CN106732842A - For micro-fluidic chip without mark high content screening and preparation method thereof - Google Patents
For micro-fluidic chip without mark high content screening and preparation method thereof Download PDFInfo
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- CN106732842A CN106732842A CN201710077109.9A CN201710077109A CN106732842A CN 106732842 A CN106732842 A CN 106732842A CN 201710077109 A CN201710077109 A CN 201710077109A CN 106732842 A CN106732842 A CN 106732842A
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- 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
- B01L3/502761—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 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
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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
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0809—Geometry, shape and general structure rectangular shaped
- B01L2300/0829—Multi-well plates; Microtitration plates
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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
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
- B01L2300/0864—Configuration of multiple channels and/or chambers in a single devices comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
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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
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0887—Laminated structure
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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
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0893—Geometry, shape and general structure having a very large number of wells, microfabricated wells
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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
- B01L2400/00—Moving or stopping fluids
- B01L2400/04—Moving fluids with specific forces or mechanical means
- B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
- B01L2400/0415—Moving fluids with specific forces or mechanical means specific forces electrical forces, e.g. electrokinetic
Abstract
The invention belongs to the technical field of micro-fluidic chip, and in particular to a kind of for micro-fluidic chip without mark high content screening and preparation method thereof；The technical problem of solution is：There is provided one kind can be captured to cell, be positioned, and can carry out intension high, micro-fluidic chip of unmarked drug screening and preparation method thereof；The technical scheme for using for：For the micro-fluidic chip without mark high content screening, the micro-fluidic chip is provided with detection cell and a plurality of fluid channel, the a plurality of fluid channel is connected with the detection cell, and be not attached to lead to each other between a plurality of fluid channel, multiple construction units are provided with the detection cell, the multiple construction unit constitutes array type structure, and the construction unit includes：At least two keeper electrodes for being used to position cell and at least one pair of growth electrode for growing nanodendrites structure.
Description
Technical field
The invention belongs to the technical field of micro-fluidic chip, and in particular to a kind of for without mark high content screening
Micro-fluidic chip and preparation method thereof.
Background technology
High content screening be mainly keep eucaryotic cell structure and it is fully functional on the premise of, while detection be screened sample pair
The influence of the aspects such as cellular morphology, cell growth, signal path and cytotoxicity, and it is subject to comprehensive analysis.In traditional height
It is usually that cell culture is carried out on microwell plate to contain screening, applies medicine irritation, and be aided with automation equipment and high-resolution optics
Test system is detected.
Conventional optical detection is based on fluorescence microscope, and it can realize high-resolution with reference to laser confocal scanning technology
Unicellular cells device is imaged.But the technology needs to use fluorescent dye to subcellular fraction unit（Nucleus, mitochondria）Enter rower
Note, and the bio-toxicity of fluorescent dye has a strong impact on the long-time observation and research of cell, additionally, the fluorescence of non-specific binding
Dye molecule can introduce background noise, reduce detection sensitivity.Different from fluorescent method, SERS technologies are without dye molecule mark
Know, multiple molecule functional groups can be detected simultaneously, to cell, the structural information of medicine and medicine in metabolic process
The change that molecular structure occurs, carries out the research of unimolecule magnitude.Additionally, micro-fluidic chip is relative to microwell plate, with life
Thing reagent and cell consumption are few, Large-scale Screening efficiency high the characteristics of, it is significant for reducing drug development cost.Cause
This, it is to carry out the effective way without mark high content screening that SERS technologies are combined with microflow control technique.
Cui Yiping（62,2014,BIOSENSORS & BIOELECTRONICS）Using drop micro-fluidic chip, obtain
The SERS of rhodanate in human serum and saliva.Optical signal is collected as ensemble average value, and experiment can
To obtain quantitative repeatability well.But, this method to cell due to can not well be captured, be positioned and training
Support, still face the problem of extracellular matrix environment change, the cell model in drop lacks mutual between cell and matrix
Effect, this may block cell-signaling pathways, suppress differential protein and related gene expression.
The content of the invention
The present invention overcomes the shortcomings of that prior art is present, and technical problem to be solved is：There is provided one kind can be to cell
Captured, positioned, enough carried out label-free, high content screening micro-fluidic chip and preparation method thereof.
In order to solve the above-mentioned technical problem, the technical solution adopted by the present invention is：For without mark high content screening
Micro-fluidic chip, the micro-fluidic chip is provided with detection cell and a plurality of fluid channel, a plurality of fluid channel with the inspection
Survey pond to be connected, and be not attached to lead to each other between a plurality of fluid channel, multiple construction units are provided with the detection cell, it is the multiple
Construction unit constitutes array type structure, and the construction unit includes：At least two keeper electrodes and at least for being used to position cell
It is used to grow the growth electrode of nanodendrites structure for a pair.
Preferably, the construction unit is three toroidals, and three annulus are the different donut of diameter, including
Annulus is provided with least two tips electrode；At least two circular electrodes are provided with middle annulus, with above-mentioned circular electrode as etc.
Branch, middle annulus is divided into many equal portions, and point is equipped with each circular electrode；Multiple tip electricity are provided with outer toroid
Pole, the point electrode in outer toroid is corresponded with the circular electrode on middle annulus；Point electrode in the interior annular is two-by-two
Between form a pair of growth electrodes, the point of point electrode in the outer toroid and the circular electrode on corresponding middle annulus
A pair of growth electrodes are formed, the non-point of the circular electrode is keeper electrode.
Preferably, the construction unit is double circular ring structures, and two annulus are the different donut of diameter, including
Annulus is provided with least two tips electrode, and at least two circular electrodes are provided with outer toroid, with above-mentioned circular electrode as etc.
Branch, outer toroid is divided into many equal portions, and the point electrode in the interior annular forms a pair of growth electrodes between any two, described
Circular electrode is keeper electrode.
Preferably, the construction unit is double circular ring structures, and two annulus are the different donut of diameter, including
Annulus is provided with least two circular electrodes, and with above-mentioned circular electrode as Along ent, outer toroid is divided into many equal portions, Mei Yiyuan
Point is equipped with shape electrode, multiple point electrodes are provided with outer toroid, on the point electrode in outer toroid and interior annular
Circular electrode correspond；The point shape of point electrode in the outer toroid and the circular electrode in corresponding interior annular
Electrode is grown in a pair, and the non-point of the circular electrode is keeper electrode.
Preferably, the micro-fluidic chip includes：Substrate, substrate and the transparent cover being bonded together successively, the knot
Structure unit is arranged in the substrate, and the detection cell and a plurality of fluid channel may be contained within the substrate, and the detection cell
Top cover be the transparent cover, the transparent cover is provided with multiple solution gateways, the multiple solution gateway and institute
State a plurality of fluid channel and correspond connection.
Preferably, described a plurality of fluid channel includes：Cell liquid fluid channel, culture medium fluid channel, medicine fluid channel, plating solution
Fluid channel, deionized water fluid channel and waste liquid fluid channel, are equipped with one-way conduction valve in above-mentioned fluid channel.
Preferably, comprise the following steps：S10, using MEMS technology, being made in substrate includes that keeper electrode and growth are electric
The array architecture unit of pole, makes detection cell and a plurality of fluid channel on substrate, multiple solution is made in transparent cover and is gone out
Entrance, substrate, substrate and transparent cover are bonded together successively；S20, by the electrolyte containing noble metal pass through transparent cover
On a solution gateway, flow through fluid channel, be passed through in detection cell；S30, the indirect current between each two growth electrode,
To grow the nanodendrites structure that growth on the nearest tip of electrode distance forms noble metal at two；S40, noble metal nano branch
After crystal structure is formed, to deionized water is passed through in detection cell, to rinse detection cell and fluid channel.
Preferably, in step S10, described utilization MEMS technology, being made in substrate includes keeper electrode and growth electricity
The array architecture unit of pole, specifically includes：S101, in substrate deposit first layer metal layer and etch, formed growth electrode
Electrical cable；S102, the first layer insulating of deposition are simultaneously etched, and expose electrode window through ray；S103, deposition second layer metal layer are simultaneously
Etching, forms the electrical cable of keeper electrode；S104, the second layer insulating of deposition are simultaneously etched, and expose electrode window through ray；S105、
Deposition third layer metal level is simultaneously etched, and forms growth electrode and keeper electrode.
Preferably, after completing a drug screening, between the growth electrode that each two growth has nanodendrites structure, apply
The alternating current of amplitude is increased, fuse nanodendrites structure, is passed through deionized water rinsing.
Preferably, the electrolyte containing noble metal is solion or complex solution, between each opposite long electrode
The amplitude of the alternating current of applying is more than 3V, and frequency values are located between 1kHz and 10MHz.
The present invention has the advantages that compared with prior art：
1st, the multiple construction units set in the detection cell on the micro-fluidic chip in the present invention, in array type structure, Ke Yishi
Existing high-throughout drug screening.Construction unit is included for positioning the keeper electrode of cell and for growing nanodendrites structure
Growth electrode.Using growing electrode, can on the position of cell capture prefabricated SERS substrates, realize cell and its drug response
SERS in situ detections.Using keeper electrode, cell can be captured, be positioned and adhere-wall culture.Finally realize slender
The medicine that born of the same parents, intension high are combined with SERS without mark and automatically screening analysis.
2nd, the present invention in, complete a drug screening after, each two growth have nanodendrites structure growth electrode it
Between, apply the alternating current of amplitude, fuse nanodendrites structure, then passes to deionized water rinsing, chip is returned to originally
The state of nanodendrites structure is not grown, to prepare for the next use, chip is reached reusable purpose.
Brief description of the drawings
The present invention will be further described in detail below in conjunction with the accompanying drawings.
Fig. 1 is that the structure for the micro-fluidic chip without mark high content screening that the embodiment of the present invention one is provided is shown
It is intended to；
Fig. 2 is the offer of the embodiment of the present invention one for construction unit in the micro-fluidic chip without mark high content screening
Structural representation；
Fig. 3 is the offer of the embodiment of the present invention two for construction unit in the micro-fluidic chip without mark high content screening
Structural representation；
Fig. 4 is the offer of the embodiment of the present invention three for construction unit in the micro-fluidic chip without mark high content screening
Structural representation；
Fig. 5 is the line for electrode in the micro-fluidic chip without mark high content screening that the embodiment of the present invention one is provided
Schematic diagram；
Fig. 6 to Figure 10 is the offer of the embodiment of the present invention one for structure in the micro-fluidic chip without mark high content screening
The fabrication processing figure of unit；
Figure 11 is the structural representation of the cell drug screening system that the embodiment of the present invention one is provided；
In figure：1 is detection cell, and 2 is fluid channel, and 3 is construction unit, and 4 is substrate, and 5 is substrate, and 6 is transparent cover, and 7 is solution
Gateway, 8 is external pad, and 9 is cell observing and controlling chip, and 10 is chip TT＆C system, and 11 is Raman test system, and 12 is fluorescence
Test system, 13 is liquid feeding system, and 31 is keeper electrode, and 32 make a living long electrode, and 41 is first layer metal layer, and 42 is first
Layer insulating, 43 is second layer metal layer, and 44 is the second layer insulating, and 45 is third layer metal level.
Specific embodiment
To make the purpose, technical scheme and advantage of the embodiment of the present invention clearer, below in conjunction with the embodiment of the present invention
In accompanying drawing, the technical scheme in the embodiment of the present invention is clearly and completely described, it is clear that described embodiment is
A part of embodiment of the invention, rather than whole embodiments；Based on the embodiment in the present invention, ordinary skill people
The every other embodiment that member is obtained under the premise of creative work is not made, belongs to the scope of protection of the invention.
Fig. 1 is that the structure for the micro-fluidic chip without mark high content screening that the embodiment of the present invention one is provided is shown
It is intended to, as shown in figure 1, for the micro-fluidic chip without mark high content screening, the micro-fluidic chip is provided with detection
Pond 1 and a plurality of fluid channel 2, a plurality of fluid channel 2 are connected with the detection cell 1, and between a plurality of fluid channel 2 each other not
It is connected, multiple construction units 3, the composition array type structure of the multiple construction unit 3, the knot is provided with the detection cell 1
Structure unit 3 includes：At least two keeper electrodes 31 for being used to position cell are used to grow nanodendrites structure with least one pair of
Growth electrode 32.
When cell is positioned, keeper electrode 31 plus traveling wave voltage, the phase difference between adjacent positioned electrode 31 is：360 degree/
All keeper electrode numbers, such as the quantity of keeper electrode 31 are two, then differ 180 degree between adjacent positioned electrode 31,
The quantity of keeper electrode 31 is three, then 120 degree are differed between adjacent positioned electrode 31, if the quantity of keeper electrode 31 is six
It is individual, then 60 degree are differed between adjacent positioned electrode 31.
Specifically, the micro-fluidic chip may include：Substrate 4, substrate 5 and the transparent cover 6 being bonded together successively, institute
State construction unit 3 to be arranged in the substrate 4, the detection cell 1 and a plurality of fluid channel 2 may be contained within the substrate 5, and institute
The top cover of detection cell 1 is stated for the transparent cover 6, the transparent cover 6 is provided with multiple solution gateways 7, the multiple molten
Liquid gateway 7 corresponds with a plurality of fluid channel 2 and connects.
More specifically, the substrate 4 is insulating materials, or to be covered with the silicon materials of insulating barrier thereon.
More specifically, the substrate 5 is curing type polymer, or it is thermoplastic polymer, or for solvent volatile-type is polymerized
Thing.
Specifically, described a plurality of fluid channel 2 may include：Cell liquid fluid channel, culture medium fluid channel, medicine fluid channel,
Plating solution fluid channel, deionized water fluid channel and waste liquid fluid channel, can be provided with one-way conduction valve, i.e. waste liquid in above-mentioned fluid channel 2
Fluid channel is used for unidirectional outflow, and other fluid channels are used to unidirectionally flow into detection cell 1.
Fig. 2 is the offer of the embodiment of the present invention one for structure list in the micro-fluidic chip without mark high content screening
The structural representation of unit, as shown in Fig. 2 the construction unit 3 is double circular ring structures, two annulus are different same of diameter
Heart annulus, is provided with least two tips electrode in interior annular, and at least two circular electrodes are provided with outer toroid, with above-mentioned circle
Shape electrode is Along ent, and outer toroid is divided into many equal portions, and the point electrode in the interior annular forms an opposite between any two
Long electrode 32, the circular electrode is keeper electrode 31.
The quantity and set location of the quantity of the annulus, the keeper electrode 31 and the growth electrode 32 can bases
It is actually needed and is set, in the present embodiment, the quantity of the keeper electrode 31 and the growth electrode 32 is six.It is described
Substrate 5 is etched with six miniflows with glass, silicon, polymetylmethacrylate or dimethyl silicone polymer PSMS as base material
Road 2 and a detection cell 1；Additionally, when construction unit 3 is made, one layer of electrode connecting line layer of each annulus correspondence, adjacent electrode
A layer insulating is provided between connecting line layer.
Fig. 5 is the offer of the embodiment of the present invention one for electrode in the micro-fluidic chip without mark high content screening
Line schematic diagram, as shown in figure 5, the electrode connecting line layer of the construction unit 3 in the present embodiment is by horizontal connecting line layer and longitudinal line
Layer is constituted, wherein horizontal connecting line layer is used for six keeper electrodes 31 of outer toroid corresponding with neighbouring detecting elements respectively determining
Position electrode 31 is connected, and longitudinal connecting line layer is used for six growth electrodes 32 of interior annular are corresponding with neighbouring detecting elements respectively
Growth electrode 32 is connected, and one end of last connecting line is connected on the external pad 8 of chip edge.
The multiple construction units 3 set in detection cell 1 on micro-fluidic chip in the present embodiment, in array type structure,
Can realize high-throughout drug screening, and construction unit 3 includes keeper electrode 31 for positioning cell and is received for growing
The growth electrode 32 of rice pine-tree structure, using growing electrode 32, can on the position of cell capture prefabricated SERS substrates, realize
Cell and its SERS in situ detections of medicine reflection, using keeper electrode 31, can be captured to cell, be positioned and adherent training
Support, finally realize multi-mode, the automatically screening analysis of the medicine that unicellular, high flux is combined with SERS.
Fig. 3 is the offer of the embodiment of the present invention two for structure list in the micro-fluidic chip without mark high content screening
The structural representation of unit, as shown in figure 3, the present embodiment is with the difference of embodiment one, the construction unit 3 can be double annulus
Structure, two annulus are the different donut of diameter, at least two circular electrodes are provided with interior annular, with above-mentioned circle
Shape electrode is Along ent, and outer toroid is divided into many equal portions, point is equipped with each circular electrode, is provided with outer toroid
Multiple point electrodes, the point electrode in outer toroid is corresponded with the circular electrode in interior annular；Point in the outer toroid
Termination electrode forms a pair of growth electrodes 32, the non-point of the circular electrode with the point of the circular electrode in corresponding interior annular
End is keeper electrode 31.
Fig. 4 is the offer of the embodiment of the present invention three for structure list in the micro-fluidic chip without mark high content screening
The structural representation of unit, as shown in figure 4, the present embodiment is with the difference of embodiment one, two, the construction unit 3 can be three
Toroidal, three annulus are the different donut of diameter, and at least two tips electrode is provided with interior annular；In
Annulus is provided with least two circular electrodes, and with above-mentioned circular electrode as Along ent, middle annulus is divided into many equal portions, Mei Yiyuan
Point is equipped with shape electrode；Multiple point electrodes are provided with outer toroid, on the point electrode in outer toroid and middle annulus
Circular electrode correspond；Point electrode in the interior annular forms a pair of growth electrodes 32 between any two, described cylindrical
Point electrode on ring forms a pair of growth electrodes 32, the circular electric with the point of the circular electrode on corresponding middle annulus
The non-point of pole is keeper electrode 31.
For the concrete structure of the construction unit 3 in embodiment one, two, three, can be designed according to actual needs, its
In, the diameter of the annulus where keeper electrode 31 according to being designed by the diameter of detected cell, for growing nanometer branch
Two of crystal structure grow the distance between the closest tip of electrodes 32 ∈（0 μm, 40 μm], and two above-mentioned tips are relative
Set.
For a cell, when be positioned finish after, can be in cell by the construction unit 3 of embodiment one
Portion's structure and its drug response carry out preferably detection observation, can be to the periphery of cell by the construction unit 3 of embodiment two
Structure and its drug response carry out preferably detection observation, and the middle part of cell can be tied by the construction unit 3 of embodiment three
Structure and its drug response and peripheral structure and its drug response carry out preferably detection observation, you can by adjusting keeper electrode 31
With the set location of growth electrode 32, detection observation is carried out come the different parts to cell.
Correspondingly, for the preparation method of the micro-fluidic chip without mark high content screening, it may include following steps：
S10, using MEMS technology, the array architecture unit for including keeper electrode 31 and growing electrode 32 is made in substrate 4
3, detection cell 1 and a plurality of fluid channel 2 are made on substrate 5, multiple solution gateways 7 are made in transparent cover 6, by substrate 4,
Substrate 5 and transparent cover 6 are bonded together successively.
S20, by the electrolyte containing noble metal by a solution gateway 7 in transparent cover 6, flow through fluid channel 2,
It is passed through in detection cell 1.
S30, the indirect current between each opposite long electrode 32, grow on the closest tip of electrode 32 with two
Growth forms the nanodendrites structure of noble metal.
After S40, noble metal nano pine-tree structure are formed, to deionized water is passed through in detection cell 1, to rinse the He of detection cell 1
Fluid channel 2.
Specifically, by taking the construction unit 3 in embodiment one in bicyclic six electrode structure as an example, in step S10, described profit
With MEMS technology, being made in substrate 4 includes the array architecture unit 3 of keeper electrode 31 and growth electrode 32, can specifically wrap
Include：
S101, deposit first layer metal layer in substrate 4 and 41 and etch, the electrical cable of growth electrode 32 is formed, such as Fig. 6 institutes
Show.
Specifically, before step S101 is carried out, first from quartz glass as substrate, respectively with acetone, alcohol, go from
Sub- water is cleaned, and dry for standby；Then photoetching and lift-off techniques processing first layer metal layer 41 are recycled.
First, spin coating and dry on the glass sheet, photoetching development is carried out using mask plate；Then, sputtering thickness is 30nm
Titanium（Ti）As sheet glass and the adhesion layer of metal, then sputter the gold that thickness is 200nm（Au）；Finally, wafer is placed on dress
There is 30min in the ultrasonic tank of acetone, complete lift-off, realize the graphical of first layer metal layer 41.
S102, the first layer insulating 42 of deposition are simultaneously etched, and expose electrode window through ray, as shown in Figure 7.
Specifically, the growth thickness in substrate is the SiO of 300nm again to use PECVD2Insulating barrier, and entered using reticle
Row exposure, and hydrofluoric acid solution etching insulating layer is used, it is spilt electrode.
S103, deposition second layer metal layer 43 are simultaneously etched, and form the electrical cable of keeper electrode 31, as shown in Figure 8.
Specifically, the processing and fabricating of second layer metal layer 43, this layer of metal level are carried out using photoetching and lift-off techniques
It it is Ti/Au layers, thickness is（30nm/200nm）, image dissolves transverse electrode lead.
S104, the second layer insulating 44 of deposition are simultaneously etched, and expose electrode window through ray, as shown in Figure 9；
Specifically, the thick SiO of another layer of 300nm are generated using the method for PECVD2Insulating barrier, company is etched by wet etching
Connect the electrode hole of keeper electrode 31 and growth electrode 32.
S105, deposition third layer metal level 45 are simultaneously etched, and form growth electrode 32 and keeper electrode 31, as shown in Figure 10.
Specifically, keeper electrode 31 and growth electrode 32, this Ti/Au thickness are processed using photoetching and lift-off techniques
It is 30nm/300nm to spend.
More specifically, in step S10, detection cell 1 and a plurality of fluid channel 2 are made on substrate 5, the technique for being used includes
The technologies such as soft lithographic, method of molding, pressure sintering, LIGA techniques and laser ablation process.
After the preliminary chip that completes, the encapsulation of chip is also carried out, during encapsulation：First design, make PCB, its
On be provided be connected with the one-to-one turmeric pad of chip and with external control devices metal row pin；Then by chip glue
It is bonded on PCB, is attached the pad on the pad and circuit board on chip using gold ball bonding technique, board metal
Row's pin is connected by wire with external control devices again.
Complete chip manufacturing out after, carry out the preparation of SERS substrates：Electrolyte containing noble metal is passed through detection
In pond 1, by controlling the amplitude and the frequency of alternating voltage of exchange and DC offset voltage, nanodendrites structure can be controlled
Pattern, position and the direction of growth, will form the nanodendrites structure of noble metal on each tip of opposite long electrode 32；It is expensive
After metal nano pine-tree structure is formed, deionized water is passed through toward detection cell 1 is interior, to rinse detection cell 1 and fluid channel 2, in case after
Continuous chip normal work.
Specifically, the alternating current for applying between electrode 32 is grown can be sine voltage, square-wave voltage, triangular wave electricity
Pressure, or bias voltage, its amplitude are more than 3V, and frequency is located between 1KHz and 10MHz；The electrolyte being passed through can be ion
Solution or complex solution, more specifically, can be AuCl3·HCl·3H2O、Pd（acetate）2、In（CH3COO）3、
HauCl4·3H2O、H2PtCl6·6H2O、K2PtCl4And Cu（NO3）2Deng solution.
During cellular localization, identical sinusoidal signal is applied on relative positioning electrode 31, applied on adjacent positioned electrode 32
Plus phase difference is（360 degree/all keeper electrode numbers）Sinusoidal signal, cell is in the presence of negative dielectric power to electric field
The relatively low position motion of middle field intensity.
After completing a drug screening, between the growth electrode 32 that each two growth has nanodendrites structure, apply big
The alternating current of amplitude, fuse nanodendrites structure, is passed through deionized water rinsing, returns to chip and did not grew nanodendrites originally
The state of structure, to prepare for the next use, makes chip reach reusable purpose.
Correspondingly, Figure 11 is the structural representation of the cell drug screening system that the embodiment of the present invention one is provided, such as Figure 11
Shown, the cell drug screening system includes：Cell observing and controlling chip 9（Micro-fluidic chip i.e. in the present invention）, chip observing and controlling
System 10, Raman test system 11, fluorometric investigation system 12 and liquid feeding system 13.Wherein, chip TT＆C system 10 be by
Arbitrary waveform signal generator is constituted, and mainly has following functions：SERS substrates prepare needed for overpotential and cellular localization signal
Produce；Raman test system 11, is mainly used to realize for cardiovascular and cerebrovascular, tumour is viral, the major disease such as neuropsychiatric
The drug screening of preventing and treating and the raman spectra in situ detection of interaction；Fluorometric investigation system 12, in experimental stage pair
Cellular morphology carries out Real Time Observation and record, and is contrasted with Raman test result, after formally being screened to medicine, can
Fluorometric investigation system 12 is not used；Liquid feeding system 13 is mainly made up of syringe pump and stream, for realizing in microchip
Various fluids are controlled, at the same can also realize it is automatic, quantitatively inject corresponding reagent.
During work, SERS substrates are first prepared, use deionized water rinsing；Then cell liquid, nutrient solution, medicine are passed through successively；
Finally observation detection；After end, fuse SERS, and rinses well；When needing to carry out the drug screening of a new round, then original
Micro-fluidic chip on prepare SERA substrates again, repeat above step.
The design that the present invention passes through cleverly many fluid channels and big detection cell, realizes high flux, the inspection of efficient cell
Survey analysis and drug screening；By special circular keeper electrode and tip-growth electrode design, using electrochemical techniques, realize
Unicellular fixation and SERS substrates are in situ prepares and detect；The connection mode of electrode independence allows the present invention by outside
Variation and automation electrode control, finally realize intension high, it is unmarked for cardiovascular and cerebrovascular, tumour, virus, nerve essence
The multi-mode of the major disease protective agents such as refreshing system, automatically screening analysis.
Finally it should be noted that：Various embodiments above is merely illustrative of the technical solution of the present invention, rather than its limitations；To the greatest extent
Pipe has been described in detail with reference to foregoing embodiments to the present invention, it will be understood by those within the art that：Its according to
The technical scheme described in foregoing embodiments can so be modified, or which part or all technical characteristic are entered
Row equivalent；And these modifications or replacement, the essence of appropriate technical solution is departed from various embodiments of the present invention technology
The scope of scheme.
Claims (10)
1. the micro-fluidic chip without mark high content screening is used for, and the micro-fluidic chip is provided with detection cell（1）With it is many
Bar fluid channel（2）, a plurality of fluid channel（2）With the detection cell（1）It is connected, and a plurality of fluid channel（2）Between each other
It is not attached to lead to, it is characterised in that：The detection cell（1）Inside it is provided with multiple construction units（3）, the multiple construction unit（3）Group
Into array type structure, the construction unit（3）Including：At least two keeper electrodes for being used to position cell（31）And at least one pair of
Growth electrode for growing nanodendrites structure（32）.
2. according to claim 1 for the micro-fluidic chip without mark high content screening, it is characterised in that：It is described
Construction unit（3）It is three toroidals, three annulus are the different donut of diameter, and at least two are provided with interior annular
Individual point electrode；At least two circular electrodes are provided with middle annulus, with above-mentioned circular electrode as Along ent, middle annulus is divided equally
It is many equal portions, point is equipped with each circular electrode；Multiple point electrodes are provided with outer toroid, the tip in outer toroid
Electrode is corresponded with the circular electrode on middle annulus；Point electrode in the interior annular forms a pair of growths electricity between any two
Pole（32）, it is electric that the point electrode in the outer toroid forms a pair of growths with the point of the circular electrode on corresponding middle annulus
Pole（32）, the non-point of the circular electrode is keeper electrode（31）.
3. according to claim 1 for the micro-fluidic chip without mark high content screening, it is characterised in that：It is described
Construction unit（3）It is double circular ring structures, two annulus are the different donut of diameter, and at least two are provided with interior annular
Individual point electrode, is provided with least two circular electrodes in outer toroid, and with above-mentioned circular electrode as Along ent, outer toroid is divided equally
It is many equal portions, the point electrode in the interior annular forms a pair of growth electrodes between any two（32）, the circular electrode is
Keeper electrode（31）.
4. according to claim 1 for the micro-fluidic chip without mark high content screening, it is characterised in that：It is described
Construction unit（3）It is double circular ring structures, two annulus are the different donut of diameter, and at least two are provided with interior annular
Individual circular electrode, with above-mentioned circular electrode as Along ent, outer toroid is divided into many equal portions, and point is equipped with each circular electrode
End, is provided with multiple point electrodes in outer toroid, a pair of the circular electrode 1 on the point electrode in outer toroid and interior annular
Should；Point electrode in the outer toroid forms a pair of growth electrodes with the point of the circular electrode in corresponding interior annular
（32）, the non-point of the circular electrode is keeper electrode（31）.
5. according to claim 1 for the micro-fluidic chip without mark high content screening, it is characterised in that：It is described
Micro-fluidic chip includes：The substrate being bonded together successively（4）, substrate（5）And transparent cover（6）, the construction unit（3）If
It is placed in the substrate（4）On, the detection cell（1）With a plurality of fluid channel（2）May be contained within the substrate（5）On, and the inspection
Survey pond（1）Top cover be the transparent cover（6）, the transparent cover（6）It is provided with multiple solution gateways（7）, it is described many
Individual solution gateway（7）With a plurality of fluid channel（2）Correspond connection.
6. according to claim 1 for the micro-fluidic chip without mark high content screening, it is characterised in that：It is described
A plurality of fluid channel（2）Including：Cell liquid fluid channel, culture medium fluid channel, medicine fluid channel, plating solution fluid channel, deionized water
Fluid channel and waste liquid fluid channel, above-mentioned fluid channel（2）Inside it is equipped with one-way conduction valve.
7. as described in any in claim 1 to 6 for without mark high content screening micro-fluidic chip making side
Method, it is characterised in that：Comprise the following steps：
S10, using MEMS technology, in substrate（4）Upper making includes keeper electrode（31）With growth electrode（32）Array knot
Structure unit（3）, in substrate（5）Upper making detection cell（1）With a plurality of fluid channel（2）, in transparent cover（6）It is upper to make multiple solution
Gateway（7）, by substrate（4）, substrate（5）And transparent cover（6）It is bonded together successively；
S20, by the electrolyte containing noble metal pass through transparent cover（6）On a solution gateway（7）, flow through fluid channel
（2）, it is passed through detection cell（1）In；
S30, in each opposite long electrode（32）Between indirect current, with each opposite long electrode（32）Closest tip
It is upper to grow the nanodendrites structure for forming noble metal；
After S40, noble metal nano pine-tree structure are formed, to detection cell（1）Deionized water is inside passed through, to rinse detection cell（1）With
Fluid channel（2）.
8. according to claim 7 for the preparation method without the micro-fluidic chip for identifying high content screening, it is special
Levy and be：In step S10, described utilization MEMS technology, in substrate（4）Upper making includes keeper electrode（31）With growth electrode
（32）Array architecture unit（3）, specifically include：
S101, in substrate（4）Upper deposition first layer metal layer（41）And etch, form growth electrode（32）Electrical cable；
S102, the first layer insulating of deposition（42）And etch, expose electrode window through ray；
S103, deposition second layer metal layer（43）And etch, form keeper electrode（31）Electrical cable；
S104, the second layer insulating of deposition（44）And etch, expose electrode window through ray；
S105, deposition third layer metal level（45）And etch, form growth electrode（32）And keeper electrode（31）.
9. according to claim 7 for the preparation method without the micro-fluidic chip for identifying high content screening, it is special
Levy and be：After completing a drug screening, there is the growth electrode of nanodendrites structure in each two growth（32）Between, apply big
The alternating current of amplitude, fuse nanodendrites structure, is passed through deionized water rinsing.
10. according to claim 7 for the preparation method without the micro-fluidic chip for identifying high content screening, it is special
Levy and be：Electrolyte containing noble metal is solion or complex solution, each opposite long electrode（32）Between apply
Alternating current amplitude be more than 3V, frequency values be located between 1kHz and 10MHz.
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| CN110394204A (en) * | 2019-08-21 | 2019-11-01 | 苏州大学 | A kind of micro-fluidic chip and preparation method thereof comprising liquid metal electrode |
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