CN105772118A - SERS (Surface Enhanced Raman Spectroscopy) microfluidic chip with enhanced substrate integrated on ITO conductive glass and preparation method for SERS microfluidic chip - Google Patents
SERS (Surface Enhanced Raman Spectroscopy) microfluidic chip with enhanced substrate integrated on ITO conductive glass and preparation method for SERS microfluidic chip Download PDFInfo
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- CN105772118A CN105772118A CN201510695042.6A CN201510695042A CN105772118A CN 105772118 A CN105772118 A CN 105772118A CN 201510695042 A CN201510695042 A CN 201510695042A CN 105772118 A CN105772118 A CN 105772118A
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components
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- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N21/65—Raman scattering
- G01N21/658—Raman scattering enhancement Raman, e.g. surface plasmons
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/10—Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
Abstract
The invention relates to a complex type multichannel SERS (Surface Enhanced Raman Spectroscopy) microfluidic chip based on a nano enhanced substrate integrated on ITO conductive glass and preparation method for the SERS microfluidic chip. According to the SERS microfluidic chip and the preparation method therefor, the ITO conductive glass is used as a substrate surface of an SERS microstructure by making full use the characteristics, i.e., smooth surface and good conductivity of the ITO conductive glass, and the morphology and size of nanoparticles are controlled through controlling the deposition potential and deposition time of the nanoparticles during nucleation and growth by adopting a two-step timed current deposition method, so that a metal-nanostructure SERS substrate with high sensitivity, good stability and uniform distribution can be effectively prepared. Further, the metal-nanostructure SERS substrate is bonded to a PDMS (Polydimethylsiloxane) cover plate containing array type microchannels so as to form and obtain the complex type high-flux SERS microfluidic chip. According to the chip, a plurality of SERS detection areas are manufactured and formed on the same ITO glass, and the nano enhanced substrate is obtained through synchronous electrochemical deposition, so that the morphology consistency is good, and the signal reproducibility is good.
Description
Technical field
The present invention relates to surface enhanced raman spectroscopy analysis (SurfaceEnhancedRamanSpectroscopy, SERS) and microfluidic chip analysis technical field.
Background technology
With it, microfluidic chip analysis method and technology have that amount of samples is little, structural design flexibly, in-situ test can be realized, be easily achieved the advantages such as microminiaturized and integrated, receive much concern in biochemical sample analysis and detection.At present, detection method conventional in micro-fluidic chip mainly has Electrochemical Detection, fluoroscopic examination and chemiluminescence detection etc., these methods have higher detection sensitivity, but the quantity of information that there is sample responses is few, it is difficult to acquire the deficiencies such as biological sample detailed structural information.Raman spectrum detects as a kind of lossless spectrum detection technique, detection speed is fast, the information of compound group and chemical bond can be provided from molecular level, informative, and its response signal is not by moisture interference in sample system, directly laser facula can be focused on the sample in the minim channel of micro-fluidic chip, it is shown that with biochemical sample detection on micro-fluidic chip, there is good matching and compatibility simultaneously.But it is weak especially to there is spectral response signal in Raman spectrum response, the problem that detection sensitivity is low.Therefore, the introducing of surface enhanced raman spectroscopy (SERS) technology becomes the first-selection of efficient analysis and the detection realizing biochemical sample on micro-fluidic chip.
At present, the MEMS based on micro photo-electro-mechanical technology is mainly had to prepare method and Chemical assembly method etc. in the method for the integrated SERS substrate in micro-fluidic SERS chip detection district.
MEMS prepares method, and to have controllability good, the advantage that the SERS substrate of preparation is uniform sequential, but it needs special installation, and preparation cost is higher.Vacuum vapour deposition is passed through as patent " a kind of micro-fluidic chip with surface reinforced Raman active and preparation method thereof " (CN103604797A) discloses one, the method of the microarray surface evaporation bi-layer metal film in PDMS micro-fluidic chip passage, structure has SERS substrate micro-fluidic chip, the method needs special instrument, preparation cost is higher, to being difficult to effective preparation in special construction microchannel on chip.Patent " a kind of micro fluid control detection device based on surface-enhanced Raman scattering activity substrate " (CN1O1792112A) prepares nano-pillar structure or nanofiber rising structure by the physical-chemical reaction of oxygen plasma Yu photoresist in silicon-based substrate, splash-proofing sputtering metal nano-particle layer again, obtain surface-enhanced Raman scattering activity substrate, it is bonded with PDMS microfluidic channel, prepares silicon-PDMS double-decker SERS micro fluid control detection device.Substrate prepared by this method has good SERS activity, but prepares the instrument that its needs are special, and preparation process is loaded down with trivial details.
Chemical assembly technology have available chemical reaction system select more, simple to operate flexibly, it is not necessary to expensive device, cost of manufacture is low, but preparation underlying structure controllability not as good as MEMS technology of preparing.Such as JuyoungLeem (Nanoscale, 2014,6,2895) et al. reporting in microfluidic channel, utilize the method that reduction of ethylene glycol method original position prepares nanometer Ag SERS substrate, the method is easy and simple to handle, but being limited by preparation process condition to be limit, the nano-particle of preparation is uniformly not good.Therefore, the SERS system that more convenient structure is more controlled prepare in the research and development of chemically based package technique, and the enhancing substrate of efficient integrated nanometer in micro-fluidic SERS chip is particularly significant and receive much concern.
Summary of the invention
Present invention aims to the difficulties of the surface enhanced active substrate preparation of current micro-fluidic SERS chip detection district and integrated chip thereof, it is provided that a kind of Combined type multi-channel SERS micro-fluidic chip strengthening substrate based on integrated nanometer on ITO electro-conductive glass and preparation method thereof.
The present invention proposes to adopt two step chrono-amperometric deposition process, and on ITO electro-conductive glass, integrated nanometer strengthens substrate, and builds the PDMS-ITO combined type SERS micro-fluidic chip containing array microchannel.The present invention makes full use of that ITO conductive glass surface is smooth, the characteristic of good conductivity, it can be used as the substrate surface of SERS micro structure, adopt two step chrono-amperometric deposition process, nanoparticle nucleated with the sedimentation potential in growth course and sedimentation time by controlling, control nano-particle pattern and size, can effectively prepare highly sensitive, good stability, equally distributed metal Nano structure SERS substrate.Further, it is bonded with the PDMS cover plate containing array microchannel, build and obtain combined type, high-throughout micro-fluidic SERS chip, this chip is with making the multiple SERS detection zones of formation on a piece of ito glass, it is that synchronous electric chemical deposition obtains that its nanometer strengthens substrate, its morphological consistency is good, and signal reproducibility is good.
The present invention realizes by the following technical solutions:
The SERS micro-fluidic chip strengthening substrate based on integrated nanometer on ITO electro-conductive glass that the present invention proposes, it is strengthened the ITO substrate of substrate by integrated SERS and is bonded with PDMS cover plate and to form.Designing a plurality of parallel microchannels on PDMS cover plate, microchannel end face is open, is being bonded rear enclosed with ITO substrate, and every passage two ends arrange the inlet with channel size matched and liquid outlet；Being integrated with SERS by synchronous electric chemical deposition on ITO substrate and strengthen substrate, on corresponding PDMS cover plate, the position of every microchannel at least forms a SERS detection zone.
The preparation method of the SERS micro-fluidic chip strengthening substrate based on integrated nanometer on ITO electro-conductive glass of the present invention comprises the following steps:
(1) preparation is with the PDMS cover plate of a plurality of parallel microchannels, inlet and liquid outlet；
(2) prepare integrated SERS and strengthen the ITO substrate of substrate:
Adopt two step chrono-amperometric deposition process, with ITO substrate for working electrode, directly prepare nano-metal particle in its surface electrochemistry deposition, obtain the metal nanoparticle diameter such as gold, silver or the copper amplified medium layer in 50～200nm scope, form SERS substrate；Two step chrono-amperometric depositions are to deposit 50s at 1.0v, deposit 2500s respectively at 0.2V.
A nanometer enhancing substrate beyond SERS detection zone is removed, only retains the enhancing substrate of SERS detection zone.
(3) by the cover plate with microchannel prepared and the ITO electro-conductive glass substrate bonding being integrated with SERS active-substrate, in metal nano SERS substrate close to PDMS microchannel, the micro-fluidic SERS chip of Combined type multi-channel will be obtained.
Beneficial effects of the present invention is as follows:
The present invention is bonded being integrated with a nanometer enhancing substrate I TO electro-conductive glass substrate with the PDMS cover plate with microchannel, prepares combined type SERS chip, has the advantage that
(1) present invention utilizes the electric conductivity of ito glass, adopt two step chrono-amperometric sedimentations, by controlling sedimentation potential and the sedimentation time of metal nanoparticle nucleation process and growth course, metal nanoparticle is made to cross fast nucleation under some position higher, when then relatively low again constant potential, the nucleus of formation is made to grow up gradually.Therefore, the metal nanoparticle of the SERS detection zone of preparation has good structural controllability and uniformity.
(2) present invention by optimizing metal metal nano-particle in sedimentation potential and the density of sedimentation time optimizing metal metal nano-particle of nucleation process, Optimal Growing current potential and growth time optimizing metal metal nano-particle particle diameter and spacing, it is thus achieved that metal nano micro structure on can obtain the high sensitivity SERS signal of biochemical sample.
(3) present invention design by parallel microchannels structure, with building a plurality of microchannel on a piece of ito glass.On same chip, the enhancing substrate of all SERS detection zones synchronizes deposition acquisition by an electrodeposition process, and the consistent appearance of metal nano micro structure is good, therefore, demonstrates, in biochemical sample analysis is tested, the repeatability that SERS signal is good.Meanwhile, the design of array microtube structure can realize SERS chip high throughput testing, promotes detection efficiency.
The combined type SERS chip preparing process of the present invention is relatively easy, and nanostructured is controlled, and its chip detection prepared is highly sensitive and signal is reproducible, cost is low, consuming time few, is designed by array microchannel, high flux test can be realized, be widely applied prospect in biochemistry detection field.
Accompanying drawing explanation
Fig. 1 combined type SERS chip structure schematic diagram
The detection zone schematic diagram of Fig. 2 ITO conductive glass surface integrated nanometer metal SERS micro structure.
The scanning electron microscope (SEM) photograph of the nanometer silver SERS micro structure of Fig. 3 embodiment 2 preparation.
Fig. 4 strengthens the preparation flow schematic diagram of the combined type SERS chip of substrate based on integrated nanometer on ITO electro-conductive glass.Wherein, a is the integrated nanometer SERS ITO electro-conductive glass substrate strengthening substrate；B is the PDMS cover plate containing array microchannel, inlet and liquid outlet；C is the combined type SERS chip prepared；
Wherein, 1 is SERS substrate integrated on ITO electro-conductive glass, and 2,3 is inlet and liquid outlet, and 4 is SERS microchannel.
Wherein, 1 is SERS substrate integrated on ITO electro-conductive glass, and 2,3 is inlet and liquid outlet, and 4 is SERS microchannel.
Fig. 5: the SERS spectra figure of the R6G of the variable concentrations detected for embodiment 4, do not having at the bottom of nano silver-group, it is impossible to the Raman signal of sample R6G detected.
Fig. 6: in embodiment 4, the SERS spectra figure of the R6G of the 10-8M that the SERS detection zone at different passages records.
Detailed description of the invention
Below in conjunction with accompanying drawing, the preferred embodiments of the present invention are described in detail.
The present invention is with ITO electro-conductive glass for substrate, the SERS set at micro-fluidic chip detects region, by the integrated SERS active-substrate preparing metal Nano structure of electrochemical deposition technique in-situ deposition, again with the polydimethylsiloxane (Polydimethylsiloxane containing microchannel, PDMS) cover plate bonding, prepares SERS micro-fluidic chip.
Embodiment 1
The present embodiment is the design of micro-fluidic SERS chip, specifically comprises the following steps that
(1) chip in the present embodiment is formed with the PDMS cover plate with a plurality of parallel channels by being integrated with the ITO substrate of metal nano SERS substrate.
SERS chip configuration designs: designing 8 straight channel in the present embodiment on the chip of 20 × 20mm, see Fig. 1, wherein 1 is SERS detection zone, and every passage sets 2 SERS detection zones, and detection zone length is 1mm, and its central point is respectively from exporting as 3mm and 5mm.2,3 is inlet and liquid outlet position, and 4 is microchannel, and the long 12mm in microchannel is wide 200 μm, high 150 μm.
Embodiment 2 the present embodiment is the preparation of PDMS cover plate, specifically comprises the following steps that the preparation of (1) SU-8 anode membrane: according to the chip configuration set, on silicon chip, and spin coating SU-8 photoresist, by UV-soft lithographic, prepares chip force plate.The long 12mm of straight channel that template upper channel is protruding is highly 150 μm, width is 200 μm.
(2) preparation of PDMS cover plate: in the present embodiment, selected patch material is PDMS, SU-8 photoetching anode membrane plate is put into a groove, polydimethylsiloxane (PDMS) elastomeric material that cast performed polymer prepares by 1:10 with solvent, it is placed in baking oven 90 DEG C of 1h that are heating and curing after vacuum outgas, the PDMS with micro-channel structure after solidification and formpiston are opened separation, must have 8 microchannel PDMS cover plates as shown in Figure 4 b.Coverslip thickness is about 1mm, the long 12mm in microchannel, be highly 150 μm, width be 200 μm.
Injection port 2 and liquid outlet 3 make: the inlet set on PDMS cover plate and the punching of liquid outlet position, then grafting silica gel catheter, prepare chip inlet and liquid outlet transfusion catheter, obtain the PDMS cover plate with microchannel and inlet and liquid outlet.
Embodiment 3:
The present embodiment is the ITO electro-conductive glass substrate prepared and be integrated with SERS active-substrate, comprises the following steps:
(1) cleaning of ITO electro-conductive glass: take the ITO electro-conductive glass (resistance is less than 10 Ω) of 20mm × 20mm successively with toluene, acetone, ethanol, ultra-pure water ultrasonic cleaning 15 minutes, in order to remove the greasy dirt on its surface.Dry, standby.
(2) in SERS detection zone electro-deposition integrated nanometer human lymph node substrate: preparing nanometer silver SERS substrate with electrochemical deposition technique in the conduction region of ITO conductive glass surface, its preparation process is shown in Fig. 2.
First, adopt VersaSTAT3 electrochemical workstation three-electrode system, with platinum electrode for electrode, saturated glycosides mercury electrode for auxiliary electrode, the ITO electro-conductive glass (10 Ω cm 2) of 20mm × 20mm) for working electrode.With the KNO containing 0.3mol/L3It is electrolyte with the mixed solution of the silver-colored ammonia of 0.8mmol/L, under room temperature (25 ± 2 DEG C), adopts two step chrono-amperometric deposition techniques, at the integrated AgNP of ITO conductive glass surface.In order to obtain the SERS micro-fluidic chip of high sensitivity activity, experiment, with R6G for probe molecule, optimizes substrate preparation condition.Under-1.0v condition, deposit 50s, make Ag+ in ITO conductive glass surface nucleation, then make nano-particle grow up under-0.2V current potential again.During the deposition 2000s under-0.2V condition～3000s, probe molecule R6G is had preferably reinforced effects by substrate, and when sedimentation time is 2500s, its reinforced effects is best.By substrate deionized water rinsing, nitrogen dries up, and obtains high density, equally distributed nanometer silver SERS substrate, and its pattern field emission scanning electron microscope characterizes.See Fig. 3.The nano-Ag particles of preparation is evenly distributed, and mean diameter is about 100nm.
Then, take the adhesive tape of 20mm × 20mm, by the method for laser ablation, the adhesive tape of SERS detection zone on adhesive tape is removed, then it have with deposition nanometer ito glass strengthening substrate align, paste (Fig. 2 c), after throwing off adhesive tape, the nanometer silver beyond SERS detection zone is stuck out, and obtains being integrated with the ITO substrate (see Fig. 2 d) at the bottom of high density nano silver-group at SERS detection zone.
Embodiment 4
The micro-fluidic chip of preparation in the present embodiment, including being integrated with nanometer silver SERS active-substrate ITO substrate and the PDMS cover plate comprising microchannel, injection port and liquid outlet, both using plasma lithographic techniques, hydroxylating is bonded.ITO electro-conductive glass substrate (Fig. 4 b) being integrated with SERS active-substrate of preparation in cover plate module (Fig. 4 a) with microchannel prepared in embodiment 1 and embodiment 2 is put in etching machine, oxygen plasma is utilized to carry out hydroxylating etching, then align them that and fit together, press, place 12 hours, obtain having completely airtight micro-fluidic SERS chip of the PDMS for passage (Fig. 4 c).
Embodiment 5
Micro-fluidic SERS chip integrated in embodiment 3 is carried out SERS performance test.Utilize flow injection pump that the R6G sample of variable concentrations is passed into micro-fluidic chip detection zone.The FT-Raman and confocal Raman instrument equipped with the He-Ne laser instrument (17mW) of 632.8nm is adopted to carry out SERS test.1800 gratings are selected during test, 10% neutral filter, adopt 50XLWD microcobjective by laser beam focusing in micro-fluidic chip, spot diameter 1 μm, it is 2s in the time of integration, when cumulative frequency is 2 times, the SERS signal (Fig. 5) of the R6G of test variable concentrations.The minimal detectable concentration of R6G is 10 by this micro-fluidic chip‐10M, by 10‐8The R6G of M is measured at the SERS detection zone of 8 microchannels respectively, spectrogram (Fig. 6), uses 1510cm‐1The peak at place calculates by force, and its RSD is 6.9%,.
These results suggest that high flux SERS micro-fluidic chip prepared by the present invention has significantly high sensitivity and repeatability.
Preferred embodiment above is only in order to illustrate technical scheme and unrestricted, although the present invention being described in detail by above preferred embodiment, but skilled artisan would appreciate that, in the form and details it can be made various change, without departing from claims of the present invention limited range.
Claims (10)
1. strengthen the SERS micro-fluidic chip of substrate based on integrated nanometer on ITO electro-conductive glass, it is bonded by the ITO substrate of PDMS cover plate with integrated SERS enhancing substrate and forms；It is characterized in that: having a plurality of parallel microchannels on PDMS cover plate, microchannel end face is open, be bonded rear enclosed with ITO substrate, every passage two ends arrange the inlet with channel size matched and liquid outlet；Being integrated with SERS by synchronous electric chemical deposition on ITO substrate and strengthen substrate, on corresponding PDMS cover plate, the position of every microchannel at least forms a SERS detection zone.
2. the SERS micro-fluidic chip strengthening substrate based on integrated nanometer on ITO electro-conductive glass according to claim 1, it is characterised in that described SERS detection zone has the diameter nano-metal particle in 50～200nm scope by synchronous electric chemical deposition.
3. the SERS micro-fluidic chip strengthening substrate based on integrated nanometer on ITO electro-conductive glass according to claim 1, it is characterised in that described microchannel width is 50 μm～300 μm, and the degree of depth is 5～200 μm.
4. the SERS micro-fluidic chip strengthening substrate based on integrated nanometer on ITO electro-conductive glass according to claim 1, it is characterised in that the SERS detection zone of corresponding same passage design is parallel to each other, and detects head of district 1mm～10mm.
5. the preparation method of the SERS micro-fluidic chip described in claim 1-4, it is characterised in that comprise the following steps:
(1) preparation is with the PDMS cover plate of a plurality of parallel microchannels, inlet and liquid outlet；
(2) prepare integrated SERS and strengthen the ITO substrate of substrate:
(2.1) two step chrono-amperometric deposition process are adopted, with ITO substrate for working electrode, directly prepare nano-metal particle in its surface electrochemistry deposition, obtain the metal nanoparticle diameter such as gold, silver or the copper amplified medium layer in 50～200nm scope, form SERS substrate；
(2.2) nanometer enhancing substrate beyond SERS detection zone is removed, only retain the enhancing substrate of SERS detection zone；
(3) by the cover plate with microchannel prepared and the ITO electro-conductive glass substrate bonding being integrated with SERS active-substrate, in metal nano SERS substrate close to PDMS microchannel, the micro-fluidic SERS chip of Combined type multi-channel will be obtained.
6. the preparation method of the SERS micro-fluidic chip strengthening substrate based on integrated nanometer on ITO electro-conductive glass according to claim 5, it is characterized in that, two step chrono-amperometric depositions are to deposit 40～60s at 10.8～1.2v, deposit 2000s～3000s respectively at 0.2V.
7. the preparation method of the SERS micro-fluidic chip strengthening substrate based on integrated nanometer on ITO electro-conductive glass according to claim 5, it is characterised in that two step chrono-amperometric depositions are to deposit 50s at 1.0v, deposit 2500s respectively at 0.2V.
8. the preparation method of the SERS micro-fluidic chip strengthening substrate based on integrated nanometer on ITO electro-conductive glass according to claim 5, it is characterized in that, adopt three-electrode system, with platinum electrode for electrode, saturated glycosides mercury electrode is auxiliary electrode, ITO electro-conductive glass (the 10 Ω cm of 20mm × 20mm‐2)) for working electrode, with the KNO containing 0.3mol/L3It is electrolyte with the mixed solution of the silver-colored ammonia of 0.8mmol/L.
9. the preparation method of the SERS micro-fluidic chip strengthening substrate based on integrated nanometer on ITO electro-conductive glass according to claim 5, it is characterised in that microchannel width is 50 μm～300 μm, and the degree of depth is 5～200 μm.
10. the preparation method of the SERS micro-fluidic chip strengthening substrate based on integrated nanometer on ITO electro-conductive glass according to claim 5, it is characterised in that the SERS detection zone of corresponding same passage design is parallel to each other, and detection zone length is 1mm～10mm.
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