relatively simple, related place is referring to embodiment of the method
Part explanation.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the scope of the present invention.It is all
Any modification, equivalent replacement, improvement and so within the spirit and principles in the present invention, are all contained in protection scope of the present invention
It is interior.
Sequence table
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<212> RNA
<213> artificial sequence
<400> 10
uggaguguga caaugguguu ugu 23
<210> 11
<211> 22
<212> RNA
<213> artificial sequence
<400> 11
uagcuuauca gacugaaguu ga 22
<210> 12
<211> 22
<212> RNA
<213> artificial sequence
<400> 12
uagcuuauca gacugaaguu ca 22
<210> 13
<211> 22
<212> RNA
<213> artificial sequence
<400> 13
uagcuuauca gacuggaguu ca 22
Claims (9)
1. the method for the DNA gel detection miRNA-21 based on blood glucose meter, which is characterized in that specifically follow the steps below:
Step S1, the synthesis of DNA polymeric chain PS- I and DNA polymeric chain PS- II；
Step S2, the DNA gel that DNA polymeric chain PS- I and DNA polymeric chain PS- II is packaged amylase is prepared with step S1
Preparation；
Step S3, miRNA-21 is measured with blood glucose meter.
2. the method for the DNA gel detection miRNA-21 according to claim 1 based on blood glucose meter, which is characterized in that institute
It states step S1 to mix the acrylamide aqueous solution that mass fraction is 25% with buffer solution, is then added to the acryloyl of 3mM
In I aqueous solution of Strands- of amine label, mixed solution is deaerated 10min in vacuum desiccator, it is 5% that mass fraction, which is added,
Ammonium persulfate aqueous solution and mass fraction be 5% N, N, N', N'- tetramethylethylenediamine aqueous solution, continue deaerate 18min,
DNA polymeric chain PS- I is obtained, 4 DEG C of preservations are placed in；
The nucleotide sequence of Strands- I is as shown in SEQ ID No.1；
The acrylamide aqueous solution that mass fraction is 25% is mixed with buffer solution, is then added to the acrylamide mark of 3mM
In II aqueous solution of Strands- of note, mixed solution is deaerated 10min in vacuum desiccator, it is 5% that mass fraction, which is added,
Ammonium persulfate aqueous solution, the N, N, N' that mass fraction is 5%, N'- tetramethylethylenediamine aqueous solution continue the 18min that deaerates, obtain
DNA polymeric chain PS- II is placed in 4 DEG C of preservations；
The nucleotide sequence of Strands- II is as shown in SEQ ID No.2.
3. the method for the DNA gel detection miRNA-21 according to claim 2 based on blood glucose meter, which is characterized in that institute
Step S2 is stated specifically to follow the steps below:
Substrate-link and EA-21 and EB-21 with DNAzyme cleavage site is mixed to form mixed liquor, 37 DEG C of temperature
30min is educated, mixed liquor is taken to be added to the centrifugation containing DNA polymeric chain PS- I, DNA polymeric chain PS- II and 0.4 μ g/ μ L amylase
Hydrogel is formed in pipe, so that Strands- I, Strands- II and the Substrate-link with DNAzyme cleavage site
The mass ratio of the material be 1:1:1；
The nucleotide sequence of DNAzyme is as shown in SEQ ID No.5；
The nucleotide sequence of Substrate-link with DNAzyme cleavage site is as shown in SEQ ID No.7.
4. the method for the DNA gel detection miRNA-21 according to claim 3 based on blood glucose meter, which is characterized in that institute
Stating step S3 will be containing 4-6 μM of Pb2+With the solution of various concentration miRNA-21 be added to step S2 preparation contain hydrogel
Centrifuge tube in, react 4h under the conditions of 37 DEG C, the amidin of 14 μ g/ μ L be added, 37 DEG C of the reaction was continued 2h take supernatant
Liquid detects the glucose content in supernatant using blood glucose meter, obtains the corresponding grape of various concentration miRNA-21
Sugared content, to determine the concentration of miRNA-21 according to glucose content.
5. the method for the DNA gel detection miRNA-21 according to claim 3 based on blood glucose meter, which is characterized in that institute
The nucleotide sequence of EA-21 in step S3 is stated as shown in SEQ ID No.3；
The nucleotide sequence of EB-21 is as shown in SEQ ID No.4.
6. the method for the DNA gel detection miRNA-21 according to claim 4 based on blood glucose meter, which is characterized in that institute
The nucleotide sequence of miRNA-21 is stated as shown in SEQ ID No.6.
7. the method for the DNA gel detection miRNA-21 according to claim 2 based on blood glucose meter, which is characterized in that institute
State the acrylamide aqueous solution that mass fraction is 25%: buffer solution: Strands- I: quality points of the acrylamide label of 3mM
The ammonium persulfate aqueous solution that number is 5%: the volume ratio of the N that mass fraction is 5%, N, N', N'- tetramethylethylenediamine aqueous solution is
4:12:8:1:1；
The acrylamide aqueous solution that mass fraction is 25%: buffer solution: the Strands- II: matter of the acrylamide label of 3mM
Measure the ammonium persulfate aqueous solution that score is 5%: the N that mass fraction is 5%, N, N', the volume of N'- tetramethylethylenediamine aqueous solution
Than for 4:12:8:1:1；
Buffer solution pH is 7.3, Na in buffer solution+Concentration and Cl-Concentration be 100-150mM.
8. the method for the DNA gel detection miRNA-21 according to claim 5 based on blood glucose meter, which is characterized in that institute
State the substance of EA-21, EB-21 amount it is equal be the Substrate-link with DNAzyme cleavage site substance amount
1/10th；
Substrate-link and EA-21 and EB-21 with DNAzyme cleavage site are mixed to form the volume of mixed liquor and contain
There is the volume of DNA polymeric chain PS- I, DNA polymeric chain PS- II and 0.4 μ g/ μ L amylase equal.
9. the method for the DNA gel detection miRNA-21 according to claim 6 based on blood glucose meter, which is characterized in that institute
State the hydrogel solution of step S2 preparation: containing 4-6 μM of Pb2+With the solution of miRNA-21: the volume ratio of amidin is
6:1:4。
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113943777A (en) * | 2021-10-29 | 2022-01-18 | 福州大学 | Construction method of self-protection DNA enzyme walker and application of self-protection DNA enzyme walker in living cell miRNA detection |
| CN114231632A (en) * | 2021-12-20 | 2022-03-25 | 深圳大学 | DNA hydrogel microneedle patch capable of stimulating response and preparation method and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103804590A (en) * | 2013-12-19 | 2014-05-21 | 沈阳药科大学 | DNA hydrogel and application thereof in detection of peroxide |
| CN103913542A (en) * | 2014-02-20 | 2014-07-09 | 厦门大学 | Instant quantitative analysis method directed at targets |
| US20160252515A1 (en) * | 2013-11-08 | 2016-09-01 | The Board Of Trustees Of The University Of Illinois | Personal glucose meters for detection and quantification of enzymes and metabolites based on coenzyme detection |
| CN107557459A (en) * | 2017-09-29 | 2018-01-09 | 沈阳药科大学 | A kind of method that DNA hydrogels and DNAzyme detections SNP is used in combination |
-
2019
- 2019-04-12 CN CN201910294545.0A patent/CN109991421A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20160252515A1 (en) * | 2013-11-08 | 2016-09-01 | The Board Of Trustees Of The University Of Illinois | Personal glucose meters for detection and quantification of enzymes and metabolites based on coenzyme detection |
| CN103804590A (en) * | 2013-12-19 | 2014-05-21 | 沈阳药科大学 | DNA hydrogel and application thereof in detection of peroxide |
| CN103804590B (en) * | 2013-12-19 | 2015-08-19 | 沈阳药科大学 | A kind of DNA hydrogel and the application in superoxide detects thereof |
| CN103913542A (en) * | 2014-02-20 | 2014-07-09 | 厦门大学 | Instant quantitative analysis method directed at targets |
| CN103913542B (en) * | 2014-02-20 | 2017-06-16 | 厦门大学 | A kind of real-time and quantification analysis method for target |
| CN107557459A (en) * | 2017-09-29 | 2018-01-09 | 沈阳药科大学 | A kind of method that DNA hydrogels and DNAzyme detections SNP is used in combination |
Non-Patent Citations (1)
| Title |
|---|
| YANMEI SI ETAL: ""Oligonucleotide Cross-Linked Hydrogel for Recognition and Quantitation of MicroRNAs Based on a Portable Glucometer Readout"", 《ACS APPL.MATER.INTERFACES》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113943777A (en) * | 2021-10-29 | 2022-01-18 | 福州大学 | Construction method of self-protection DNA enzyme walker and application of self-protection DNA enzyme walker in living cell miRNA detection |
| CN114231632A (en) * | 2021-12-20 | 2022-03-25 | 深圳大学 | DNA hydrogel microneedle patch capable of stimulating response and preparation method and application thereof |
| CN114231632B (en) * | 2021-12-20 | 2023-09-22 | 深圳大学 | DNA hydrogel microneedle patch capable of stimulating response and preparation method and application thereof |
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