CN206599554U - A kind of unicellular organism detection chip - Google Patents
A kind of unicellular organism detection chip Download PDFInfo
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- CN206599554U CN206599554U CN201720199936.0U CN201720199936U CN206599554U CN 206599554 U CN206599554 U CN 206599554U CN 201720199936 U CN201720199936 U CN 201720199936U CN 206599554 U CN206599554 U CN 206599554U
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Abstract
A kind of unicellular organism detection chip, the biological detection chip surface carries micrometer structure, and its micron of graphic structure can be inverted pyramid, taper or hole post, groove array.The utility model creates a kind of biological detection chip applied towards biological Single cell analysis, the chip is made up of polymeric material, using micro-structural, the mechanism of microchannel, the micro-structural that its surface is carried can both control passing through for macromolecular material, can make to be detected cell again in microchannel, micro-structural.Graphic parameter on the chip is variable, and the unicellular organism detection of realization has high flux, controllable, visual advantage, has a extensive future.
Description
Technical field
The utility model is related to the micrometer structure substrate applied towards biological Single cell analysis, and particularly a kind of auxiliary is slender
Born of the same parents' visualization, drug screening, the biogelatin material micron fabric chip of toxicity detection.
Mainly by imprint process twice, high-precision micron order knot is gone out in the Surface Machining of bioactive materials film
Composition shape, the culture observation microorganism individual on these micrometer structures, it is possible to achieve biological Single cell analysis can also be to big point
Sub- medicine is screened, and detects toxicity.
Background technology
Different types of microorganism is typically all to exist in the form of colony in nature, by some special between individual
Method carries out information interchange and communication.In recent years, researcher is to information exchange method between the individual in microbial population
Research is more and more extensive.Due to information exchange method generally existing and extensive use in nature between this individual, therefore lead to
The behavioural characteristic crossed between microorganisms individual and information interchange, it is possible to achieve drug screening, toxicity detection etc., referring to
Parsek,M.R.and E.P.Greenberg, Sociomicrobiology:the connections between
quorum sensing and biofilms.Trends in Microbiology,2005.13(1):p.27-33..With life
The fast development of thing microoperation technology, 3D printing, referring to Connell, J.L., et al., 3D printing of
microscopic bacterial communities. Proceedings of the National Academy of
Sciences of the United States of America, 2013.110(46):P.18380-18385., fluid channel,
The technology such as microcavity and drop generator, is widely used in biological Single cell analysis field, and these technologies are realized to microorganism
The high accuracy observation of information interchange and behavioural characteristic change between individual.Techniques described above is although realize biological unicellular inspection
Survey, but be required for the equipment and higher processing cost of costliness, it is difficult to which applying is needing high-throughout biological Single cell analysis
Field, and do not possess visual advantage when carrying out observation statistics to the behavioral trait of microorganism individual in the later stage yet.
With the fast development of MEMS technology, it is widely used in every field.Micro-nano technology is examined for unicellular organism
Survey field provides a kind of new method, and micro-nano embossing has good application because its is with low cost, simple operation and other advantages
Prospect.Thermal imprint process is a kind of low and fireballing method of cost that parallel replicated architecture is obtained in micro-nano-scale, it is only necessary to
One mould, identical structure can be copied on big surface on demand.Hot padding is by Stephen Y.Chou in 1995
Year proposes and applied for a patent first, referring to patent US5772905 " Nanoimprint lithography ".
At present, the micro-embossing process based on bioactive materials, very big problem is still run into technology realization, this
It is because temperature in micro-embossing process and knockout course, pressure condition is single and impression block is fixed, it is easy to damage biological material
The activity of material, is unfavorable for the culture of follow-up bacterium or cell.
The content of the invention
The utility model is in order to overcome difficulty and shortcoming encountered in above-mentioned cell detection process, for living based on biology
The micro-embossing process of property material is difficult to this technical barrier, employs two step stamping techniques with low cost, simple to operate,
It has developed a kind of biological Single cell analysis chip based on micro-structural.
Technical solutions of the utility model：
A kind of unicellular organism detection chip, including biological detection chip；
The micron graphic structure carried including biological detection chip surface；
Wherein described micron graphic structure is inverted pyramid, taper or hole post, groove array；
Between described graphic structure at equal intervals or be changing distance distribution.It is micro- that biological detection chip and its surface are carried
Rice graphic structure is structure as a whole.
The utility model biological detection chip material is biological gelatin film.
Implementation step is as follows：
1st, micrometer structure conduct is prepared using etching method, growth method, galvanoplastic, photoetching process or sedimentation in substrate surface
Template, prepared micrometer structure is inverted pyramid, taper or hole post array；Described base material is silicon, nickel, copper or zinc etc.
Hard sheet；
2nd, before thermal imprint process, to make the demoulding simple, in above-mentioned micrometer structure template surface using spin-coating method, spraying
Method, infusion method, evaporation or sputtering method prepare hydrophobic coating, and hydrophobic coating material is times that intrinsic static contact angle is more than 90 degree
Meaning hydrophobic material, obtains the micrometer structure template with hydrophobic coating；
3rd, based on the micrometer structure template that hydrophobic coating is carried described in above-mentioned 2nd step, thermoplastic, polymeric materials are carried out
A thermal imprint process, imprint temperature is slightly above the fusing point of thermoplastic, polymeric materials, and passes through in moulding process wafer
Tweezers apply pressure manually, and keep the uniformity of pressure applied, by 2min-6min imprint time, to template and poly-
Compound material is rapidly cooled to room temperature, then carries out releasing process, and the figure of thermal imprint process of experience is carved again, obtains surface
Polymeric material with micro-structural.
4th, using polymeric material of above-mentioned 3rd step with micro-structural as template, the secondary painting of bioactive materials is carried out
Cover imprint process.One layer of bioactive materials configured are coated in polymer in-mold plate surface with spin coating, spraying or wetting method,
This bioactive materials are for realizing bacterium or cell culture medium during biological Single cell analysis, by 2 DEG C -8 DEG C
After low temperature environment refrigeration 8h-14h, releasing process is carried out, it is thin that the micrometer structure of polymeric material is carved into bioactive materials again
Film surface.
The beneficial effects of the utility model are：The utility model has innovatively used a kind of two steps imprint process and figure
Time thermal imprint process of the technology carved again, i.e., one and secondary coating imprint process.The silicon template of micro-structural is carried due to being directly based upon
Or in the single method for stamping of the bioactive materials of metal hard template, releasing process is relatively difficult, and is easily damaged life
The activity of thing material, therefore, the technology used according to the utility model, first carry out based on the silicon template with micro-structural or
Microstructure graph in template, is carved into thermoplastic by thermal imprint process of the thermoplastic, polymeric materials of metal hard template again
Property thin polymer film on, then using obtained thin polymer film as template, carry out bioactive materials secondary impressing and the demoulding
Technique, is intactly carved on bioactive materials film again with the micro-structural on this silicon template or metal hard template.This
Utility model is realized simple and convenient, and it is high that figure carves precision again, good in economic efficiency, and does not damage the activity of biomaterial, realization
Unicellular organism detection has high flux, controllable, visual advantage, has a extensive future.
Brief description of the drawings
Fig. 1 (a)-(f) is inverted pyramid silicon template preparation technology flow chart.
Fig. 2 (a)-(f) is imprint process flow chart twice.
Fig. 3 (a)-(c) is 3-dimensional structural representation, wherein (a) is inverted pyramid structure silicon template, after (b) is the demoulding
Teflon FEP templates, (c) is gelatin film after the demoulding.
Fig. 4 (a)-(c) SEM results of imprint process for etching and twice, wherein (a) is inverted pyramid silicon template SEM
Figure, (b) is the SEM figures on the polymer Teflon FEP surfaces after hot padding, and (c) is the biogelatin material list after coating impressing
The SEM figures in face.
In figure, 1 silicon base, 2 inverted pyramid structures, 3 Teflon FEP substrates, 4 positive pyramid structures, 5 biogelatins
Substrate, 6 inverted pyramid structures.
Embodiment
It is explicitly bright to become apparent from the purpose of this utility model, technical scheme and advantage, below just combine it is specific
The utility model is explained embodiment, it is no intended to which expression can be built or using the unique of the utility model example
Form.It should be appreciated that specific embodiment described herein illustrates the utility model example only to explain the utility model
Function, and sequence the step of to build and operate the utility model example is not used to limit of the present utility model
Scope.
Embodiment 1
A kind of unicellular organism detection chip, realizes that step is as follows：
(1) micrometer structure is locally prepared using etching method on the surface of silicon base 1, the material of substrate 1 is silicon chip, prepared
Micrometer structure be inverted pyramid structure 2, preparation technology is specific as follows：1. clean, four cun of silicon chips and mask plate are respectively put into
In acetone, it is cleaned by ultrasonic 15 minutes, is then placed in isopropanol, continues to be cleaned by ultrasonic 10 minutes, finally by silicon chip and mask plate
It is put into deionized water to be cleaned by ultrasonic 10 minutes, is dried up with nitrogen gun standby；2. grown using LPCVD techniques in above-mentioned silicon chip surface
Layer of silicon dioxide is as mask, and thickness is 1 micron, as shown in Fig. 1 (a)；3. it is glutinous in silicon chip surface coating using evaporation
Attached dose, the above-mentioned 2. middle silicon chip cleaned up is put into binder HDMS casings, 10 minutes time was set；4. photoetching, will be applied
The silicon chip for having binder is placed on glue spreader, instills photoresist, and photoresist uses AZ5214, rotating speed 4000r/s, spin coating 30s, so
After be placed on hot plate and dry 90s, temperature is 95 DEG C；Exposure machine exposes 6.5s, is then placed in 3038 developer solutions the 45s that develops, and takes out
After being cleaned and being dried up with nitrogen with deionized water, it is placed on hot plate and dries 2min, temperature is 110 DEG C, as shown in Fig. 1 (b) (c)；
5. using RIE techniques etching 20min, the silicon dioxide layer of growth is etched away, as shown in Fig. 1 (d)；6. clean, by above-mentioned warp
The silicon chip for crossing RIE etchings is put into acetone, is cleaned by ultrasonic 15 minutes, then pours out acetone, adds isopropanol, continues to be cleaned by ultrasonic
15 minutes, finally by silicon chip extracting, it is cleaned by ultrasonic 5-15 minutes with deionized water, is dried up with nitrogen gun；7. KOH is etched, will be upper
State the silicon chip cleaned and be put into concentration to etch 20min in 30% KOH solution, temperature is maintained at 80 DEG C, such as institute in Fig. 1 (e)
Show；8. clean, the above-mentioned process KOH silicon chips etched are put into acetone, is cleaned by ultrasonic 15 minutes, then pours out acetone, are added
Isopropanol, continues to be cleaned by ultrasonic 15 minutes, finally by silicon chip extracting, is cleaned by ultrasonic 5-15 minutes with deionized water, uses nitrogen gun
Drying；9. remove photoresist, the good silicon chip of above-mentioned etching be sequentially placed into acetone and isopropanol and soaks 10min respectively, then spend from
Sub- water cleaning, and dried up with nitrogen, the silicon chip cleaned up is put into dry method in plasma degumming machine cleans, and power is 600w,
Time 15min, is made inverted pyramid micrometer structure, shown in gained inverted pyramid structure silicon formwork structure such as Fig. 3 (a), its SEM is such as
Shown in Fig. 4 (a).
(2) hydrophobic coating, wherein hydrophobic coating are prepared using spin-coating method on the surface of above-mentioned gained inverted pyramid silicon template
Material be concentration 0.05wt% Teflon AF1600 solution, obtain the inverted pyramid silicon template with hydrophobic coating, specifically
Method is as follows：First, the solvent FC40 by quality for 50mg solute Teflon AF1600 and 100g is well mixed, and is matched
To concentration be 0.05wt% Teflon AF1600 solution, using magnetic stirrer 24h, then by step 1) in obtain
Inverted pyramid silicon template be cut to the square piece that length and width are 1cm, the Teflon that obtained concentration is 0.05wt% will be configured
AF1600 solution is spin-coated on the surface of the inverted pyramid silicon template by cutting, finally carries out drying operation, is in temperature by it
5min is dried on 170 DEG C of hot plate, temperature is increased to 340 DEG C of FC40 quilts continued in drying 30min, such mixed solution afterwards
Evaporation, Teflon AF1600 are uniformly coated in the surface of inverted pyramid silicon template, so that follow-up releasing process is easily operated.
(3) it is same to cut the polymer Teflon that thickness is 3.5mm according to the size of above-mentioned inverted pyramid silicon template
FEP films are 1cm*1cm square piece, and Teflon FEP films are clipped between inverted pyramid silicon template and sheet glass first, this
Sample make it that Teflon FEP film stress is heated evenly in moulding process, and the Teflon FEP films clipped are then placed on hot plate
On, temperature is set in 280 DEG C, and a constant middle pressure is applied in glass either manually or by wafer tweezers during heating
On piece, and keep the uniformity of pressure applied, so that the Teflon FEP in glassy state can fully penetrate into down gold
In the groove of the inverted pyramid structure of word tower silicon template surface, as shown in Fig. 2 (c).By 5min hot embossing process, make rapidly
Teflon FEP are transferred in stainless steel cold bench with flat-nose pliers, handled by sufficient cooling, then carry out releasing process, due to
The V-structure of the inverted pyramid of inverted pyramid silicon template surface, and because the utility model is in step 2) the middle hydrophobic coating carried out
Pretreatment so that this method releasing process is relatively easy, the positive pyramid structure 4 on the obtained surface of Teflon FEP substrates 3
After flat smooth, the demoulding shown in Teflon FEP formwork structures such as Fig. 3 (b), shown in its SEM such as Fig. 4 (b).
(4) 3g gelatin particle and 0.3g bovine serum albumin are weighed first, are then blended in 10ml distilled water, are stirred
Mix uniform, then 100 DEG C of heating water bath 20min, and period is stirred continuously mixed solution so that and gelatin and bovine serum albumin are mixed
Close uniform, then dip three drop gelatin mixed solutions using stirring rod, the polymer after hot padding in coating in step (3)
The surface of Teflon FEP materials, coating is uniform, as shown in Fig. 2 (e), finally Teflon FEP deepfreeze 12h, temperature
Degree is arranged on 4 DEG C.It is stripped after the solidification of gelatin mixed solution, due to the low-adhesion and the bullet of gelatin materials of polymeric material
Property, therefore releasing process is relatively easy.Thus the surface being integrated with biogelatin substrate 5, which is made, has inverted pyramid structure 6
Biogelatin material, inverted pyramid structure carves completely again, and shown in its structure such as Fig. 3 (c), its SEM, can as shown in Fig. 4 (c)
For in the unicellular organism detection technique of next step.In the experiment of unicellular organism culture and observation is subsequently carried out, this
The inverted pyramid structure gelatin culture medium bottom of utility model preparation is extraordinary to realize unicellular organism detection, experiment success rate
Close to 100%.
The description of embodiments herein be intended only as example provide and those skilled in the art various modifications may be made.
Described above, example and data are there is provided the structure to each exemplary embodiment of the present utility model and the comprehensive description used.
Only the utility model is made further instructions using inverted pyramid structure as embodiment in present embodiment, but this practicality
Micron graphic structure in new bio detection chip is not limited to inverted pyramid structure, can be equally taper or hole post, groove battle array
Row, its effect and preparation are identical with the inverted pyramid structure in the present embodiment, do not repeat one by one herein.Although above with one
Fixed level of detail describes each embodiment of the present utility model with reference to one or more single embodiments, still, without departing from
In the case of spirit or scope of the present utility model, those skilled in the art can make to the disclosed embodiments and much repair
Change.
Claims (3)
1. a kind of unicellular organism detection chip, it is characterised in that：Including biological detection chip；
The micron graphic structure carried including biological detection chip surface；
Wherein described micron graphic structure is inverted pyramid, taper or hole post, groove array；
Between described graphic structure at equal intervals or be changing distance distribution.
2. unicellular organism detection chip according to claim 1, it is characterised in that：Biological detection chip material is biology
Gelatin film.
3. unicellular organism detection chip according to claim 1, it is characterised in that：Biological detection chip and its surface band
Some micron graphic structures are structure as a whole.
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| CN201720199936.0U CN206599554U (en) | 2017-03-03 | 2017-03-03 | A kind of unicellular organism detection chip |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111218400A (en) * | 2018-11-23 | 2020-06-02 | 中国科学院大连化学物理研究所 | Culture method for development of waste embryo blastula |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111218400A (en) * | 2018-11-23 | 2020-06-02 | 中国科学院大连化学物理研究所 | Culture method for development of waste embryo blastula |
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