CN105203508B - The preparation method of fluorescence nano on-gauge plate - Google Patents
The preparation method of fluorescence nano on-gauge plate Download PDFInfo
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- CN105203508B CN105203508B CN201510551458.0A CN201510551458A CN105203508B CN 105203508 B CN105203508 B CN 105203508B CN 201510551458 A CN201510551458 A CN 201510551458A CN 105203508 B CN105203508 B CN 105203508B
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Abstract
This case is related to the preparation method of fluorescence nano on-gauge plate, including：1) electron-beam vapor deposition method or magnetron sputtering method is used to form metal layer in substrate surface；2) in layer on surface of metal spin coating photoresist layer；Photoresist layer is equidistantly exposed using E-beam lithography, forms equidistant photoresist lines after development；3) photoresist lines are soaked in progress surface silanization processing in silylating reagent, form silylation layer；4) silylation layer is soaked in fluorescent dye solution and reacts 30min in the surface modification fluorescent dye layer of silylation layer；5) in fluorescent dye layer surface spin coating transparent protective layer.This preparation method step is few, simple for process, and reaction condition is mildly pollution-free, technique favorable reproducibility；By accurately adjusting the structure size of fluorescence nano on-gauge plate, realizes the proper alignment of fluorescence linear, intuitively reflect the resolution capability of system；Meanwhile the on-gauge plate of this structure can realize batch making, stability is high, high resolution.
Description
Technical field
The present invention relates to a kind of preparation methods of fluorescence nano on-gauge plate, more particularly to a kind of for characterizing super-resolution fluorescence
The preparation method of the fluorescence nano on-gauge plate of microscopic system resolution ratio.
Background technology
For a long time, optical microscopy imaging technology plays always important work during the mankind explore microcosmos
With.However, being constrained by Abbe diffraction limit, traditional optical microtechnic can not take life science to nanoscale.
With the continuous development of fluorescent microscopic imaging and perfect so that break through diffraction limit and be possibly realized.In recent years, it has emerged and has been permitted
More super-resolution fluorescence microtechnics, such as unimolecule positioning microscopy (Single Molecule Localization
Microscopy, SMLM), Structured Illumination microscopy (Structured Illumination Microscopy, SIM) and
Stimulated emission depletion microscopy (Stimulated Emission Depletion, STED) etc..These Induced Fluorescence Microscopies
Bring scientific research into " nanometer " field, allow the mankind can it is " real-time " observation living cells in molecular motion rule, ground for disease
Study carefully and brings revolutionary variation with medicament research and development.Compared with other technologies, STED is based on Confocal laser endomicroscopy, is excited by
Fluorescent material be limited to less than in the range of diffraction limit to realize ultrahigh resolution, image taking speed is fast, can observe living thin
The process of intracellular real-time change.
Resolution ratio is the evaluation most important standard of optical microscope system, is different from ordinary optical microscopic system, and super-resolution is glimmering
The resolution ratio of light microscopic system can not directly give, but need to carry out complicated image procossing by testing standard plate, with
The factors such as optical system parameter (such as optical source wavelength, intensity), data processing technique and fluorescent dye type are closely related, and this
A little requirements all substantially increased to testing standard plate.Therefore, the resolution ratio of accurate characterization super-resolution fluorescence microscopic system seems
It is extremely important.But in the prior art, the structure and size of on-gauge plate are difficult to obtain the accurate processing of repeatability, fluorescence lines
Arrangement it is not neat enough, manufacturing process is complicated, cannot achieve mass production, and the difficulty of this processing is mainly originated to mark
The structure design of quasi- plate is scientific and reasonable not enough, also fails to accomplish perfection in the arrangement of some process procedures.
Invention content
For the shortcomings of the prior art, the purpose of the present invention is to provide a kind of fluorescence nano on-gauge plates
It is aobvious to can be used for super-resolution fluorescence by redesign to plate construction and to the redesign of process detail for preparation method
The measurement of micro-system resolution ratio, preparation process is simple, reusable, and product stability is high, is easy to produce in batches.
The technical solution adopted by the present invention is as follows：
A kind of preparation method of fluorescence nano on-gauge plate, including：
Step 1) forms one layer of metal layer using electron-beam vapor deposition method or magnetron sputtering method in substrate surface；
Step 2) is in the layer on surface of metal spin coating photoresist layer；Photoresist layer is equidistantly exposed using E-beam lithography
Light forms equidistant photoresist lines after development；
The photoresist lines are soaked in progress surface silanization processing in silylating reagent by step 3), in photoresist line
Surface forms silylation layer；
The silylation layer is soaked in fluorescent dye solution by step 4) reacts 30min to be repaiied on the surface of the silylation layer
Adorn fluorescent dye layer；After being rinsed with ethyl alcohol, nitrogen drying；
Step 5) is used to protect the transparent protective layer of fluorescent dye in the fluorescent dye layer surface spin coating.
Preferably, the preparation method of the fluorescence nano on-gauge plate, wherein the basal layer is silicon or silicon nitride.
Preferably, the preparation method of the fluorescence nano on-gauge plate, wherein the basal layer is silicon.
Preferably, the preparation method of the fluorescence nano on-gauge plate, wherein the metal layer be selected from chromium, titanium, aluminium,
Gold or combinations thereof.
Preferably, the preparation method of the fluorescence nano on-gauge plate, wherein the metal layer is chromium.
Preferably, the preparation method of the fluorescence nano on-gauge plate, wherein the silylating reagent is 3- aminopropyls
Three second methoxy silanes or 3- aminopropyl triethoxysilanes.
Preferably, the preparation method of the fluorescence nano on-gauge plate, wherein the thickness of the metal layer be 5~
50nm。
Preferably, the preparation method of the fluorescence nano on-gauge plate, wherein the thickness of the photoresist layer be 50~
130nm。
Preferably, the preparation method of the fluorescence nano on-gauge plate, wherein the thickness of the transparent protective layer is 30
~100nm.
Preferably, the preparation method of the fluorescence nano on-gauge plate, wherein the fluorescent dye is that isothiocyanic acid is glimmering
Light element or fluorescin ATTO 488.
The beneficial effects of the invention are as follows：The preparation method step for the on-gauge plate that this case refers to is few, simple for process, reaction condition
Mild pollution-free, technique favorable reproducibility passes through the improvement to standard harden structure so that it can be used for super-resolution fluorescence microscopic system
The accurate measurement of resolution ratio；By accurately adjusting the structure size of fluorescence nano on-gauge plate, the neat of fluorescence linear is realized
Arrangement, intuitively reflects the resolution capability of system；Meanwhile the on-gauge plate of this structure can realize batch making, operation letter
Easily convenient, reusable, stability is high, high resolution.
Description of the drawings
Fig. 1 is the structural schematic diagram using fluorescence nano on-gauge plate made from preparation method of the present invention.
Specific implementation mode
Present invention will be described in further detail below with reference to the accompanying drawings, to enable those skilled in the art with reference to specification text
Word can be implemented according to this.
Referring to Fig.1, this case proposes the preparation method of the fluorescence nano on-gauge plate of an embodiment, including：
Step 1) forms one layer of metal layer 2 using electron-beam vapor deposition method or magnetron sputtering method on 1 surface of basal layer；
Step 2) is in 2 surface spin coating photoresist layer 3 of metal layer；Photoresist layer 3 is equidistantly exposed using E-beam lithography
Light forms equidistant photoresist lines after development；
Photoresist lines are soaked in progress surface silanization processing in silylating reagent by step 3), form silylation layer 4；It is aobvious
The photoresist lines of movie queen are quartz-like (SiO2) structure, surface has abundant hydroxyl group, can directly utilize silanization
Reagent carries out silanization to linear surface, and surface is made to have functionalization group.Layer on surface of metal is not conducive to silanization
Hydroxyl group, so selective modification can be realized.
Silylation layer 4 is soaked in fluorescent dye solution by step 4) reacts 30min in the surface modification fluorescence of silylation layer 4
Dye coating 5；After being rinsed with ethyl alcohol, nitrogen drying；
Step 5) is used to protect the transparent protective layer 6 of fluorescent dye in the 5 surface spin coating of fluorescent dye layer.Transparent protective layer 6
For transparent macromolecule resin material, its effect is protection fluorescent dye, and own not will produce fluorescence.
In the above-described embodiments, basal layer 1 is preferably silicon or silicon nitride, most preferably silicon.
In the above-described embodiments, metal layer 2 is preferably selected from chromium, titanium, aluminium, gold or combinations thereof, most preferably chromium.
In the above-described embodiments, silylating reagent is preferably three second methoxy silane of 3- aminopropyls or three ethoxy of 3- aminopropyls
Base silane.
In the above-described embodiments, the thickness of metal layer 2 is preferably 5~50nm.The study found that the thickness of metal layer 2 is to mark
The precision of quasi- the surveyed resolution ratio of plate has a certain impact, if resolution accuracy will be caused to deviate optimal value beyond preferred range.
In the above-described embodiments, the thickness of photoresist layer 3 is preferably 50~130nm.The study found that the thickness of photoresist layer 3
The precision of degree resolution ratio surveyed to on-gauge plate has a certain impact, if resolution accuracy will be caused to deviate most beyond preferred range
The figure of merit.
In the above-described embodiments, the thickness of transparent protective layer 6 is preferably 30~100nm.The study found that transparent protective layer 6
The precision of thickness resolution ratio surveyed to on-gauge plate have a certain impact, if resolution accuracy will be caused inclined beyond preferred range
From optimal value.
In the above-described embodiments, fluorescent dye is preferably fluorescein isothiocynate or fluorescin ATTO 488, most preferably
For fluorescein isothiocynate.Fluorescein isothiocynate and the on-gauge plate matching degree of this structure are best, and the two matching coordinative can make mark
Quasi- plate obtains best resolution test precision.
Although the embodiments of the present invention have been disclosed as above, but its is not only in the description and the implementation listed
With it can be fully applied to various fields suitable for the present invention, for those skilled in the art, can be easily
Realize other modification, therefore without departing from the general concept defined in the claims and the equivalent scope, the present invention is simultaneously unlimited
In specific details and legend shown and described herein.
Claims (8)
1. a kind of preparation method of fluorescence nano on-gauge plate, including：
Step 1) forms one layer of metal layer using electron-beam vapor deposition method or magnetron sputtering method in substrate surface；
Step 2) is in the layer on surface of metal spin coating photoresist layer；Photoresist layer is equidistantly exposed using E-beam lithography,
Equidistant photoresist lines are formed after development；
The photoresist lines are soaked in progress surface silanization processing in silylating reagent by step 3), in photoresist lines table
Face forms silylation layer；
The silylation layer is soaked in fluorescent dye solution by step 4) reacts 30min with glimmering in the surface modification of the silylation layer
Photoinitiator dye layer；After being rinsed with ethyl alcohol, nitrogen drying；
Step 5) is used to protect the transparent protective layer of fluorescent dye in the fluorescent dye layer surface spin coating.
2. the preparation method of fluorescence nano on-gauge plate according to claim 1, which is characterized in that the basal layer be silicon or
Silicon nitride.
3. the preparation method of fluorescence nano on-gauge plate according to claim 1, which is characterized in that the metal layer is selected from
Chromium, titanium, aluminium, gold or combinations thereof.
4. the preparation method of fluorescence nano on-gauge plate according to claim 1, which is characterized in that the silylating reagent is
3- aminopropyl trimethoxysilanes or 3- aminopropyl triethoxysilanes.
5. the preparation method of fluorescence nano on-gauge plate according to claim 1, which is characterized in that the thickness of the metal layer
For 5~50nm.
6. the preparation method of fluorescence nano on-gauge plate according to claim 1, which is characterized in that the thickness of the photoresist layer
Degree is 50~130nm.
7. the preparation method of fluorescence nano on-gauge plate according to claim 1, which is characterized in that the transparent protective layer
Thickness is 30~100nm.
8. the preparation method of fluorescence nano on-gauge plate according to claim 1, which is characterized in that the fluorescent dye is different
Thiocyanic acid fluorescein or fluorescin ATTO 488.
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| CN110095441B (en) * | 2019-04-19 | 2021-12-10 | 中国科学院苏州生物医学工程技术研究所 | Fluorescent nanometer scale component and preparation and application thereof |
| CN111006772A (en) * | 2019-12-31 | 2020-04-14 | 上海市计量测试技术研究院 | Standard plate group and method for detecting minimum detectable size of thermal infrared imager |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1844300A (en) * | 2006-04-04 | 2006-10-11 | 吉林大学 | Method for making same dye molecule emit light with different colors by substrate induction |
| CN103712965A (en) * | 2013-12-23 | 2014-04-09 | 中国科学院苏州生物医学工程技术研究所 | Resolution testing standard board manufacturing method for super-resolution fluorescence microscope system |
| CN103954600A (en) * | 2014-05-12 | 2014-07-30 | 国家纳米科学中心 | Fluorescent nano-scale component and manufacturing method thereof |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1844300A (en) * | 2006-04-04 | 2006-10-11 | 吉林大学 | Method for making same dye molecule emit light with different colors by substrate induction |
| CN103712965A (en) * | 2013-12-23 | 2014-04-09 | 中国科学院苏州生物医学工程技术研究所 | Resolution testing standard board manufacturing method for super-resolution fluorescence microscope system |
| CN103954600A (en) * | 2014-05-12 | 2014-07-30 | 国家纳米科学中心 | Fluorescent nano-scale component and manufacturing method thereof |
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