CN102410996A - Plane parallel fluorescent biological detecting chip based on total reflection and preparation method thereof - Google Patents
Plane parallel fluorescent biological detecting chip based on total reflection and preparation method thereof Download PDFInfo
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- CN102410996A CN102410996A CN2011102480902A CN201110248090A CN102410996A CN 102410996 A CN102410996 A CN 102410996A CN 2011102480902 A CN2011102480902 A CN 2011102480902A CN 201110248090 A CN201110248090 A CN 201110248090A CN 102410996 A CN102410996 A CN 102410996A
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- optical fiber
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Abstract
The invention discloses a plane parallel fluorescent biological detecting chip based on total reflection and a preparation method thereof. In the chip, the sample table is bonded by glass sheets and PDMS chips and located in the top surface of the whole chip; a prism, a lens, an optical fiber stopping part, an optical fiber supporting part and optical fiber that are on the same level are arranged below the sample table; the prism, the lens, the optical fiber stopping part and the optical fiber supporting part are all made from PDM integrally; when laser comes into the single-mode fiber after being coupled, the light beam transmits in the optical fiber; the emergent light is corrected by the lens and is reflected when the emergent light reaches the air/prism interface; then the reflected light reaches the prism/glass interface and is further reflected; the reflected light is fully reflected on the glass/water interface when the reflected light comes into glass; and evanescent wave is generated in the full-reflecting zone for generating fluorescence reaction of the sample in water. The chip is easy to be integrated with mini-analyzing system without laser source. Therefore, the detecting chip is low in biological material consumption, low fluorescent background noise and high sensitivity.
Description
Technical field
the present invention relates to a kind of chip structure that biological detects that is used for, particularly a kind of parallel biological detection chip in plane based on total reflection and preparation method thereof.
Background technology
total reflection fluorescent microscope in recent years thereby have received extensive concern owing to be fit to the nearly surface molecular dynamics research of single molecules level.The principle of work of total reflection fluorescent microscope depends on the evanescent wave that total reflection produces.With respect to traditional fluorescent microscope, utilize evanescent wave fluorescence excitation group, it is very little background noise to be dropped to, thereby makes micro-effect more clear.
are found through the literature search to prior art; " TIR Fluorescence Reader for Selective Detection of Cell Membranes " (the total reflection fluorescence reader that is used for selectivity detection of cells film) that Thomas Bruns etc. delivered in 2005; The reader experimental detection of describing in this article the T47D breast cancer cell; Structure is following: the no base plate of one 96 well (8 * 12); Bonding agent and the thick glass sheet that has than low absorptivity of 2mm through no cytotoxicity are bonded together; Laterally cohere the glass bar of a square-section again at the two ends of glass base, be used for the coupling of incident light and emergent light.Concrete to detect step following: at first, the ventilation type Argon ion laser that has single mode fiber system sends the laser that wavelength is 488nm, and light beam just is divided into 8 bundles after through 3 spectroscopes; Then this 8 bundle laser gets into glass bar through reflecting after the direct reflection again; The incident angle of light that get into the glass film plates of d=2mm by glass bar this moment is 66 °, and (glass refraction is 1.525; The tenuigenin refractive index is 1.37; Between two wells is 9mm apart from s, Θ=arctan (s/2d)=66 °), greater than 64 ° of the critical angles between glass and the tenuigenin; At last, light beam gets into glass film plates afterwards through penetrating from another glass bar after 23 total reflections, carries out the fluorescent brightness observation of each well simultaneously through charge coupling camera (ccd video camera).The size of this reader is unfavorable for integrated more greatly; Biomaterial consumption is bigger; Each use all needs to aim at; If light beam gets into tenuigenin through refraction and may produce some influence by pair cell under the situation that does not have aligning, and when observing with ccd video camera, need reduce background noise with filter.
Summary of the invention
the objective of the invention is to solve the deficiency of prior art; Parallel biological detection chip in a kind of plane based on total reflection and preparation method thereof has been proposed; This chip is beneficial to micro-analysis system integrated, does not need the volumetric laser source, and biomaterial consumes low; The fluorescence background noise is little, and is highly sensitive.
The present invention realizes through following technical scheme:
The parallel biological detection chip in the plane based on total reflection that
the present invention relates to comprises fiber support, optical fiber, and optical fiber stops, lens, prism and sample stage.Wherein, sample stage is formed by glass sheet and PDMS (dimethyl silicone polymer) chip bonding, is positioned at the top of entire chip.Be prism under the sample stage, on same horizontal line is lens, and optical fiber stops, and fiber support, and these parts are all made by the PDMS one, succinct, compactness.In addition, optical fiber is bonded on the fiber support through bonding agent.
biological detection chip of the present invention, when laser when being coupled into single-mode fiber, light beam is propagated in optical fiber; Proofread and correct by lens after the outgoing; Arriving air/prism boundary (horizontal sextant angle is 54.7 °, and air refraction is 1, and the prismatic refraction rate is 1.46) time reflects; Incident angle is 35.3 °, and the refraction angle is 23.3 °.Refract light prism/the glass interface of arriving soon after further reflects; Incident angle is 78 °; The refraction angle is 69.97 °; This refraction angle is greater than 61 ° of the cirtical angles of total reflection of glass/water termination, so refracted ray can total reflection take place at the interface surely, and refract light gets into behind the glass in glass/water termination generation total reflection.In fact, when total reflection took place, not all light wave all had been reflected back toward glass; Still understand some ripple; Be that evanescent wave can appear glass/water termination, penetration depth is 100-300nm, and the enough fluorescence excitation groups of this wave energy reside near medium/liquid; Not only impel sample to send fluorescence but also be unlikely to illuminate whole sample, thereby reduce the background interference of fluorescence imaging greatly.
The preparation method based on the biological detection chip of total reflection who the present invention relates to comprises the steps:
At first prepare (a 100) silicon chip, thermal oxide growth layer of silicon dioxide film then, coating one deck photoresist on this substrate is placed on the mask plate of making directly over the substrate, exposure, develop after with the design transfer of mask plate to substrate,
utilize BHF that substrate is carried out etching then, remove silicon dioxide.Then this structure is carried out the silicon wet etching; The etching liquid that uses mainly carries out anisotropic etching to silicon as TMAH (TMAH), and the etch rate of (111) crystal face is slow; Therefore the side can form 54.7 ° inclination angle, thereby has formed fiber support and prism structure.
form the lens window through fine process again, and concrete steps are for plated metal aluminium (Al) layer at first and form the lens window, then carry out DRIE (deep reaction ion etching) and wear silicon chip quarter.
Remove the Al layer at last, SiO
2
Layer combines Si chip and glass substrate with epoxy glue again, just processes full die, adopts PDMS to carry out transcription, just obtains the core component of this chip, fiber support, and optical fiber stops (Stopper), lens and prism.
the present invention compare with existing biological detection technique, and with fiber support, optical fiber stops, collector lens, and prism four is together partly integrated, and chip volume is little, is beneficial to micro-analysis system integrated; In the MEMS technical field, cost of manufacture is low, and the silicon mould that makes is reusable, only needs to carry out transcription with PDMS; Do not need the volumetric laser source; Biomaterial consumes low, and the fluorescence background noise is little, and is highly sensitive, can be used for the detection of molecular level; Three samples of parallel detection (can according to concrete needs parallel fiber support of increase and decrease when the template construct) simultaneously.
Description of drawings
Fig. 1 is the structural representation of sample stage part in the embodiment of the invention;
Fig. 2 is a sample stage below structural representation in the embodiment of the invention;
Fig. 3 is the sectional view of the embodiment of the invention;
Fig. 4 is the process chart of the embodiment of the invention.
Embodiment
elaborate to embodiments of the invention below in conjunction with accompanying drawing: present embodiment is being to implement under the prerequisite with technical scheme of the present invention; Provided detailed embodiment and process, but protection scope of the present invention is not limited to following embodiment.
Shown in Fig. 1-3, the biological detection chip in the present embodiment is to come the fluorescence excitation reaction by the evanescent wave that total reflection produces
.Concrete structure comprises: optical fiber 1, and fiber support 2, optical fiber stops 3, lens 4, prism 5, sample stage 6.Said sample stage 6 is formed by glass sheet and PDMS chip bonding; Sample stage 6 is positioned at the top of entire chip, is the prism 5 that is positioned at same surface level below the sample stage 6, lens 4, and optical fiber stops 3, fiber support 2, optical fiber 1.Wherein, optical fiber 1 is sticked on fiber support 2 by bonding agent, prism 5, and lens 4, optical fiber prevention 3 and fiber support 2 materials all are PDMS, make the Si mould via micro fabrication, again by the PDMS transcription.The entire chip integrated level is high, and compact conformation, Si mould can be used for repeating preparation.
PDMS has duplicated the structure on the silicon mould through transcription, and then bonding is two 100
μThe glass sheet that m is thick combines with it through van der waals force, strengthens physical strength and as sample stage 6, and is as shown in Figure 1.It is 250 that single-mode fiber embeds external diameter
μThe glass tube blended rubber of m is bonded in the fiber support, has so just avoided follow-up alignment work.The width that optical fiber stops is 115
μM is convenient to fiber orientation and is installed, and is as shown in Figure 2.Fig. 3 is the sectional view of present embodiment.
The main process that
present embodiment is accomplished: laser is divided into three beams through light-splitting device and is coupled into single-mode fiber; The light beam that penetrates is calibrated by lens, arrives air/prism boundary and reflects, and refract light arrives prism/glass interface and reflects once more; Refract light in the incident angle at glass/water (sample is in water) interface greater than critical angle; Therefore total reflection takes place, produce evanescent wave in the total reflection zone and excite the sample generation fluorescence reaction in the water, observed the fluorescence phenomenon of three samples more simultaneously by ccd video camera 7; One of them can be used as reference, paired observation two other.
above-mentioned biological detection chip core based on total reflection comprises two steps in addition in the PDMS middle layer of preparation chip, the glass sheet bonding, and optical fiber is bonding.
As shown in Figure 4, the PDMS middle layer preparation of present embodiment comprises following step:
The first step: prepare (a 100) Si sheet, thickness is 300
μM.Put into oxidation furnace then silicon chip is carried out thermal oxide, the temperature of oxidation furnace is 1100 degrees centigrade, and oxidization time is 5 minutes, and the silica membrane thickness of generation is 600nm, like Fig. 4 (a).
Second step: spin coating photoresist 3 PMMA (polymethylmethacrylate), thickness are 1
μM utilizes set mask plate, adopts electron beam that substrate is made public; The electric current of electron beam is 10pA; Time shutter is 30sec, so just transfer to the square chamber pattern on the mask plate (optical fiber align and prism window) on the substrate, again with BHF with the SiO in the window
2
Remove.Adopt TMAH solution (electromagnetic agitation speed is 250rpm for 20%wt, 90 ° of C) to carry out anisotropic wet etch 6 hours, etching depth is 270
μM.Etch rate is about 0.75
μM min
1
, lower etch rate Shi De<111>Crystal face is very level and smooth.Clean, remove etching liquid and photoresist, as Fig. 4 (b, c).
Four flute length side by side during fiber support is promptly schemed, the wide 4mm that is respectively, 1mm, separation are 115
μM.Prism is long, the wide 8mm that is respectively, 9mm.
the 3rd step: deposition one deck Al, photoetching forms optical fiber and stops and the lens window, removes Al and SiO2 in the window with BHF, like Fig. 4 (d).
Optical fiber stops part front opening 115
μM, 50 ° of inside openings, the circular arc radius-of-curvature is 450
μM.The lens component radius-of-curvature is 450
μM, thickness are 436
μM, the lens center is apart from fiber support 463
μM, the i.e. focal length of lens.
the 4th step: adopt vertical quarter of DRIE to wear lenticule and optical fiber prevention window.Remove Al layer and SiO
2
The layer, just silicon mould complete, like Fig. 4 (e).
the 5th step: mould is carried out the PDMS transcription, and base plate thickness is 0.5mm, and the weight ratio of hardening agent and prepolymer is 1:10; Heating makes the PDMS hardening, and the demoulding just obtains core component of the present invention; Fiber support, optical fiber stops, lens and prism such as Fig. 4 (f).
Although content of the present invention has been done detailed introduction through above-mentioned preferred embodiment, will be appreciated that above-mentioned description should not be considered to limitation of the present invention.After those skilled in the art have read foregoing, for multiple modification of the present invention with to substitute all will be conspicuous.Therefore, protection scope of the present invention should be limited appended claim.
Claims (6)
1. the parallel biological detection chip in the plane based on total reflection is characterized in that comprising fiber support, optical fiber, and optical fiber stops, lens, prism and sample stage; Wherein: said sample stage is formed by glass sheet and PDMS chip bonding, is positioned at the top of entire chip, is the prism that is positioned at same surface level below the sample stage; Lens; Optical fiber stops, fiber support and optical fiber, said prism; Lens, optical fiber stops and fiber support is all made by the PDMS one; When laser when being coupled into single-mode fiber; Light beam is propagated in optical fiber; Proofreaied and correct by lens after the outgoing, reflect when arriving air/prism boundary, the refract light prism/glass interface of arriving soon after further reflects; In glass/water termination generation total reflection, produce evanescent wave in the total reflection zone and excite the sample generation fluorescence reaction in the water behind the refract light entering glass.
2. the parallel biological detection chip in the plane based on total reflection according to claim 1 is characterized in that said optical fiber is sticked on fiber support by bonding agent.
3. the parallel biological detection chip in the plane based on total reflection according to claim 1 is characterized in that, when said light beam arrives air/prism boundary; Wherein horizontal sextant angle is 54.7 °; The prismatic refraction rate is 1.46, and incident angle is 35.3 °, and the refraction angle is 23.3 °.
4. the parallel biological detection chip in the plane based on total reflection according to claim 1 is characterized in that said light beam arrives prism/glass interface and further reflects, and incident angle is 78 °, and the refraction angle is 69.97 °.
5. the preparation method based on the parallel biological detection chip in plane of total reflection is characterized in that, comprises the steps:
At first prepare (a 100) silicon chip, thermal oxide growth layer of silicon dioxide film then, coating one deck photoresist on this substrate is placed on the mask plate of making directly over the substrate, exposure, develop after with the design transfer of mask plate to substrate;
Utilize BHF that substrate is carried out etching then, remove silicon dioxide, then this structure is carried out the silicon wet etching, thereby formed fiber support and prism structure;
Form the lens window through fine process again, plated metal aluminium Al layer and form the lens window at first then carries out DRIE and carves and wear silicon chip;
Remove the Al layer at last, SiO
2Layer combines Si chip and glass substrate with epoxy glue again, just processes full die, carries out transcription with PDMS, obtains the fiber support of this chip, and optical fiber stops, lens and prism.
6. the preparation method of the parallel biological detection chip in the plane based on total reflection according to claim 5; It is characterized in that; Said silicon wet etching is meant: the etching liquid that uses is TMAH TMAH, and silicon is carried out anisotropic etching; (111) etch rate of crystal face is slow, and the side forms 54.7 ° inclination angle.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102998234A (en) * | 2012-12-14 | 2013-03-27 | 江苏苏净集团有限公司 | Micro liquid grain counter chip |
| JP2019522814A (en) * | 2016-05-31 | 2019-08-15 | サントル ナショナル ドゥ ラ ルシェルシュ シアンティフィック | Apparatus and method for providing illumination for total reflection fluorescence microscopy |
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| CN1306631A (en) * | 1997-12-01 | 2001-08-01 | 艾利森电话股份有限公司 | Method and device for fixing optical fibres |
| CN1569271A (en) * | 2003-12-26 | 2005-01-26 | 中国科学院理化技术研究所 | Minisize solid silicon needle array chip and its preparation method and use |
| CN101796394A (en) * | 2007-07-12 | 2010-08-04 | 纳米识别技术股份公司 | Optoelectronic sensor system |
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2011
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| CN1306631A (en) * | 1997-12-01 | 2001-08-01 | 艾利森电话股份有限公司 | Method and device for fixing optical fibres |
| CN1569271A (en) * | 2003-12-26 | 2005-01-26 | 中国科学院理化技术研究所 | Minisize solid silicon needle array chip and its preparation method and use |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102998234A (en) * | 2012-12-14 | 2013-03-27 | 江苏苏净集团有限公司 | Micro liquid grain counter chip |
| JP2019522814A (en) * | 2016-05-31 | 2019-08-15 | サントル ナショナル ドゥ ラ ルシェルシュ シアンティフィック | Apparatus and method for providing illumination for total reflection fluorescence microscopy |
| US11047799B2 (en) | 2016-05-31 | 2021-06-29 | Centre National De La Recherche Scientifique | Device and method for providing illumination for total-internal-reflection fluorescence microscopy using opaque mask |
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Application publication date: 20120411 |