CN106932372A - The detection architecture and structure being combined with surface-enhanced fluorescence technology based on optical tweezer - Google Patents
The detection architecture and structure being combined with surface-enhanced fluorescence technology based on optical tweezer Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
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Abstract
The present invention relates to plant the detection architecture being combined with surface-enhanced fluorescence technology based on optical tweezer, using SiO2@Ag complex microspheres and Ag films, while using SiO2@Ag complex microspheres and Ag nano thin-films enhancing fluorescence molecule signal, by SiO2@Ag complex microspheres, in carried metal nano particle above, SiO are manipulated with optical optical tweezers system as handle2@Ag complex microspheres, capture laser is the Submillineter Wave Technology of 975nm, and fluorescing fractions actively find fluorescence molecule to be measured, carry out the scanning analysis and real-time monitoring of ad-hoc location using the exciting light of 532nm.Construction method the invention further relates to be based on the detection architecture that optical tweezer is combined with surface-enhanced fluorescence technology.Detected with optical tweezer fluorescence detecting system, the fluorescence signal of ad-hoc location can be detected.Compared with prior art, this invention structure is relatively simple, can be with the Fluorescence Increasing signal of the hypofluorescence material of real-time detection ad-hoc location.
Description
Technical field
Detection architecture and structure that the present invention is combined based on optical tweezer with surface-enhanced fluorescence technology, are related to fluoroscopic examination skill
Art, it is specific for precisely strengthening in surface-enhanced fluorescence based on the method that optical tweezer technology is combined with surface-enhanced fluorescence technology
The signal of the fluorescence molecule of position simultaneously realizes analysis in real time, improves the sensitivity of fluoroscopic examination.
Background technology
Fluoroscopic examination has the advantages that sensitivity is high, method is various as a kind of important modern spectral technique, extensive
It is applied to medical diagnosis and biological technical field.But, for trace analysis or hypofluorescence molecular system, detection technique of fluorescence
Remolding sensitivity is relatively low, limits its range of application, so, the sensitivity for improving fluoroscopic examination is the heat of fluorescent technique current research
Point.
Early in the seventies in last century, K.H.Drexhage has found metal surface enhancing fluorescence phenomenon and has carried out detailed solution
Release.It is international research focus in recent years to the study on the modification that Electromagnetic enhancement effect carries out molecular fluorescence using metal Nano structure
One of.At present, numerous researchers realizes fluorescence in nanostructured surfaces such as metal nanometer thin film, metal nanoparticles
Enhancing.The general principle of foundation：To metal nanometer thin film, beam of laser is radiated at smooth metal film surfaces, metal from
Intercoupled resonance by electronics and incident field, a kind of electromagnetic wave along surface transmission of formation, we term it surface etc. from
Polariton.This CGCM has two most obvious features, i.e. surface local and near field enhancing.Surface local refers to table
Face etc. is tied to metallic surface from polariton, exists in the form of surface plasma wave.Near field enhancing refers to this
The electromagnetic field intensity of coupling can be more than original incident light for being used for exciting and producing surface plasmon-polarition near metal surface
Field intensity, when fluorescence molecule is in this near metal surface, fluorescence signal is remarkably reinforced；To metal nanoparticle, due to
The yardstick very little of metal nanoparticle, incident light is applied to whole nano particle when on metal nanoparticle and will all be in light field
In effect.Therefore under the electric field action of incident light, the electron cloud inside nano particle will deviate with atomic nucleus, due to
There is storehouse storehouse power between the two, therefore electron cloud ceaselessly will back and forth vibrate under the effect of extraneous light field around atomic nucleus, this
It is exactly local surface plasma vibration to plant vibration.Particularly, when the frequency of incident light is identical with the frequency that electron cloud vibrates,
Local surface plasma resonance will occur, local surface phasmon is produced.When resonance occurs, metal nanoparticle will be big
Amount absorbs the energy of incident field and by its confinement in its near surface, so that very strong local fields are produced, so, when fluorescence point
When son is in vicinity of metal nanoparticles, fluorescence signal enhancing.
Gap（gap）Structure is one of very extensive composite metal structures for applying in recent years, is primarily referred to as metal nano
There is nano level distance between structure, and this distance typically uses metal oxide, Si02, PMMA, or polyelectrolyte etc.
Realized Deng as separation layer.In interstitial structure, by taking metal nanoparticle and metallic film as an example, metal nanoparticle surface
Local surface of generation etc. occurs consumingly to couple from the surface plasmon-polarition that polariton and metal film surfaces are produced
Effect so that in interstitial structure the fluorescence signal of fluorescence molecule greatly strengthened.
What early start systematically studied metal-enhanced fluorescence signal is the Lakowicz groups from the U.S., and they will be only
The DNA molecular solution that week fluorescent can be sent out pours into the parallel glass composition that two pieces of surfaces deposited Ag island film (Argent grain of raw sugar)
Container in, due to surface plasma Localized field enhancement act on, when light irradiation is excited with 287nm find DNA fluorescence
Light strength ratio does not have to increased 80 times in the presence of Ag island film.J.H. Song of Brown universities et al. are using local surface etc. from pole
Change excimer and surface plasmon-polarition coupling effect, realize enhancement effect higher.They with CdSe/ZnS quantum dots with
PMMA mixed films carve candle technology and have obtained cylindrical type pores array as mask using electron beam, then deposit Ag, obtain
The cyclic array and film composite structure of Ag.By detection, fluorescence signal is 50 times or so in the presence of no Ag arrays, this
Just explanation fluorescence signal enhancing has inseparable relation with the size of metal Nano structure.But, use electron beam lithography
Technology, cost is higher, and the region of one piece of very little can only be operated every time, so, this method is in interstitial structure
In using limited.The PMMA media of the RhB that adulterates are placed on Ag nanometer blocks and Ag films by the Ming Hai professor seminars of Chinese University of Science and Technology of China
Between, cause that the fluorescence intensity of RhB is obtained from polariton and the close coupling of surface plasmon-polarition using local surface etc.
To 521 times of enhancings.Here, Ag nanometer blocks form interstitial structure with the film of Ag, by ceaselessly preparing the mixed of different-thickness
Dielectric layer is closed, the enhancing effect of maximum is obtained, the cycle that operates is long.Also there is researcher with diameter 70nm or so, length
The Ag nano wires of 1-3um and smooth Ag films constitute an interstitial structure, and, in 15-70nm, this gap is by 1-2 layers for clearance distance
The nanocrystalline controls of PbS, use the laser excitation of 633nm, focused on 100 times of object lens and collect signal, in this gap
The nanocrystalline fluorescence signals of PbS greatly strengthened.But, for this interstitial structure, it is desirable to accurately deexcitation gap
Fluorescence molecule in structure is relatively difficult.
To sum up, for interstitial structure, the treatment generally to sample is comparatively laborious, and electron beam lithography skill is also used sometimes
Art, cost is higher, and the cycle is more long；Additionally, we can't control the distance between metal nanoparticle and testing molecule, i.e.,
Can not be positioned, it is impossible to precisely excite and determine, range of application is very limited.In order to overcome these not enough, some researchers are just
Optical tweezer technology is introduced in interstitial structure, for detecting Raman.
Optical tweezer can be manipulated and captured, briefly, just to nanometer also known as single beam particle traps to micron-sized particle
It is the technology for capturing, manipulating control fine particle with the three-dimensional gradient potential well of the laser formation of a branch of height convergence.But, it is right
In the detection architecture that optical tweezer is combined with surface-enhanced fluorescence, the focus of research is the single metal nanoparticle of optical tweezer capture,
Such as Ag nano particles strengthen the fluorescence molecule of ad-hoc location, and the enhancing due to single nanoparticle to fluorescence signal has
Limit, so enhancing effect is unsatisfactory.
The content of the invention
It is an object of the invention to：A kind of detection architecture being combined with surface-enhanced fluorescence technology based on optical tweezer is provided.
Another object of the present invention is：There is provided and be based on the detection architecture that optical tweezer is combined with surface-enhanced fluorescence technology
Construction method.
The object of the invention is achieved through the following technical solutions：It is a kind of to be combined with surface-enhanced fluorescence technology based on optical tweezer
Detection architecture, using SiO2@Ag complex microspheres and Ag films, wherein, while using SiO2@Ag complex microspheres and Ag nano thin-films increase
Hyperfluorescence molecular signal, by SiO2@Ag complex microspheres, in carried metal nano particle above, are grasped as handle with optical optical tweezers system
Control SiO2@Ag complex microspheres, capture laser is the Submillineter Wave Technology of 975nm, and fluorescing fractions use the exciting light of 532nm, actively
Fluorescence molecule to be measured is found, the scanning analysis of ad-hoc location is carried out and is realized real-time monitoring.
Described optical optical tweezers system is purchased from Thorlabs, is Thorlabs systems.
The present invention provides a kind of construction method of the detection architecture being combined with surface-enhanced fluorescence technology based on optical tweezer, adopts
With sandwich structure, using upper and lower sheet glass as upper and lower substrate, it is successively from top to bottom：Upper substrate, Ag nano thin-films, poly- electricity
Solution matter layer（PE layers）, fluorescence molecule layer, subtegulum, at least with two-sided glued together, centre is SiO for two pieces of substrates2@Ag are combined
Microspheres solution.
Further, described SiO2@Ag complex microsphere solution is 20uL, and upper subtegulum is enclosed in double faced adhesive tape and vaseline
Between.
On the basis of such scheme, one layer of individual layer Ag nano thin-film is directly loaded on described upper substrate, concrete operations are：
12mL Ag colloidal sols, 6mL hexamethylenes, 5mL absolute ethyl alcohols are added in beaker, most of hexamethylene is then removed, with clean glass
Piece picks up Ag nano thin-films, and drying must be loaded with Ag nano thin-film substrates.
On the basis of such scheme, polyelectrolyte layer is loaded on described Ag nano thin-films, step is：
（1）It is loaded with the PDDA that Ag nano thin-films substrate immersion 1mol/L NaCl solution is prepared（0.003mol/L）In solution,
Taken out after 10min, the deionized water rinsing substrate surface is for several times；
（2）The PSS that Ag nano thin-films substrate immersion 1mol/L NaCl solution is prepared will be loaded with（0.003mol/L）In solution,
Taken out after 10min, the deionized water rinsing substrate surface number；
（3）Repeat（1）、（2）Twice, repeat（1）Once, 7 strata electrolyte individual layers of Ag nano thin-films surface eutectoid content, i.e.,
It is successively PDDA, PSS, PDDA, PSS, PDDA, PSS, PDDA layer, totally 3.5 polyelectrolyte layers of bilayer.
On the basis of such scheme, fluorescence molecule is loaded on described polyelectrolyte layer, method is：Poly- electricity will have been adsorbed
The Ag nano thin-films substrate immersion 2.5*10 of solution matter layer-5The Cy3 fluorescence molecules of the positively charged anionic in mpl/L surfaces（Band
Negative electrical charge）30min in solution, deionized water rinsing is multiple, N2Drying.
It is of the invention compared with traditional interstitial structure because introduce optical tweezer, can carry out detection and localization and in real time analysis；With
Optical tweezer manipulation single metal receives particle to carry out Fluorescence Increasing and compares, in SiO2More metal nano is loaded on@Ag complex microspheres
Particle, can obtain enhancing effect higher while detection and localization.Present invention structure is relatively simple, can be special with real-time detection
The Fluorescence Increasing signal of the hypofluorescence material that positioning is put.
Brief description of the drawings
The sandwich knot of the structure of the detection architecture that Fig. 1 present invention is combined based on optical tweezer with surface-enhanced fluorescence technology
Structure schematic diagram；
Fig. 2 is the close-up schematic view of Fig. 1；
Fig. 3 fluoroscopic examination figures；
Label declaration in figure：
In Fig. 1 and Fig. 2
10 --- sandwich structure；
11st, 12 --- upper and lower sheet glass；
13 --- Ag nano thin-films；
14 --- polyelectrolyte layer；
15 --- fluorescence molecule layer；
16——SiO2@Ag complex microsphere solution
In Fig. 3
1 --- without 1SiO at Ag films2@Ag；
2 --- without 1SiO at Ag films2@Ag；
3 --- 1SiO at Ag films2@Ag。
Specific embodiment
A kind of detection architecture being combined with surface-enhanced fluorescence technology based on optical tweezer, using SiO2@Ag complex microspheres and
Ag films, while using SiO2@Ag complex microspheres and Ag nano thin-films enhancing fluorescence molecule signal, by SiO2@Ag complex microsphere conducts
Handle, in carried metal nano particle above, SiO is manipulated with optical optical tweezers system2@Ag complex microspheres, capture laser is remote for 975nm's
Infrared laser, fluorescing fractions actively find fluorescence molecule to be measured using the exciting light of 532nm, carry out the scanning point of ad-hoc location
Analyse and realize real-time monitoring.
Optical optical tweezers system in detection architecture is purchased from Thorlabs, and the capture laser for using is the Submillineter Wave Technology of 975nm, glimmering
Light part uses the exciting light of 532nm.Build as depicted in figs. 1 and 2：
The structure of the detection architecture being combined with surface-enhanced fluorescence technology based on optical tweezer, using sandwich structure, with upper and lower glass
Glass piece 11,12 is successively from top to bottom as upper and lower substrate：Upper substrate, Ag nano thin-films 13, polyelectrolyte layer 14（PE layers）、
Fluorescence molecule layer 15, subtegulum, at least with two-sided glued together, centre is SiO for two pieces of substrates2@Ag complex microspheres solution 16.
Build in the steps below：
1）Chemical reduction method prepares Ag nano particles：In 500mL beakers, 0.0600g AgNO3,300mL deionized water, boiling are added
6mL1% citric acid three sodium solutions are added after rising（0.0600g trisodium citrates are dissolved in 6mL deionized waters）, ebuillition of heated 1h.
After cooling, centrifugation washing 3 times obtains the negatively charged Ag nano particles in surface standby.
2）SiO2The alkylation of microballoon：Using silane coupling A PTMS to SiO2Microballoon is alkylated, and makes its surface band
Upper positive charge, concrete operations：In 4m centrifuge tubes, sequentially add 150uL amido SiO2 microballoons (4um), 1.3mL absolute ethyl alcohols,
1.5uL APTMS, shaking table reaction（40 °, 200r/min）6 hours.It is centrifuged after end, is first washed 6 times with absolute ethyl alcohol, then wash 3
It is secondary, deionized water is added, obtain the positively charged SiO in surface2Microballoon is standby.
3）SiO2The preparation of@Ag complex microspheres：SiO after alkylation2Microsphere surface is positively charged, and Ag nanometers for preparing
Particle surface is negatively charged, sets the SiO of different proportion2:AgNPs, after mixing, shaking table reaction 5min（Room temperature, 70 r/min）,
Centrifugation washing 3 times, adds deionized water standby.
4）The surface cleaning of glass substrate：The ultrasound 30min in following liquid, 2% Hellmanex washing lotions successively；It is ultrapure
Water；Acetone；Ultra-pure water；Methyl alcohol；Ultra-pure water, removes organic impurities such as grease, rosin, the wax of surface of glass slide etc., N2After drying
12h, ultra-pure water ultrasound 30min, N are soaked in the concentrated sulfuric acid2Drying is standby.
5）The preparation of Ag nano thin-films：12mL Ag colloidal sols, 6mL hexamethylenes, the anhydrous second of 5mL are sequentially added in 50mL beakers
Alcohol, Ag NPs are transferred quickly to the interface of the aqueous solution and hexamethylene；Upper strata major part hexamethylene is removed, due to interfacial tension, shape
Into the monofilm of Ag nano particles；The monofilm that the Ag nano particles for picking up liquid level surface with clean glass substrate are formed, in
Dried in ventilating kitchen standby.
6）Fluorescence molecule is carried on Ag nano thin-films：Ag nano thin-films are negatively charged, the fluorescence used in the present invention point
Sub- Cy3 is the fluorescent dye of anionic, in order to fluorescence molecule is fixed on Ag nano thin-films, the first group on Ag nano thin-films
Polyelectrolyte layer is filled, step is：
（1）Negatively charged Ag film substrates are immersed into the 0.003mol/L PDDA solution positively charged with it（The NaCl of 1mol/L
Solution is prepared）In, taken out after 10min, now due to Electrostatic Absorption, Ag film surface charges are reversed to positive charge, deionized water punching
Wash substrate surface for several times；
（2）By Ag film transfers to electronegative 0.003mol/L PSS solution（The NaCl solution of 1mol/L is prepared）In, after 10min
Take out, Ag films surface will adsorb a strata anion electrolyte, its surface is taken negative electrical charge, deionized water rinsing base again
Piece surface number；
（3）Repeat（1）（2）Twice, repeat（1）Once, i.e., last Ag films adsorption is PDDA layers, positively charged.It is poly-
After dielectric substrate is completed, 2.5*10 is placed in-530min in mol/L Cy3 fluorescence molecule solution, deionized water rinsing, now,
The fluorescent dye Cy3 of anionic has been carried on Ag nano thin-films, N2Drying is standby.
7）Prepare " sandwich " sample structure：The upper substrate and subtegulum that will load fluorescence molecule using double faced adhesive tape are bonded at
Together, the middle SiO that 20uL is added dropwise2@Ag complex microsphere solution, carries out positioning fluoroscopic examination.
Testing result：
As shown in figure 3,1 is without 1SiO at Ag films2@Ag；2 is without 1SiO at Ag films2@Ag；3 is 1SiO at Ag films2@Ag, without Ag films
Place 1SiO2Without 1SiO at@Ag and Ag films2@Ag wavelength is little on fluorescence intensity influence, is level of approximation line；At Ag films of the present invention
1SiO2The wavelength of@Ag3 is responded to fluorescence intensity, is computed, and the present invention is combined based on optical tweezer with surface-enhanced fluorescence technology
Detection architecture, Fluorescence Increasing 41 times.
Claims (8)
1. a kind of detection architecture being combined with surface-enhanced fluorescence technology based on optical tweezer, using SiO2@Ag complex microspheres and Ag
Film, it is characterised in that：Use SiO simultaneously2@Ag complex microspheres and Ag nano thin-films enhancing fluorescence molecule signal, by SiO2@Ag are answered
Microballoon is closed as handle, in carried metal nano particle above, SiO is manipulated with optical optical tweezers system2@Ag complex microspheres, capture laser
It is the Submillineter Wave Technology of 975nm, fluorescing fractions actively find fluorescence molecule to be measured, carry out certain bits using the exciting light of 532nm
The scanning analysis and real-time monitoring put.
2. the detection architecture being combined with surface-enhanced fluorescence technology based on optical tweezer according to claim 1, its feature is existed
In：Described optical optical tweezers system is Thorlabs systems.
3. a kind of detection architecture being combined with surface-enhanced fluorescence technology based on optical tweezer according to claim 1 and 2
Build, using sandwich structure, it is characterised in that：Using upper and lower sheet glass as upper and lower substrate, it is successively from top to bottom：Upper base
Piece, Ag nano thin-films, polyelectrolyte layer（PE layers）, fluorescence molecule layer, subtegulum, two pieces of substrates are at least adhesive in one with two-sided
Rise, centre is SiO2@Ag complex microsphere solution.
4. the structure side of the detection architecture being combined with surface-enhanced fluorescence technology based on optical tweezer according to claim 3
Method, it is characterised in that：Described SiO2@Ag complex microsphere solution is 20uL, between being enclosed in upper subtegulum with double faced adhesive tape and vaseline.
5. the structure side of the detection architecture being combined with surface-enhanced fluorescence technology based on optical tweezer according to claim 3
Method, it is characterised in that：One layer of individual layer Ag nano thin-film is directly loaded on described upper substrate, concrete operations are：Add in beaker
Enter 12mL Ag colloidal sols, 6mL hexamethylenes, 5mL absolute ethyl alcohols, then remove most of hexamethylene, pick up Ag with clean slide and receive
Rice film, drying must be loaded with Ag nano thin-film substrates.
6. the structure side of the detection architecture being combined with surface-enhanced fluorescence technology based on optical tweezer according to claim 5
Method, it is characterised in that：Polyelectrolyte layer is loaded on described Ag nano thin-films, step is：
(1) it is loaded with the PDDA that Ag nano thin-films substrate immersion 1mol/L NaCl solution is prepared（0.003mol/L）In solution,
Taken out after 10min, the deionized water rinsing substrate surface is for several times；
(2) PSS that Ag nano thin-films substrate immersion 1mol/L NaCl solution is prepared will be loaded with（0.003mol/L）In solution,
Taken out after 10min, the deionized water rinsing substrate surface number；
（3）Repeat（1）、（2）Twice, repeat（1）Once, 7 strata electrolyte individual layers of Ag nano thin-films surface eutectoid content, i.e.,
It is successively PDDA, PSS, PDDA, PSS, PDDA, PSS, PDDA layer, totally 3.5 polyelectrolyte layers of bilayer.
7. the structure side of the detection architecture being combined with surface-enhanced fluorescence technology based on optical tweezer according to claim 6
Method, it is characterised in that：Fluorescence molecule is loaded on described polyelectrolyte layer, method is：The Ag of polyelectrolyte layer will have been adsorbed
Nano thin-film substrate immerses 2.5*10-5The Cy3 fluorescence molecules of the positively charged anionic in mpl/L surfaces（It is negatively charged）Solution
Middle 30min, deionized water rinsing is multiple, N2Drying.
8. the structure side of the detection architecture being combined with surface-enhanced fluorescence technology based on optical tweezer according to claim 3
Method, it is characterised in that the SiO2The preparation of@Ag complex microsphere solution：
1）Chemical reduction method prepares Ag nano particles：In 500mL beakers, 0.0600g AgNO3,300mL deionized water, boiling are added
6mL1% citric acid three sodium solutions are added after rising（0.0600g trisodium citrates are dissolved in 6mL deionized waters）, ebuillition of heated 1h,
After cooling, centrifugation washing 3 times obtains the negatively charged Ag nano particles in surface standby；
2）SiO2The alkylation of microballoon：Using silane coupling A PTMS to SiO2Microballoon is alkylated, and its surface is taken just
Electric charge, concrete operations：In 4m centrifuge tubes, 150uL amido SiO2 microballoons (4um), 1.3mL absolute ethyl alcohols, 1.5uL are sequentially added
APTMS, shaking table reaction（40 °, 200r/min）6 hours.It is centrifuged after end, is first washed 6 times with absolute ethyl alcohol, then washed 3 times, is added
Deionized water, obtains the positively charged SiO in surface2Microballoon is standby.
3）SiO2The preparation of@Ag complex microspheres：SiO after alkylation2Microsphere surface is positively charged, and the Ag nano particles for preparing
Surface is negatively charged, sets the SiO of different proportion2:AgNPs, after mixing, shaking table reaction 5min（Room temperature, 70 r/min）, centrifugation
Washing 3 times, adds deionized water to obtain SiO2@Ag complex microsphere solution.
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| WO2020021154A1 (en) * | 2018-07-27 | 2020-01-30 | Universidad Autónoma de Madrid | Method for detection of marked structures |
| CN113588615A (en) * | 2021-07-29 | 2021-11-02 | 东北大学秦皇岛分校 | SPR-based fluorescence enhancement device of nano-hemispherical structure array |
| CN114018881A (en) * | 2021-10-25 | 2022-02-08 | 南京大学 | Method for detecting local acoustic vibration by plasmon enhanced monomolecular fluorescence system |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108254632A (en) * | 2017-12-22 | 2018-07-06 | 同济大学 | Based on SiO2The method that microballoon movable information analyzes its surface charge density |
| WO2020021154A1 (en) * | 2018-07-27 | 2020-01-30 | Universidad Autónoma de Madrid | Method for detection of marked structures |
| CN113588615A (en) * | 2021-07-29 | 2021-11-02 | 东北大学秦皇岛分校 | SPR-based fluorescence enhancement device of nano-hemispherical structure array |
| CN114018881A (en) * | 2021-10-25 | 2022-02-08 | 南京大学 | Method for detecting local acoustic vibration by plasmon enhanced monomolecular fluorescence system |
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