CN110452961A - It is a kind of for detecting the preparation method and application of the hydrogel of miRNA - Google Patents
It is a kind of for detecting the preparation method and application of the hydrogel of miRNA Download PDFInfo
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- CN110452961A CN110452961A CN201910738718.3A CN201910738718A CN110452961A CN 110452961 A CN110452961 A CN 110452961A CN 201910738718 A CN201910738718 A CN 201910738718A CN 110452961 A CN110452961 A CN 110452961A
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J3/00—Processes of treating or compounding macromolecular substances
- C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
- C08J3/03—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media
- C08J3/075—Macromolecular gels
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J2371/00—Characterised by the use of polyethers obtained by reactions forming an ether link in the main chain; Derivatives of such polymers
- C08J2371/02—Polyalkylene oxides
Abstract
The present invention provides a kind of for detecting the preparation method of the hydrogel of miRNA, it is a kind of technology for producing hydrogel in batches, use the method for stopping flowing photoetching, and the shape by changing mask plate change synthesis hydrogel shape with reach different shapes represent different miRNA. encoding of graphs technology replace it is traditional fluorescence-encoded, so that code capacity greatly improves, and it does not need additional light supply apparatus to be excited, identification is carried out to hydrogel of different shapes by way of image procossing and solves the time-consuming of traditional detection method, laborious expense and high drawback, the processing of image is combined with mobile phone terminal APP simultaneously, realize the intelligent portability of detection, user is set to stay indoors the prevention and detection that can be realized to cancer, there is important meaning in disease detection field.
Description
Technical field
It is especially a kind of for detecting the hydrogel of miRNA the present invention relates to hydrogel preparation and image processing techniques
Preparation method and application.
Background technique
MiRNA is a kind of Microrna for being present in human body, and the function in relation to miRNA in cancer has had a large amount of text
Research is offered, multiple studies have shown that there is also miRNA in human peripheral blood, and the peripheral blood miRNA of kinds cancer patient is expressed
Spectrum, which occurs specifically to sexually revise more and more evidences and shows to recycle miRNA, can be used as a kind of novel molecular marker and is applied to cancer
Diagnosis and the traditional detection based on miRNA such as gene sequencing, quantitative fluorescent PCR of prognosis evaluation etc., but these detection modes
Large-scale instrument cooperation is required without exception, it is not only time-consuming, laborious, but also some testing agencies such as be only suitable for hospital, it can not
Each family is benefited to realize that portable, Huimin is popular.
Summary of the invention
It is a kind of for detecting the preparation method of the hydrogel of miRNA technical problem to be solved by the present invention lies in providing.
Another technical problem to be solved by this invention is to provide a kind of miRNA detection technique based on hydrogel.
In order to solve the above technical problems, the technical scheme is that
It is a kind of for detecting the preparation method of the hydrogel of miRNA, the specific steps are as follows:
1) design of light transmission mask: exposure mask board diameter is 30mm, with a thickness of 1mm；
2) stop building for flowing photoetching platform；
3) hydrogel detects；
4) preparation is connected with the hydrogel fines of miRNA aptamers.
Preferably, above-mentioned for detecting the preparation method of the hydrogel of miRNA, the pattern of step 1) the light transmission mask
Design, the specific steps are as follows: use L-Edit software, selecting diameter is the region of 30mm as working region, in this workspace
Another figure layer is done in the region that a length of 5-7mm is chosen in the center in domain, width is 3-5mm, then subtracts this figure layer with working region
Just obtaining central area is rectangular mask plate, continues to synthesize rectangle, square, circle or three furthermore with such method
It is angular.
Preferably, above-mentioned for detecting the preparation method of the hydrogel of miRNA, the step 2) stops flowing photoetching platform
Build, the specific steps are as follows:
(1) above-mentioned mask plate is placed in microscopical optical path, adjusts angle and focal position；
(2) by the punching two ends of (60 μm high, the long 1cm) carrier gas passage respectively of PDMS chip, one end is sample holes, and one section is
Sample outlet hole；
(3) ultraviolet source is connected into adjustment focal length in microscopical optical path makes light-resource fousing into channel, and adjustment eyepiece is put
Big multiple is 10 ×, object lens magnification is 40 ×.
Preferably, above-mentioned for detecting the preparation method of the hydrogel of miRNA, the step 4) preparation is connected with miRNA
The hydrogel fines of aptamers, the specific steps are as follows:
(1) prepare pre-polymer solution: by 3.5 milliliters of 700 liquid of PEG-DA, 2 milliliters of 200 liquid of PEG, 0.5ml light draws
It sends out 1173 liquid of agent Darocur and 4 milliliters of 3 × TE buffer mixes, it is stand-by as pre-polymer solution；
(2) by prepolymer inject injection port at syringe in, syringe is packed into syringe pump, set injection speed as
500-1000μm/min；
(3) syringe pump is opened, makes prepolymer full of channel, stops injection pump work, open ultraviolet source and expose 50-
100ms reopens syringe pump and goes out particle after forming particle, repeats above step until obtaining sufficient amount of hydrogel
Particle；
(4) by the particle being collected into 1 × TET buffer solution for cleaning for several times, be finally resuspended in 1 × TET of 300-1000 μ l
In buffer, 4 DEG C are saved for use.
Preferably, above-mentioned for detecting the preparation method of the hydrogel of miRNA, ultraviolet light is utilized in the step 4) (3)
The length-width ratio for solidifying the hydrogel fines of synthesis miRNA aptamers (by taking miRNA21 as an example) is (50-100) μ m (30-50) μ
m。
A kind of miRNA detection technique based on hydrogel is the miRNA detection based on hydrogel particle, is covered by changing
Hydrogel of different shapes may be implemented in the shape of the shape control light transmission of diaphragm plate, different coding modes is realized, in conjunction with image
Processing is to identify that different shapes achievees the purpose that the more inspections of mixed inspection.
Preferably, the above-mentioned miRNA detection technique based on hydrogel, concrete application method is:
(1) TET (sodium chloride containing 700 mMs every liter) of 50 microlitres of 50 microlitres of hydrogel particle addition is taken；
(2) take 5 microlitres (10-1000amol) of miRNA to be checked (every containing 500 mMs in 95 microlitres of TE Buffer
The sodium chloride risen)；
(3) above-mentioned two solution is mixed in metal bath 90 minutes (55 DEG C, 1500r/min)；
(4) reaction solution is cleaned three times in TET；
(5) it is added after 900r/min is centrifuged 3 minutes and takes 100 microlitres of lower liquid, the hybridization buffer of 245 μ l is added
(900TET, 0.031 gram of NaCl, 100 microlitres of NEB Buffer, 2 microlitres of ATP, 0.33 microlitre of generic linker, 1.88 microlitres
T4DNA ligase)；
(6) (21.5 DEG C, 1500r/min) 30 minutes in metal bath；
(7) SAPE (phycoerythrin connecting with Avidin) that 5 μ l dilute 100 times is added.
Structure of the invention has the advantages that:
Above-mentioned for detecting the preparation method of the hydrogel of miRNA, hydrogel fines preparation process is simple, and by particle
Preparation connect step completion with aptamers, time needed for shortening detection；The shape of synthesis particle can be precisely controlled
With pattern, to realize encoding of graphs function when the mixed inspection of a variety of miRNA；Realize the popular of disease initial survey.
The preparation method is a kind of technology for producing hydrogel in batches, using the method for stopping flowing photoetching, and is passed through
Change the shape of mask plate to change the shape of the hydrogel of synthesis to reach different shapes and represent different miRNA. figures
The technology of shape coding replaces traditional fluorescence-encoded, so that code capacity greatly improves, and does not need additional light supply apparatus
It is excited, the expense that identification solves traditional detection method is carried out to hydrogel of different shapes by way of image procossing
When, laborious expense high drawback again, while the processing of image is combined with mobile phone terminal APP, and it is portable to realize detection intelligence
Change, so that user is stayed indoors the prevention and detection that can be realized to cancer, there is important meaning in disease detection field.
Detailed description of the invention
Fig. 1: the hydrogel fines microscope photograph via bright field for stopping flowing photolithography method preparation is utilized.
Fig. 2: the figure after being reacted using the hydrogel for stopping flowing photolithography method preparation with various concentration object to be checked.
Fig. 3: hydrogel particle fluorescence field (control) figure after non-object to be checked is added.
Specific embodiment
Embodiment 1
Prepared by the hydrogel of the detection for miRNA, specific step is as follows:
1) design method of the design of light transmission mask: using L-Edit software, selectes the region that diameter is 30mm
As working region, another figure layer is done in the region that we choose a length of 5mm, width is 3mm in the center of this working region, so
Subtracting this figure layer just to obtain central area with working region afterwards is rectangular mask plate.
2) stop flowing photoetching platform to build: above-mentioned mask plate be placed in microscopical optical path, adjust angle with
Focal position.By the punching two ends of (60 μm high, the long 1cm) carrier gas passage respectively of PDMS chip, one end is sample holes, and one section is out
Sample hole.Ultraviolet source, which is connected into adjustment focal length in microscopical optical path, makes light-resource fousing into channel, adjusts eyepiece amplification factor
For 10 ×, object lens magnification is 40 ×.
3) hydrogel detects: hydrogel being injected in chip, ultraviolet light is introduced into the above-mentioned optical path regulated.It adjusts thick
Thin quasi- burnt spiral, until can see purpose shape hydrogel (Fig. 1 is left).
4) it is connected with the preparation of the hydrogel fines of miRNA aptamers: the commercialization probe (purchase of target miRNA will be captured
From the raw work in Shanghai) it is mixed in hydrogel solution by 1:100, hydrogel particle can be obtained by method irradiation 3) (Fig. 1 is left).
Embodiment 2
Prepared by the hydrogel of the detection for miRNA, specific step is as follows:
1) design method of the design of light transmission mask: using L-Edit software, selectes the region that diameter is 30mm
As working region, another figure layer is done in the region that we choose a length of 3mm, width is 3mm in the center of this working region, so
This figure layer, which is subtracted, with working region afterwards just obtains the mask plate being square to central area.
2) stop flowing photoetching platform to build: above-mentioned mask plate be placed in microscopical optical path, adjust angle with
Focal position.By the punching two ends of (60 μm high, the long 1cm) carrier gas passage respectively of PDMS chip, one end is sample holes, and one section is out
Sample hole.Ultraviolet source, which is connected into adjustment focal length in microscopical optical path, makes light-resource fousing into channel, adjusts eyepiece amplification factor
For 10 ×, object lens magnification is 40 ×.
3) hydrogel detects: hydrogel being injected in chip, ultraviolet light is introduced into the above-mentioned optical path regulated.It adjusts thick
Thin quasi- burnt spiral, until can see purpose shape hydrogel (in Fig. 1).
4) it is connected with the preparation of the hydrogel fines of miRNA aptamers: the commercialization probe (purchase of target miRNA will be captured
From the raw work in Shanghai) it is mixed in hydrogel solution by 1:100, hydrogel particle can be obtained by method irradiation 3) (in Fig. 1).
Embodiment 3
Prepared by the hydrogel of the detection for miRNA, specific step is as follows:
1) design method of the design of light transmission mask: using L-Edit software, selectes the region that diameter is 30mm
As working region, another figure layer is done in the region that we choose that radius is 3mm in the center of this working region, then recruitment
Subtracting this figure layer just to obtain central area as region is circular mask plate.
2) stop flowing photoetching platform to build: above-mentioned mask plate be placed in microscopical optical path, adjust angle with
Focal position.By the punching two ends of (60 μm high, the long 1cm) carrier gas passage respectively of PDMS chip, one end is sample holes, and one section is out
Sample hole.Ultraviolet source, which is connected into adjustment focal length in microscopical optical path, makes light-resource fousing into channel, adjusts eyepiece amplification factor
For 10 ×, object lens magnification is 40 ×.
3) hydrogel detects: hydrogel being injected in chip, ultraviolet light is introduced into the above-mentioned optical path regulated.It adjusts thick
Thin quasi- burnt spiral, until can see purpose shape hydrogel (Fig. 1 is right).
4) it is connected with the preparation of the hydrogel fines of miRNA aptamers: the commercialization probe (purchase of target miRNA will be captured
From the raw work in Shanghai) it is mixed in hydrogel solution by 1:100, hydrogel particle can be obtained by method irradiation 3) (Fig. 1 is right).
Embodiment 4
Based on the detection technique of hydrogel miRNA, specific step is as follows for detection:
1) detection based on hydrogel particle miRNA:
Take 50 microlitres of hydrogel particle that 50 microlitres of TET (sodium chloride containing 700 mMs every liter) is added,
Take five microlitres (10-1000amol) of miRNA to be checked in 95 microlitres of TE Buffer (containing 500 mmoles
Your every liter of sodium chloride), by above-mentioned two solution mix with 90 minutes in metal bath (55 DEG C, 1500r/min), by reaction solution in
It is cleaned in TET three times, 900r/min is added after being centrifuged 3 minutes and takes one hectolambda of lower liquid, and the hybridization buffer of 245 μ l is added
(900 TET, 0. 031 grams of NaCl, hectolambda NEB Buffer, two microlitres of ATP, 0. 33 microlitres of general connections
Son, 1 point 88 microlitre of T4DNA ligase).100 times of 5 μ l dilution is added in (21.5 DEG C, 1500r/min) 30 minutes in metal bath
SAPE (phycoerythrin being connect with Avidin) (Fig. 2).
Embodiment 5
Based on the detection technique of hydrogel miRNA, specific step is as follows for detection:
1) take 50 microlitres of hydrogel particle that 50 microlitres of TET (sodium chloride containing 700 mMs every liter) is added,
Take five microlitres (10-1000amol) of miRNA to be checked in 95 microlitres of TE Buffer (containing 500 mmoles
Your every liter of sodium chloride), by above-mentioned two solution mix with 90 minutes in metal bath (55 DEG C, 1500r/min), by reaction solution in
It is cleaned in TET three times, 900r/min is added after being centrifuged 3 minutes and takes one hectolambda of lower liquid, and the hybridization buffer of 245 μ l is added
(900 TET, 0. 031 grams of NaCl, hectolambda NEB Buffer, two microlitres of ATP, 0. 33 microlitres of general connections
Son, 1 point 88 microlitre of T4DNA ligase).100 times of 5 μ l dilution is added in (21.5 DEG C, 1500r/min) 30 minutes in metal bath
SAPE (phycoerythrin being connect with Avidin) (Fig. 2).
Embodiment 6
Based on the detection technique of hydrogel miRNA, specific step is as follows for detection:
1) take 50 microlitres of hydrogel particle that 50 microlitres of TET (sodium chloride containing 700 mMs every liter) is added,
Take five microlitres (10-1000amol) of miRNA to be checked in 95 microlitres of TE Buffer (containing 500 mmoles
Your every liter of sodium chloride), by above-mentioned two solution mix with 90 minutes in metal bath (55 DEG C, 1500r/min), by reaction solution in
It is cleaned in TET three times, 900r/min is added after being centrifuged 3 minutes and takes one hectolambda of lower liquid, and the hybridization buffer of 245 μ l is added
(900 TET, 0. 031 grams of NaCl, hectolambda NEB Buffer, two microlitres of ATP, 0. 33 microlitres of general connections
Son, 1 point 88 microlitre of T4DNA ligase).30 minutes in metal bath (21.5 DEG C, 1500r/min), 5 μ l are added and dilute 100 times
SAPE (phycoerythrin being connect with Avidin) (Fig. 2).
Embodiment 7
Embodiment 6 is repeated, but being added is non-miRNA to be checked as control, specific step is as follows；
1) take 50 microlitres of hydrogel particle that 50 microlitres of TET (sodium chloride containing 700 mMs every liter) is added,
Take five microlitres (10-1000amol) of non-miRNA to be checked in 95 microlitres of TEBuffer (containing 500 mmoles
Your every liter of sodium chloride), by above-mentioned two solution mix with 90 minutes in metal bath (55 DEG C, 1500r/min), by reaction solution in
It is cleaned in TET three times, 900r/min is added after being centrifuged 3 minutes and takes one hectolambda of lower liquid, and the hybridization buffer of 245 μ l is added
(900 TET, 0. 031 grams of NaCl, hectolambda NEB Buffer, two microlitres of ATP, 0. 33 microlitres of general connections
Son, 1 point 88 microlitre of T4DNA ligase).30 minutes in metal bath (21.5 DEG C, 1500r/min), 5 μ l are added and dilute 100 times
SAPE (phycoerythrin being connect with Avidin) it is non-to only have very weak fluorescence signal proof with Fig. 2 compared with as a result such as Fig. 3
Object to be checked is very weak in conjunction with herein described hydrogel particle, also illustrates that such method has great exploitativeness.
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.
Claims (7)
1. a kind of for detecting the preparation method of the hydrogel of miRNA, it is characterised in that: specific step is as follows:
1) design of light transmission mask: exposure mask board diameter is 30mm, with a thickness of 1mm；
2) stop building for flowing photoetching platform；
3) hydrogel detects；
4) preparation is connected with the hydrogel fines of miRNA aptamers.
2. according to claim 1 for detecting the preparation method of the hydrogel of miRNA, it is characterised in that: the step
1) design of light transmission mask, the specific steps are as follows: use L-Edit software, selecting diameter is the region of 30mm as work
Make region, does another figure layer in the region that a length of 5-7mm is chosen in the center of this working region, width is 3-5mm, then recruitment
Subtracting this figure layer just to obtain central area as region is rectangular mask plate, continues to synthesize furthermore with such method rectangular
Shape, square, circle or triangle.
3. according to claim 1 for detecting the preparation method of the hydrogel of miRNA, it is characterised in that: the step
2) stop building for flowing photoetching platform, the specific steps are as follows:
(1) above-mentioned mask plate is placed in microscopical optical path, adjusts angle and focal position；
(2) by the punching two ends of PDMS chip difference carrier gas passage, one end is sample holes, and one section is sample outlet hole；
(3) ultraviolet source is connected into adjustment focal length in microscopical optical path makes light-resource fousing into channel, adjusts eyepiece times magnification
Number for 10 ×, object lens magnification is 40 ×.
4. according to claim 1 for detecting the preparation method of the hydrogel of miRNA, it is characterised in that: the step
4) preparation is connected with the hydrogel fines of miRNA aptamers, the specific steps are as follows:
(1) pre-polymer solution is prepared: by 3.5 milliliters of 700 liquid of PEG-DA, 2 milliliters of PEG200 liquid, 0.5ml photoinitiator
1173 liquid of Darocur and 4 milliliters of 3 × TE buffer mix, stand-by as pre-polymer solution；
(2) prepolymer is injected in the syringe at injection port, syringe is packed into syringe pump, sets injection speed as 500-
1000μm/min；
(3) syringe pump is opened, makes prepolymer full of channel, stops injection pump work, open ultraviolet source and expose 50-100ms, shape
Particle is gone out at syringe pump is reopened after particle, repeats above step until obtaining sufficient amount of hydrogel particle；
(4) by the particle being collected into 1 × TET buffer solution for cleaning for several times, be finally resuspended in 300-1000 μ l 1 × TET buffering
In liquid, 4 DEG C are saved for use.
5. according to claim 4 for detecting the preparation method of the hydrogel of miRNA, it is characterised in that: the step
It 4) is (50- using the length-width ratio that UV light is combined to the hydrogel fines of miRNA aptamers (by taking miRNA21 as an example) in (3)
100)μm×(30-50)μm。
6. a kind of miRNA detection technique based on hydrogel, it is characterised in that: be based on hydrogel particle described in claim 1
MiRNA detection, the shape that the shape by changing mask plate controls light transmission may be implemented hydrogel of different shapes, realizes not
Same coding mode identifies that different shapes achievees the purpose that the more inspections of mixed inspection in conjunction with image procossing.
7. the miRNA detection technique according to claim 6 based on hydrogel, it is characterised in that: concrete application method is:
(1) TET of 50 microlitres of 50 microlitres of hydrogel particle addition is taken；
(2) take 5 microlitres (10-1000amol) of miRNA to be checked in 95 microlitres of TE Buffer；
(3) above-mentioned two solution is mixed in metal bath 90 minutes；
(4) reaction solution is cleaned three times in TET；
(5) it is added after 900r/min is centrifuged 3 minutes and takes 100 microlitres of lower liquid, the hybridization buffer of 245 μ l is added, it is described miscellaneous
Hand over buffer containing 900TET, 0.031 gram of NaCl, 100 microlitres of NEB Buffer, 2 microlitres of ATP, 0.33 microlitre of generic linker,
1.88 microlitres of T4DNA ligases；
(6) 30 minutes in metal bath；
(7) SAPE that 5 μ l dilute 100 times is added.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20150079669A1 (en) * | 2013-09-18 | 2015-03-19 | Senseonics, Incorporated | Critical point drying of hydrogels in analyte sensors |
| CN105784664A (en) * | 2016-05-10 | 2016-07-20 | 南京工业大学 | Hydrogel microsphere fluorescence sensor as well as preparation method and application thereof |
| CN106520964A (en) * | 2016-11-18 | 2017-03-22 | 南京中医药大学 | Double-recognition quantitative detection method for microRNA |
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- 2019-08-12 CN CN201910738718.3A patent/CN110452961A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20150079669A1 (en) * | 2013-09-18 | 2015-03-19 | Senseonics, Incorporated | Critical point drying of hydrogels in analyte sensors |
| CN105784664A (en) * | 2016-05-10 | 2016-07-20 | 南京工业大学 | Hydrogel microsphere fluorescence sensor as well as preparation method and application thereof |
| CN106520964A (en) * | 2016-11-18 | 2017-03-22 | 南京中医药大学 | Double-recognition quantitative detection method for microRNA |
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