CN109507414A - The microtrabeculae and its preparation method and application of microballon is fixed in cavity - Google Patents
The microtrabeculae and its preparation method and application of microballon is fixed in cavity Download PDFInfo
- Publication number
- CN109507414A CN109507414A CN201810852641.8A CN201810852641A CN109507414A CN 109507414 A CN109507414 A CN 109507414A CN 201810852641 A CN201810852641 A CN 201810852641A CN 109507414 A CN109507414 A CN 109507414A
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- China
- Prior art keywords
- microtrabeculae
- microballon
- cavity
- prepolymer
- photopolymerizable composition
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
- G01N2333/4701—Details
- G01N2333/4737—C-reactive protein
Abstract
The present invention provides a kind of methods that microballon is fixed in the cavity of translucency microtrabeculae, it include: 1) photopolymerizable composition to be added in the cavity of the microtrabeculae, the photopolymerizable composition is the aqueous solution comprising prepolymer and photoinitiator, and the photoinitiator is used to generate free radicals the polymerization to promote the prepolymer；2) under photomask assistance, at least part cavity that irradiation is filled with the photopolymerizable composition is used up, so that the prepolymer cures at least part cavity, form perforated baffle；3) microtrabeculae is cleaned, to remove unreacted residue；4) microballon to be fixed is added；5) step 1) is repeated to 3), and the microballon is fixed between the perforated baffle, microballon section is formed.The present invention also provides the application of the microtrabeculae prepared by this method and the microtrabeculae in terms of immune detection.Microtrabeculae of the invention uses microballon fixation in situ mode, has many advantages, such as that embedding in advance, position are fixed, convenient transportation and are easy to save.
Description
Technical field
The present invention relates to microtrabeculaes and preparation method thereof, especially can be applied to immunology trace detection and quickly detect micro-
Column and preparation method thereof.
Background technique
Immunologic test is the detection project that hospital carries out extensively, by the interaction of antigen and antibody, detect serum or
Biomarker in blood plasma.At present using it is more there is enzyme to exempt from, put exempt from, time-resolved fluorescence, the methods of magnetic bead chemiluminescence.
Wherein, microballon is since it has the advantages that large specific surface area is widely used in immune response.But microballon is being immunoreacted at present
In use be mostly floated reaction, such as in magnetic bead chemiluminescence, magnetic bead will be coupled at its table by the random collision of physics
Sample to be tested in the capture molecule and reaction system in face is combined.This reactive mode, the required reaction time is longer,
And it is difficult to carry out absolute abundant reaction.If microballoon is tightly fixed in a long and narrow reaction channel, due to micro-
Gap is smaller between pearl, and reaction channel has certain length, and sample is slowly passed sequentially through to this channel at this time, will make capture point
The collision probability of son and sample to be tested greatly improves, and can shorten the reaction time and improve detection sensitivity.
Micro- reaction system has the few advantage of sample size in need.This can reduce patient suffering, especially for needs
The patient to take a blood sample repeatedly.Therefore, micro- reaction system is an important development direction of immune detection.The other reaction column of micro/nano level
(i.e. microtrabeculae) can perform well in the foundation of micro- reaction system.But preparing the other reaction column of micro/nano level has certain hardly possible
Degree.Traditional column bed fills column method, is that the spilling that a porous dam structure is used to stop filler is arranged in the channel end.
But since current micro-processing technology minimum process precision is usually at 10 μm or so, i.e. minimum 10 μm of channel end dam aperture
Left and right, in order to guarantee that microballon does not overflow channel, it is desirable that the bead diameter used is at least at 20 μm or more.Bead diameter is bigger,
Specific surface area is with regard to smaller, and the diameter of common highly sensitive immune microballon is between 0.3 to 3 μm, therefore traditional column bed
The column that declines is not used to some with higher sensitivity detection projects.
Summary of the invention
In order to overcome the problems referred above, on the one hand, the present invention provides a kind of fixes microballon in the cavity of translucency microtrabeculae
Method, comprising:
1) photopolymerizable composition is added in the cavity of the microtrabeculae, the photopolymerizable composition is to include prepolymer and light
The aqueous solution of initiator, the photoinitiator are used to generate free radicals the polymerization to promote the prepolymer；
2) under photomask assistance, at least part cavity that irradiation is filled with the photopolymerizable composition is used up, so that
The prepolymer cures form perforated baffle at least part cavity；
3) microtrabeculae is cleaned, to remove unreacted residue；
4) microballon to be fixed is added,
5) step 1) is repeated to 3), and the microballon is fixed between the perforated baffle, microballon section is formed.
In some embodiments, this method further include repeated using another kind or more microballon step 4) and
5), to form multiple microballon sections in the microtrabeculae.
In some embodiments, the bead surface coupling has capture molecule.
In preferred embodiments, the prepolymer is ENTG3800, and the photoinitiator is 2- hydroxyl -4 '-(2- hydroxyl
Ethyoxyl) -2- methyl phenyl ketone.
In a more preferred embodiment, volume ratio of the ENTG3800 in the photopolymerizable composition be 10% to
70%, 2- hydroxyl -4 '-volume ratio of (2- the hydroxy ethoxy) -2- methyl phenyl ketone in the photopolymerizable composition be 1% to
10%.
In some embodiments, the internal diameter of the microtrabeculae is in 0.1mm to 10mm.
In some embodiments, the light irradiation uses the ultraviolet light of 365nm wavelength, with 100mW/cm22 points of irradiation
Clock.
On the other hand, the present invention also provides the microtrabeculaes for being fixed with microballon in the cavity prepared by the above method.
On the other hand, the application the present invention also provides the microtrabeculae in immune detection.
Microtrabeculae of the invention uses microballon fixation in situ mode, and there is embedding in advance, position to fix, convenient transportation and be easy to
The advantages that preservation.
Detailed description of the invention
Fig. 1 shows the schematic diagram of an embodiment of microtrabeculae of the present invention, includes a microballon section in the microtrabeculae.
Fig. 2 shows the schematic diagram of the another embodiment of microtrabeculae of the present invention, includes multiple microballon sections in the microtrabeculae.
Fig. 3 shows with microtrabeculae of the present invention the curve graph for detecting c reactive protein (CRP).
Specific embodiment
Unless otherwise indicated, all technical and scientific terms used herein has those of ordinary skill in the art institute usually
The meaning of understanding.
As shown in Figure 1, microtrabeculae provided by the invention include cavity 1, microtrabeculae section 2 and perforated baffle 3 (in figure also schematically
Show the part enlarged structure of microtrabeculae section 2 and perforated baffle 3).Microballon section 2 and perforated baffle 3 are located in cavity 1, and micro-
Pearl section 2 is between two perforated baffles 3.Cavity 1 itself is also the flow channel of measuring samples, cleaning solution etc..The length of microballon section 2
Degree is generally in 1mm to 10mm, and the length of perforated baffle 3 is generally in 0.1mm between 10mm.
In another embodiment, as shown in Fig. 2, microtrabeculae provided by the invention includes multiple microtrabeculae sections 2, each microballon
Section 2 may include different microballon, such as these microballons have different capture molecules (such as antibody).
The material for preparing microtrabeculae can be cyclic olefine copolymer (COC), polymethyl methacrylate (PMMA), polystyrene
(PS) the preferable optical material of translucency such as.Microtrabeculae can morphologically be the elongated tubular product object of both ends open, or can be
The elongated passageway of the one or more formed on above-mentioned material two openings.The cross section of the cavity 1 of microtrabeculae can be it is round,
Square, rectangle etc. can accommodate the regular shape of liquid.The length of cavity 1 can be 1mm to 10cm.
Microballon section 2 is formed by being filled in the microballon in cavity 1 between two perforated baffles 3.The microballon can be magnetic microsphere,
The common miniature bead of the biology such as latex beads, silicon dioxide microsphere.Bead diameter range is can be at 500nm to 100 μm
Between.Bead surface, which is generally coupled, capture molecule, such as antibody and includes the albumen etc. including streptavidin.Coupling method
Generally include coupling modes and the physical absorptions such as carboxyl, hydroxyl, amino, toluenesulfonic acid base.These capture molecules are used for sample
Test substance in product is captured to bead surface, then carries out qualitative or quantitative analysis using corresponding characterizing method.
Perforated baffle 3 is formed by photopolymerizable composition.The photopolymerizable composition is the water comprising prepolymer and photoinitiator
Property solution.Prepolymer, which refers to, can occur the partially polymerized chemical combination object that radically curing reacts and generates porous channel.These compounds
The characteristics of be that it in molecular end contains double bond, such as ENTG3800.The use ratio of prepolymer is 10% to 70% (volume
Than), the ratio of prepolymer is higher, and the hole for being formed by perforated baffle is finer and close, and the required photopolymerization time is also shorter.Cause
Agent is some compounds that can be generated free radicals through ultraviolet light, such as 2- hydroxyl -4 '-(2- hydroxy ethoxy) -2- methylbenzene
Acetone.Initiator use ratio is 1% to 10% (volume ratio), and initiator amount is bigger, and the photopolymerization time is shorter.The photopolymerization
The other ingredients of composition are, for example, water, phosphate buffered saline (PBS), physiological saline etc..Ultraviolet light can pass through ultraviolet light generator
It generates, such as 365nm can be set by the wavelength of transmitting.Set power is higher, and the photopolymerization time is shorter.Pass through
The use ratio for adjusting prepolymer, can form the perforated baffle with different pore size.For purposes of the invention, it is formed
The pore size of perforated baffle can prevent microballon from passing through on the one hand, on the other hand should allow large biological molecule such as albumen etc.
It passes freely through.
Microtrabeculae of the invention can be made in the following way.First photopolymerizable composition is added in the cavity 1 of microtrabeculae, is used
Photomask will only need cured microtrabeculae position to be exposed under ultraviolet light, and photopolymer compositions (are formed in the solidification of this position
Perforated baffle 3) after, unreacted residue is washed away, then fixed microballon will be needed to fill to the side of the perforated baffle 3,
Microballon filling finishes, and a perforated baffle 3 is re-formed in the open sides (i.e. without perforated baffle side) of microballon, so that microballon is fixed on
Between two perforated baffles 3, microballon section 2 is formed.When referring to microballon, the word " fixation " of this paper refers to will be micro- by perforated baffle 3
Pearl group is generally maintained at the specific position of microtrabeculae, they do not pass through perforated baffle 3 and overflow, but for each specific
For microballon, they can be moved in microballon section 2.Multiple microballon sections 2 can be formed in this way, these microballon sections 2 can be with
Be it is continuous, i.e., they are only separated by perforated baffle 3, be also possible to it is discontinuous, i.e., other than perforated baffle 3, they it
Between further include one section of blank cavity.Microtrabeculae with multiple microballon sections 2 can be used for the joint-detection of multiple projects in sample.
Photomask mentioned herein for example can be a kind of transparent material that can be used for printing, the part that needs are protected from light
With printer processes at black, it is also possible to a kind of opaque material that can be micro machined into engraved structure, will needs to expose
The part of light is carved into the structure of hollow out.
Microtrabeculae of the invention can cooperate sample injector and detector, be loaded, washed and detected manually, also can integrate
It is used into the immunoassay module of full-automatic instrument.
The present invention is illustrated below in conjunction with specific embodiment.
The preparation of 1 perforated baffle of embodiment
Compare initiator 2- hydroxyl -4 '-(2- hydroxy ethoxy) -2- methyl phenyl ketone of same amount through this embodiment
The permeability of perforated baffle prepared by prepolymer ENTG3800 (buying from Japanese Kansai Broadband Trial Network coating) with different amounts.Firstly,
Prepare two groups of photopolymer compositions: high concentration group (initiator volume ratio: 3%, prepolymer volume ratio: 45%, residue supplement
PBS) and low concentration group (initiator volume accounting: 3%, prepolymer volume accounting: 7%, residual volume supplement PBS).Then from
5 μ l the compositions are added in microtrabeculae one end, and the masking foil of hollow out is used, on above-mentioned microtrabeculae, to make hollow out position as photomask
Set the position that (1mm × 1mm) alignment needs to polymerize.Then ultraviolet curing equipment (wavelength: 365nm, power: 100mW/ is used
Cm2) treatment with irradiation 2min.Microsyringe is finally used, perforated baffle 30min is washed with PBS with the flow velocity of 5 μ l/min, to remove
Go uncured component.
With the permeability of fluorescent labeled antibody detection perforated baffle: by above-mentioned two prepared kind with the micro- of perforated baffle
Column (is bought certainly using the CRP antibody GR26 that micro-fluidic sample injector is marked from 0.1 μ g/ml DyLight650 of one end sample introduction respectively
Xiamen is with benevolence Bioisystech Co., Ltd) (buffer: the PBS containing 1%BSA), sample introduction speed is 5 μ l/min.Sample introduction 5min,
And 20 μ l are continuously collected from the other end of microtrabeculae and flow through liquid.Liquid will be flowed through to be transferred in 96 orifice plates, use Multifunction fluorescent enzyme mark
The 620nm/680nm optical filter of instrument detects and records fluorescent value, while being marked using 0.1 μ g/ml DyLight650 of same volume
Remember antibody and PBS as a control group.Testing result is as shown in table 1, and the fluorescence antibody of two groups of perforated baffles flows through the fluorescent value of liquid
All with DyLight650 labelled antibody control group no significant difference, above-mentioned two groups of porous gears can be passed freely through by illustrating antibody
Plate.
With the permeability of the green fluorescent microspheres detection perforated baffle of partial size 400nm: above-mentioned two prepared kind are had
The microtrabeculae of perforated baffle (is respectively degree, buffering purchased from Suzhou from the green fluorescent microspheres of 1 μ g/ml partial size 400nm of one end sample introduction
Liquid: the PBS containing 1%BSA), sample introduction speed is 5 μ l/min.Sample introduction 5min continuously collects 20 μ l from the other end of microtrabeculae and flows through
Liquid.Liquid will be flowed through to be transferred in 96 orifice plates, detect and record glimmering using the 485nm/525nm optical filter of Multifunction fluorescent microplate reader
Light value, while as a control group using 1 μ g/ml green fluorescent microspheres of same volume and PBS.Testing result is as shown in table 1, high
For concentration group perforated baffle since prepolymer content is higher, the baffle hole of formation is small, causes to flow through in liquid that fluorescence is not detected is micro-
< 400nm is answered in ball, i.e. high concentration group baffle aperture, can successfully retain the microballoon of 400nm and diameter above.And low concentration group by
It is larger in baffle hole, overwhelming majority 400nm microballoon is passed freely through, i.e. low concentration group baffle hole average diameter >
400nm。
1 perforated baffle permeabilization assay of table
The preparation of 2 immune detection microtrabeculae of embodiment and c reactive protein (CRP) test and analyze
The microtrabeculae of small-bore baffle of the one end prepared in Example 1 with high concentration group, uses micro-fluidic sample injector
From 1 μ g/ml streptavidin of other end sample introduction coating magnetic bead, (magnetic bead is purchased from Thermo, diameter: 1 μm, buffer: containing 1%BSA
PBS), 5 μ l/min of sample introduction speed, until magnetic bead be full of entire microtrabeculae (a little surpluses is slightly stayed to be used to prepare another perforated baffle)
When stop sample introduction, then using embodiment 1 high concentration group perforated baffle preparation method, in the other side of microballon (without porous
Baffle side) perforated baffle is formed, microballon 30min is finally washed with PBS with the flow velocity of 5 μ l/min, to remove uncured component.
By CRP antibody GR25 (purchase is from Xiamen with benevolence Bioisystech Co., Ltd) (concentration: 1 μ g/ of biotin labeling
Ml, buffer: the PBS containing 1%BSA), it is equipped with using micro-fluidic feeder to above-mentioned both ends with perforated baffle, centre
Streptavidin is coated in the microtrabeculae cavity of magnetic bead, sample introduction speed 5 μ l/min, sample introduction 2min.Sample introduction terminates, using PBST with phase
Same flow velocity washs 2min.By gradient dilution CRP antigen (initial concentration: 500ng/ml, 2 times of gradient dilutions, totally 10 concentration,
Buffer: the PBS containing 1%BSA), with the flow velocity of 5 μ l/min into the above-mentioned reaction channel containing biotinylated antibody sample introduction,
Sample introduction 2min altogether.Sample introduction terminates, and washs 2min using PBST with identical flow velocity.Washing terminates, with identical 1 μ of flow velocity sample introduction
The CRP antibody GR26 (buffer: the PBS containing 1%BSA), sample introduction 2min of g/ml DyLight650 label.Sample introduction terminates, and uses
PBST washs 2min with identical flow velocity.Washing terminates, and photographs to record under laser confocal fluorescence microscope, uses ImageJ
Software carries out Fluorescence Intensity Assays, and as a result as shown in Table 2 and Fig. 3, CRP detection range is 1ng/ml to 500ng/ml.
2 CRP Analysis of test results of table
In immune detection application, for the multiple projects of joint-detection, the microtrabeculae with multiple microballon sections can be used.This
Pearl section contains the microballon that coupling has different capture molecules slightly.These capture molecules can bind directly to be checked point in sample
Son, or to be checked point can be combined by middle element (for example, CRP antibody GR25 of the biotin labeling in embodiment 2)
Son.
It is described the invention in detail above by specific embodiment, it is noted that for those skilled in the art
For, under the premise of not departing from spirit and scope of the invention, several changes can also be made to these embodiments or embodiment,
They also should be regarded as within the scope of the present invention.
Claims (9)
1. a kind of method for fixing microballon in the cavity of translucency microtrabeculae, comprising:
1) photopolymerizable composition is added in the cavity of the microtrabeculae, the photopolymerizable composition is comprising prepolymer and light-initiated
The aqueous solution of agent, the photoinitiator are used to generate free radicals the polymerization to promote the prepolymer；
2) under photomask assistance, at least part cavity that irradiation is filled with the photopolymerizable composition is used up, so that described
Prepolymer cures form perforated baffle at least part cavity；
3) microtrabeculae is cleaned, to remove unreacted residue；
4) microballon to be fixed is added,
5) step 1) is repeated to 3), and the microballon is fixed between the perforated baffle, microballon section is formed.
It further include repeating step 4) and 5) using another kind or more microballon 2. the method as described in claim 1, with
Multiple microballon sections are formed in the microtrabeculae.
3. it is method according to claim 1 or 2, wherein bead surface coupling has capture molecule.
4. method according to claim 1 or 2, wherein the prepolymer is ENTG3800, the photoinitiator is 2- hydroxyl-
4 '-(2- hydroxy ethoxy) -2- methyl phenyl ketones.
5. method as claimed in claim 4, wherein volume ratio of the ENTG3800 in the photopolymerizable composition be 10% to
70%, 2- hydroxyl -4 '-volume ratio of (2- the hydroxy ethoxy) -2- methyl phenyl ketone in the photopolymerizable composition be 1% to
10%.
6. it is method according to claim 1 or 2, wherein the internal diameter of the microtrabeculae is in 0.1mm to 10mm.
7. method as claimed in claim 5, wherein the ultraviolet light using 365nm wavelength is penetrated in the illumination, with 100mW/cm2According to
It penetrates 2 minutes.
8. the microtrabeculae for being fixed with microballon in the cavity of the preparation of method described in any one of claims 1 to 7.
9. application of the microtrabeculae according to any one of claims 8 in immune detection.
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| CN201810852641.8A CN109507414A (en) | 2018-07-30 | 2018-07-30 | The microtrabeculae and its preparation method and application of microballon is fixed in cavity |
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| CN201810852641.8A CN109507414A (en) | 2018-07-30 | 2018-07-30 | The microtrabeculae and its preparation method and application of microballon is fixed in cavity |
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