CN105772118B - The SERS micro-fluidic chips and preparation method of integrated enhancing substrate on ITO electro-conductive glass - Google Patents
The SERS micro-fluidic chips and preparation method of integrated enhancing substrate on ITO electro-conductive glass Download PDFInfo
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- 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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- 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
A kind of Combined type multi-channel SERS micro-fluidic chips for strengthening substrate based on integrated nanometer on ITO electro-conductive glass and preparation method thereof.The present invention makes full use of that ITO conductive glass surfaces are smooth, good conductivity characteristic, as the substrate surface of SERS micro-structurals, using two step chrono-amperometric deposition process, by controlling nanoparticle nucleated sedimentation potential and sedimentation time with growth course, nano particle pattern and size are controlled, can effectively prepare that sensitivity is high, stability is good, equally distributed metal Nano structure SERS substrates.Further, it is bonded with the PDMS cover plates of the microchannel containing array, build and obtain combined type, high-throughout micro-fluidic SERS chips, the chip is forming multiple SERS detection zones with making on a piece of ito glass, its nanometer of enhancing substrate is that synchronous electrochemical deposition is obtained, its morphological consistency is good, and signal reproducibility is good.
Description
Technical field
The present invention relates to SERS analysis (Surface Enhanced Raman Spectroscopy,
) and microfluidic chip analysis technical field SERS.
Background technology
Microfluidic chip analysis methods and techniques have that amount of samples is small with it, structure design flexibly, can realize in-situ test,
The advantages of being easily achieved miniaturization and be integrated, receives much concern in biochemical sample is analyzed and is detected.At present, in micro-fluidic chip
Conventional detection method mainly has Electrochemical Detection, fluoroscopic examination and chemiluminescence detection etc., and these methods have higher inspection
Sensitivity is surveyed, but it is few to there is the information content of sample responses, it is difficult to acquire the deficiencies such as biological sample detailed structural information.Raman light
Spectrum detection is as a kind of lossless spectrum detection technique, and detection speed is fast, can provide compound group and chemical bond from molecular level
Information, informative, and its response signal is not by moisture interference in sample system, while directly laser facula can be focused on
On sample in the minim channel of micro-fluidic chip, show that with biochemical sample detection on micro-fluidic chip there is good
With property and compatibility.But Raman spectrum response has a spectral response signal especially weak, the problem of detection sensitivity is low.Therefore, table
The introducing of face enhancing Raman scattering (SERS) technology, which turns into, realizes the efficient analysis of biochemical sample and detection on micro-fluidic chip
It is preferred.
At present, mainly have based on micro photo-electro-mechanical technology in the method for the integrated SERS substrates of micro-fluidic SERS chips detection zone
MEMS prepares method and Chemical assembly method etc..
MEMS, which prepares method, has controllability good, the uniform sequential advantage of the SERS substrates of preparation, but it needs special installation,
Prepare cost higher.Such as patent " a kind of micro-fluidic chip with surface reinforced Raman active and preparation method thereof " (CN
103604797 A) disclose a kind of by vacuum vapour deposition, the microarray surface evaporation in PDMS micro-fluidic chip passages is double
The method of layer metal film, building has SERS substrate micro-fluidic chips, and this method needs special instrument, and preparation cost is higher,
To being difficult to effectively preparation in special construction microchannel on chip." one kind is based on surface-enhanced Raman scattering activity base to patent
The micro fluid control detection device at bottom " (CN1O1792112A) is served as a contrast by the physical-chemical reaction of oxygen plasma and photoresist in silicon substrate
Nanometer rod structure or nanofiber rising structure, then splash-proofing sputtering metal nano-particle layer are prepared on bottom, surface-enhanced Raman is obtained and dissipates
Active substrate is penetrated, is bonded with PDMS microfluidic channels, silicon-PDMS double-decker SERS micro fluid control detection devices are prepared.This side
Substrate prepared by method has preferable SERS activity, but prepares the special instrument of its needs, and preparation process is cumbersome.
Chemical assembly technology has selects more using chemical reaction system, simple to operate flexible, without expensive device,
Low manufacture cost, but the underlying structure controllability prepared is not as good as MEMS technologies of preparing.Such as Juyoung Leem (Nanoscale,
2014,6,2895) et al. report in microfluidic channel, the reduction of ethylene glycol method original position of utilization prepares nanometer Ag SERS substrates
Method, this method is easy to operate, but is limited by preparation process condition and limited, and the nano particle of preparation is not uniform good.Therefore, base
Prepare more convenient structure more controllable SERS systems in Chemical assembly technical research, in micro-fluidic SERS chips efficiently
Integrated nanometer enhancing substrate is particularly significant and receives much concern.
The content of the invention
It is an object of the invention to being prepared for micro-fluidic SERS chips detection zone surface enhanced active substrate at present and its
The difficulties of integrated chip strengthen the Combined type multi-channel of substrate based on integrated nanometer on ITO electro-conductive glass there is provided a kind of
SERS micro-fluidic chips and preparation method thereof.
The present invention proposes that using two step chrono-amperometric deposition process integrated nanometer strengthens substrate on ITO electro-conductive glass, and
Build the PDMS-ITO combined type SERS micro-fluidic chips of the microchannel containing array.The present invention makes full use of ITO electro-conductive glass tables
Face is smooth, good conductivity characteristic, as the substrate surface of SERS micro-structurals, using two step chrono-amperometric deposition process,
By controlling nanoparticle nucleated sedimentation potential and sedimentation time with growth course, control nano particle pattern and size,
It can effectively prepare that sensitivity is high, stability is good, equally distributed metal Nano structure SERS substrates.Further, by its with containing battle array
The PDMS cover plates bonding of column microchannel, builds and obtains combined type, high-throughout micro-fluidic SERS chips, the chip is with a piece of
Made on ito glass and form multiple SERS detection zones, its nanometer of enhancing substrate is that synchronous electrochemical deposition is obtained, its consistent appearance
Property is good, and signal reproducibility is good.
The present invention is realized using following technical scheme：
Proposed by the present invention to strengthen the SERS micro-fluidic chips of substrate based on integrated nanometer on ITO electro-conductive glass, it is by collecting
It is bonded and forms with PDMS cover plates into the SERS ITO substrates for strengthening substrate.A plurality of parallel microchannels are designed on PDMS cover plates, it is micro- logical
Road top surface is open, rear enclosed is being bonded with ITO substrates, every passage two ends set the inlet with channel size matched with going out liquid
Mouthful；Being integrated with SERS by synchronous electrochemical deposition on ITO substrates strengthens substrate, every microchannel on correspondence PDMS cover plates
Position at least form a SERS detection zone.
Preparation side of the present invention based on the SERS micro-fluidic chips of integrated nanometer enhancing substrate on ITO electro-conductive glass
Method comprises the following steps：
(1) the PDMS cover plates with a plurality of parallel microchannels, inlet and liquid outlet are prepared；
(2) preparing integrated SERS strengthens the ITO substrates of substrate：
Using two step chrono-amperometric deposition process, using ITO substrates as working electrode, directly deposit and make in its surface electrochemistry
Standby nano-metal particle, obtains amplified medium layer of the metal nanoparticle such as the gold, silver or copper diameter in 50~200nm scopes, shape
Into SERS substrates；Two step chrono-amperometrics deposition is to deposit 50s in -1.0v, and 2500s is deposited respectively in -0.2V.
Nanometer enhancing substrate beyond SERS detection zones is removed, only retains the enhancing substrate of SERS detection zones.
(3) by the cover plate prepared with microchannel and the ITO electro-conductive glass substrate keys for being integrated with SERS active-substrate
Close, the micro-fluidic SERS chips of Combined type multi-channel in metal nano SERS substrate closes to PDMS microchannels, will be obtained.
Beneficial effects of the present invention are as follows：
The present invention will be integrated with nanometer enhancing substrate I TO electro-conductive glass substrate and is bonded with the PDMS cover plates with microchannel, make
Standby combined type SERS chips, with advantages below：
(1) present invention utilizes the electric conductivity of ito glass, using two step chrono-amperometric sedimentations, by controlling metal nano
The sedimentation potential and sedimentation time of particulate nucleation process and growth course, make metal nanoparticle quick under the higher position of point excessively
Nucleation, then makes the nucleus to be formed gradually grow up under the conditions of relatively low constant potential again.Therefore, the gold of the SERS detection zones of preparation
Metal nano-particle has good structural controllability and uniformity.
(2) present invention is received by optimizing metal nanoparticle in the sedimentation potential of nucleation process with sedimentation time optimization metal
Density, Optimal Growing current potential and the growth time of rice grain optimize metal nanoparticle particle diameter and spacing, the metal nano of acquisition
The high sensitivity SERS signal of biochemical sample can be obtained in micro-structural.
(3) present invention builds a plurality of microchannel by the design of parallel microchannels structure on a piece of ito glass.Together
By an electrodeposition process, synchronously deposition is obtained the enhancing substrate of all SERS detection zones on one chip, metal nano micro-structural
Consistent appearance it is good, therefore, biochemical sample analysis test in show the good reappearance of SERS signal.Meanwhile, array
The detection of SERS chips high flux can be achieved in the design of microtube structure, lifts detection efficiency.
The combined type SERS chip preparing process of the present invention is relatively easy, and nanostructured is controllable, and its chip prepared is examined
Survey sensitivity high reproducible with signal, cost is low, takes less, is designed by array microchannel, high flux test can be achieved,
There is wide application prospect in biochemistry detection field.
Brief description of the drawings
Fig. 1 combined type SERS chip structure schematic diagrames
The detection zone schematic diagram of Fig. 2 ITO conductive glass surface integrated nanometer metal SERS micro-structurals.
The scanning electron microscope (SEM) photograph of Nano Silver SERS micro-structurals prepared by Fig. 3 embodiments 2.
Preparation flow schematic diagrames of the Fig. 4 based on the combined type SERS chips of integrated nanometer enhancing substrate on ITO electro-conductive glass.
Wherein, a is that integrated nanometer SERS strengthens the ITO electro-conductive glass substrates of substrate；B is microchannel containing array, inlet and goes out liquid
The PDMS cover plates of mouth；C is the combined type SERS chips prepared；
Wherein, 1 is SERS substrates integrated on ITO electro-conductive glass, and 2,3 be inlet and liquid outlet, and 4 be SERS micro- logical
Road.
Wherein, 1 is SERS substrates integrated on ITO electro-conductive glass, and 2,3 be inlet and liquid outlet, and 4 be SERS micro- logical
Road.
Fig. 5：The R6G of the various concentrations detected for embodiment 4 SERS spectra figure, at no nano silver-group bottom, it is impossible to
Detect sample R6G Raman signal.
Fig. 6：For in embodiment 4, the 10-8M measured in the SERS detection zones of different passages R6G SERS spectra figure.
Embodiment
Below in conjunction with accompanying drawing, the preferred embodiments of the present invention are described in detail.
The present invention is the SERS detection zones set in micro-fluidic chip using ITO electro-conductive glass as substrate, passes through electrochemistry
The integrated SERS active-substrate for preparing metal Nano structure of deposition technique in-situ deposition, then with the polydimethylsiloxanes containing microchannel
Alkane (Polydimethylsiloxane, PDMS) cover plate is bonded, and prepares SERS micro-fluidic chips.
Embodiment 1
The present embodiment is the design of micro-fluidic SERS chips, is comprised the following steps that：
(1) chip in the present embodiment is by being integrated with the ITO substrates of metal nano SERS substrates and with a plurality of parallel channels
PDMS cover plates composition.
SERS chip configurations are designed：8 straight channels are designed in the present embodiment on 20 × 20mm chip, Fig. 1 is seen, wherein
1 is that 2 SERS detection zones are set on SERS detection zones, every passage, and detection section length is 1mm, and its central point is respectively away from outlet
For 3mm and 5mm.2nd, 3 be inlet and liquid outlet position, and 4 be microchannel, and the long 12mm in microchannel is wide 200 μm, high 150 μm.
The present embodiment of embodiment 2 is the preparation of PDMS cover plates, is comprised the following steps that：(1) preparation of SU-8 anode membranes：According to setting
Fixed chip configuration, on silicon chip, spin coating SU-8 photoresists, by UV- soft lithographics, prepare chip force plate.Template upper channel
The raised long 12mm of straight channel is highly 150 μm, width is 200 μm.
(2) preparation of PDMS cover plates：Selected patch material is PDMS in the present embodiment, and SU-8 photoetching anode membrane plates are put into one
In individual groove, cast performed polymer presses 1 with solvent:10 dimethyl silicone polymer prepared (PDMS) elastomeric materials, vacuum takes off
It is placed into 90 DEG C of 1h that are heating and curing in baking oven after gas, PDMS and formpiston with micro-channel structure after solidification is opened into separation,
As shown in Figure 4 b there are 8 microchannel PDMS cover plates.Coverslip thickness about 1mm, the long 12mm in microchannel are highly 150 μm, wide
Spend for 200 μm.
Injection port 2 and liquid outlet 3 make：The inlet set on PDMS cover plates is punched with liquid outlet position, then grafting
Silica gel catheter, prepares chip inlet and liquid outlet transfusion catheter, obtains the PDMS lids with microchannel and inlet and liquid outlet
Piece.
Embodiment 3：
The present embodiment is to prepare the ITO electro-conductive glass substrates for being integrated with SERS active-substrate, is comprised the following steps：
(1) cleaning of ITO electro-conductive glass：20mm × 20mm ITO electro-conductive glass (resistance is less than 10 Ω) is taken to use first successively
Benzene, acetone, ethanol, ultra-pure water are cleaned by ultrasonic 15 minutes, to remove the greasy dirt on its surface.Drying, it is standby.
(2) in SERS detection zone electro-deposition integrated nanometer human lymph node substrates：With electrochemical deposition technique in ITO electro-conductive glass
The conduction region on surface prepares Nano Silver SERS substrates, and its preparation process is shown in Fig. 2.
First, using VersaSTAT3 electrochemical workstation three-electrode systems, using platinum electrode as to electrode, saturation glycosides mercury electricity
Extremely auxiliary electrode, 20mm × 20mm ITO electro-conductive glass (10 Ω cm-2)) it is working electrode.With the KNO containing 0.3mol/L3
Mixed solution with 0.8mmol/L silver-colored ammonia is electrolyte, under room temperature (25 ± 2 DEG C), and skill is deposited using two step chrono-amperometrics
Art, in the integrated Ag NP of ITO conductive glass surfaces.In order to obtain the SERS micro-fluidic chips of high sensitivity activity, test with R6G
For probe molecule, optimize substrate preparation condition.50s is deposited under the conditions of -1.0v, makes Ag+ in ITO conductive glass surface nucleation,
Then nano particle is made to grow up under -0.2V current potentials again.In deposition 2000s~3000s under the conditions of -0.2V, substrate is to probe
Molecule R6G has preferably enhancing effect, and when sedimentation time is 2500s, its enhancing effect is best.Substrate is rushed with deionized water
Wash, nitrogen drying obtains high density, equally distributed Nano Silver SERS substrates, its pattern is characterized with field emission scanning electron microscope.See
Fig. 3.The nano-Ag particles of preparation are evenly distributed, average grain diameter about 100nm.
Then, 20mm × 20mm adhesive tape is taken, with the method for laser ablation by the adhesive tape of SERS detection zones on adhesive tape
Remove, the ito glass that itself and deposition then are had into nanometer strengthens substrate aligns, and pastes (Fig. 2 c), throws off after adhesive tape, SERS detections
Nano Silver beyond area is stuck out, and obtains being integrated with the ITO substrates (see Fig. 2 d) at high density nano silver-group bottom in SERS detection zones.
Embodiment 4
The micro-fluidic chip prepared in the present embodiment, including be integrated with Nano Silver SERS active-substrate ITO substrates and comprising
The PDMS cover plates of microchannel, injection port and liquid outlet, both using plasma lithographic techniques, hydroxylating bonding.By embodiment 1
In the cover plate module (Fig. 4 a) with microchannel for preparing and the ITO for being integrated with SERS active-substrate prepared in embodiment 2
Electro-conductive glass substrate (Fig. 4 b) is put into etching machine, is carried out hydroxylating etching using oxygen plasma, then be align them that patch
It is combined, presses, place 12 hours, obtains that there is the micro-fluidic SERS chips (Fig. 4 c) of the complete closed PDMS for passage.
Embodiment 5
SERS performance tests are carried out to micro-fluidic SERS chips integrated in embodiment 3.Using flow injection pump by difference
The R6G samples of concentration are passed through micro-fluidic chip detection zone.Using the laser of the He-Ne lasers (17mW) equipped with 632.8nm
Confocal laser-scanning microscopy instrument carries out SERS tests.1800 gratings are selected during test, 10% neutral filter is aobvious using 50X LWD
Speck mirror is by laser beam focusing in micro-fluidic chip, and 1 μm of spot diameter is 2s in the time of integration, and cumulative frequency is 2 times
Under the conditions of, test the R6G of various concentrations SERS signal (Fig. 5).It is to R6G minimal detectable concentration on the micro-fluidic chip
10‐10M, by 10‐8SERS detection zones of the M R6G respectively 8 microchannels is measured, and spectrogram (Fig. 6) uses 1510cm‐1Place
Peak intensity is calculated, and its RSD is 6.9%,.
It these results suggest that the high flux SERS micro-fluidic chips of the invention prepared with very high sensitivity and reappearance.
Preferred embodiment above is merely illustrative of the technical solution of the present invention and unrestricted, although being preferable to carry out by above-mentioned
The present invention is described in detail for example, it is to be understood by those skilled in the art that can be in the form and details
Various changes are made to it, without departing from claims of the present invention limited range.
Claims (9)
1. strengthening the SERS micro-fluidic chips of substrate based on integrated nanometer on ITO electro-conductive glass, it strengthens substrate by integrated SERS
ITO substrates be bonded and form with PDMS cover plates；It is characterized in that：There are a plurality of parallel microchannels, microchannel top on PDMS cover plates
Face is open, rear enclosed is being bonded with ITO substrates, every passage two ends set the inlet and liquid outlet with channel size matched；
Being integrated with SERS by synchronous electrochemical deposition on ITO substrates strengthens substrate, every microchannel on correspondence PDMS cover plates
Position at least forms a SERS detection zone；
The SERS detection zones have nano-metal particle of the diameter in 50~200nm scopes by synchronous electrochemical deposition.
2. according to claim 1 strengthen the SERS micro-fluidic chips of substrate based on integrated nanometer on ITO electro-conductive glass, its
It is characterised by, the microchannel width is 50 μm ~ 300 μm, depth is 5 ~ 200 μm.
3. according to claim 1 strengthen the SERS micro-fluidic chips of substrate based on integrated nanometer on ITO electro-conductive glass, its
It is characterised by, the SERS detection zones of the same passage design of correspondence are parallel to each other, detection head of district 1mm ~ 10mm.
4. the preparation method of the SERS micro-fluidic chips described in claim 1-3, it is characterised in that comprise the following steps：
（1）Prepare the PDMS cover plates with a plurality of parallel microchannels, inlet and liquid outlet；
（2）Preparing integrated SERS strengthens the ITO substrates of substrate：
（2.1) two step chrono-amperometric deposition process are used, using ITO substrates as working electrode, are directly deposited in its surface electrochemistry
Nano-metal particle is prepared, the amplified medium layer of gold, silver or copper metal diameter of nano particles in 50~200nm scopes, shape is obtained
Into SERS substrates；
（2.2）Nanometer enhancing substrate beyond SERS detection zones is removed, only retains the enhancing substrate of SERS detection zones；
（3）The cover plate with microchannel prepared is bonded with the ITO electro-conductive glass substrate for being integrated with SERS active-substrate, will
Metal nano SERS substrate closes obtain the micro-fluidic SERS chips of Combined type multi-channel in PDMS microchannels.
5. it is according to claim 4 based on the SERS micro-fluidic chips of integrated nanometer enhancing substrate on ITO electro-conductive glass
Preparation method, it is characterised in that two step chrono-amperometrics deposition is to deposit 40 ~ 60s in -10.8 ~ 1.2v, is deposited respectively in -0.2V
2000s~3000s。
6. it is according to claim 4 based on the SERS micro-fluidic chips of integrated nanometer enhancing substrate on ITO electro-conductive glass
Preparation method, it is characterised in that two step chrono-amperometrics deposition is to deposit 50s in -1.0v, and 2500s is deposited respectively in -0.2V.
7. it is according to claim 4 based on the SERS micro-fluidic chips of integrated nanometer enhancing substrate on ITO electro-conductive glass
Preparation method, it is characterised in that use three-electrode system, using platinum electrode as to electrode, saturation glycosides mercury electrode is auxiliary electrode,
20mm × 20mm ITO electro-conductive glass is working electrode, with the KNO containing 0.3mol/L3With the mixing of 0.8mmol/L silver-colored ammonia
Solution is electrolyte.
8. it is according to claim 4 based on the SERS micro-fluidic chips of integrated nanometer enhancing substrate on ITO electro-conductive glass
Preparation method, it is characterised in that microchannel width is 50 μm ~ 300 μm, depth is 5 ~ 200 μm.
9. it is according to claim 4 based on the SERS micro-fluidic chips of integrated nanometer enhancing substrate on ITO electro-conductive glass
Preparation method, it is characterised in that the SERS detection zones of correspondence same passage design are parallel to each other, detection section length for 1mm ~
10mm。
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