CN109991423A - Efficient single cell capture and rapid single cell secretory protein detection platform and detection method - Google Patents
Efficient single cell capture and rapid single cell secretory protein detection platform and detection method Download PDFInfo
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Abstract
The invention discloses a high-efficiency single cell capture and rapid single cell secretory protein detection platform and a detection method; the detection platform consists of a single cell capture and droplet incubation microfluidic chip, a secreted protein capture glass plate and a chip clamp; the micro-fluidic chip for cell capture and droplet incubation has two layers of structures, namely a single cell capture and droplet incubation layer and an isolation valve layer. The platform can efficiently and quickly capture single cells, micro liquid drops containing single cells can be quickly and stably generated by adding the isolation phase, long-term liquid drop incubation can be carried out, the high activity of the single cells is ensured, and the chip is combined with an antibody array glass plate for detecting the secretory protein, so that the high-flux, quick and high-sensitivity detection of the secretory protein of the single cells can be realized.
Description
Technical field
The present invention relates to efficiently unicellular captures and quick unicellular secretory protein detection platform, belong to micro-fluidic unicellular
Analysis method technical field.
Background technique
Traditional cell research method, is group's Journal of Sex Research based on a large amount of cells, and obtained result is often all thin
The average result of born of the same parents.And some researches show that same type of cell, its expression conditions of different cell individuals and phenotypes
There is certain difference (Zhang, Y et al. (2014) Journal of the American Chemical Society
136:15257), the referred to as heterogeneity of cell.The experimental result that population experiment obtains can mask the heterogeneity of cell, influence
The treatment of cancer, prognosis and recurrence probability.Unicellular research, which refers to, is dispersed in independent experimental considerations unit one by one for cell
In and studied, obtain independent experimental result, will not influence each other, therefore cell heterogeneity can adequately be ground
Study carefully, obtain it is more accurate, with guiding significance as a result, but single celled research want flux with higher that could have very
Good statistical significance.Secretory protein has ability (Konty, T the at al. (2011) for promoting cancer cell multiplication, transfer
Biosensors&bioelectronics 26:2707), unicellular secretory protein is carried out to cancer cell and is studied, is facilitated
Understand the relationship between the transfer ability and cancer cell and immune system of cancer cell.
Currently, micro-fluidic chip is mainly combined reality with secretory protein capturing tools by the detection of unicellular secretory protein
The detection of cell secretory protein in existing unicellular, many cells or blood plasma, and exported by fluorescence signal.It is examined for secretory protein
The micro-fluidic chip of survey includes microwell array formula chip (Lu, Y at al. (2013) Anal.Chem.85:2548), fishbone liquid
Drop generates chip two major classes；Its principle be all single celled capture is realized by Poisson distribution, therefore its there are cell utilizations
The problems such as rate is low, unicellular capture rate is low, reagent waste is serious, test chamber volume is big, detection sensitivity is low, because of detection
Cavity volume is larger so chip needs the incubation more than 12 hours that secretory protein could be allowed adequately to be captured.Lack one kind to gather around
There is lesser detection volume that it is made to be able to achieve quickly unicellular secretory protein capture in shorter incubation time, cell can be allowed
Microlayer model prolonged incubation, and can guarantee single celled high activity, realize that unicellular secretory protein is high-throughput, quick, highly sensitive
The detection platform of degree.
Summary of the invention
The present invention low, secretory protein test chamber for the existing unicellular unicellular capture rate of secretory protein detection platform
It is unicellular efficient to propose that one kind is able to achieve for the problems such as volume is big, unicellular secretory protein detection sensitivity is low, reagent waste is serious
The capture rate of rate, secretory protein test chamber are small in size, secretory protein detection sensitivity is high, quickly unicellular secretory protein is caught
Obtain, save the efficient unicellular capture and quick unicellular secretory protein detection platform of reagent.
The technical solution of the present invention is as follows:
Efficiently unicellular capture and quick unicellular secretory protein detection platform comprising cell capture is incubated for micro- with drop
Fluidic chip and secretory protein capture glass plate；Wherein, it includes: positioned at upper layer that unicellular capture, which is incubated for micro-fluidic chip with drop,
Isolation valve layer (23) and unicellular capture positioned at lower layer and drop be incubated for layer (24)；
It includes multiple units that unicellular capture, which is incubated for layer with drop, and each unit includes being located at cell flow channel or so two
The straight cell flow channel (7) at end uses U-shaped or arc cell flow channel between two straight cell flow channels (7)
(11) it connects, further includes cell capture cavity (6), the cell capture cavity (6) is connected with 3 channels, and first channel is
Cell flow channel, connects an arm end of U-shaped or arc cell flow channel (11), and the width in the channel is greater than cell to be captured
Diameter；Article 2 channel be interface channel (8), width be less than cell dia to be captured, for connect cell capture cavity (6) with
Another arm end of U-shaped or arc cell flow channel (11)；Article 3 channel is sense channel (9), and leads to solution channel
(10)；There are a solution inlet (2) and a taphole (4) in solution channel (10) both ends respectively；
Be isolated valve layer include the opposite isolation valve passage (16) in two flow directions, two isolation valve passage respectively with
Sense channel (9) intersects；Diaphragm (29) are set between isolation valve passage (16) and sense channel (9)；Cell solution entrance (1) with go out
For mouth (3) at cell flow channel both ends, there are a solution inlet (2) and a taphole (4) in solution channel both ends respectively；Every
Entrance (5) and cell flow channel (7,11) joining distal ends from phase channel；
Secretory protein capture glass plate includes: the glass plate (26) that surface modification has poly-D-lysine, secretory protein capture
Antibody array band (20) is modified on poly-D-lysine glass plate (26)；Secretory protein capture antibody array band (20) is located at
In sense channel (9), and between two crosspoints that two isolation valve passage (16) and sense channel are formed, secretory protein
Capture antibody array band (20) is directly contacted towards sense channel (9) and with the solution in sense channel (9).
In the present invention, between multiple units, it can adopt and be connected serially, parallel way can also be used
Connection can also be series-parallel hybrid mode connection.
Preferably, the cell passage (7,11), solution channel (10), isolation phase channel (5) width are that 5-1000 is micro-
Rice, depth are 5-1000 microns；Sense channel width is 5-1000 microns, and length is 5-1000 microns, highly micro- for 5-200
Rice.
Preferably, a cell capture cavity (6) and a sense channel (9) form a hatch unit, are adding one
Group secretory protein capture antibody band (20) forms a cell secretory protein detection unit；Secretory protein captures antibody band
(20) it is contacted with sense channel (9) interior solution.
Preferably, the number of secretory protein detection unit is 1-50000 in each secretory protein detection platform.
Preferably, isolation valve passage (16) width is 5-1000 microns, is highly 5-1000 microns, two channel isolation intervals
(14) distance is 5-1000 microns.
Preferably, efficiently unicellular capture and quick unicellular secretory protein detection platform further include chip fixture, described
Chip fixture include fixture upper plate and fixture lower plate, the isolation valve layer (23) and unicellular capture are incubated for layer with drop
(24) it is located between fixture upper plate and fixture lower plate
Preferably, chip fixture upper plate opening width is 0.1-2 centimetres, and length is 0.1-2 centimetres.
Preferably, chip fixture upper plate is equipped with screw permanent opening (30) and 7 chip channel openings, respectively carefully
Solution channel opening (32,34), isolating valve access portal (35,36) and phase entrance opening is isolated in born of the same parents' access portal (31,33)
(37)。
The present invention also provides foregoing efficiently unicellular captures and quick unicellular secretory protein detection platform
Using applying to the detection and/or analysis of unicellular secretory protein/intracellular protein.
The present invention also provides a kind of unicellular secretory protein detection methods, include the following steps:
1) it according to efficiently unicellular capture above-mentioned and quick unicellular secretory protein detection platform structure, prepares cell and catches
It obtains and is incubated for micro-fluidic chip and secretory protein capture glass plate with drop；
2) it is passed through capture antibody in the modification chip cavity for capturing glass plate thermal bonding with poly-D-lysine, is secreted
The modification of albumen capture antibody array；After modification is good, is closed, be washed out using buffer, finally caught secretory protein
Glass plate is obtained to be spin-dried for；
3) unicellular capture is incubated for micro-fluidic chip with drop and unicellular secretory protein captures glass plate working region
It is aligned, posts and fixes；Chip is immersed in ultrapure water；It is captured to unicellular；
A, ultrapure water is first injected in isolation valve passage (16), so that its pressure is become larger, is caused diaphragm (29) downward deformation, it will
Sense channel (9) is kept apart with cell capture cavity (6) and solution channel (10)；
B, it is reversely passed through from isolation phase entrance (5) with solution channel outlet (4) and phase is isolated, in cell capture cavity (6)
It generates and has single celled drop one by one,
C, air pump switch should be closed after droplet array generation, stands, then extracts tracheae, by isolating valve feeder connection
The water pipe of (12,13) is extracted, and release isolation valve passage (16) pressure, diaphragm (29) pattern restores, there is no isolation effect, makes to examine
It surveys channel (9) to be connected to again with cell capture cavity (6), forms unicellular hatch unit, the entrance of chip is being used into glue
Band closing；.
4) then platform is put into cell incubator, carries out the incubation of cell drop, it is slender during entire be incubated for
Born of the same parents are in cell capture cavity, and secretory protein is transferred to cell detection channel by diffusion, are captured by secretory protein anti-
Volume array is captured；Unicellular secretory protein after being incubated for is captured into glass plate, is dismantled from chip, and detect list
Cell secretory protein captures the signal on glass plate, analyzes the secretion situation of unicellular secretory protein.
Preferably, step (3) is captured to unicellular, and cell concentration is controlled in 5*105-5*107/ mL, cell phase with it is molten
Liquid phase flow rate is 0.005-0.05mL/h, and isolation phase phase flow velocity is 0.5-2.0mL/h when being reversely passed through isolation phase, to droplet array
Air pump switch should be closed after generation, stood 1-10min and then extracted tracheae, the entrance of chip is closed using adhesive tape.
Preferably, the unicellular secretory protein after being incubated for is captured into glass plate, is dismantled from chip, and immediately
It with buffer blind, is then incubated for 45 minutes with the detection antibody of 4 kinds of secretory proteins with biotin group with it, detection is anti-
Body dilutes 200 times；Then it is washed using the PBS of 1%BSA, captures glass using the SA-APC and secretory protein that dilute 100 times
Plate is incubated for 20 minutes；Gradient wash finally is carried out using PBS, 50%PBS, ultrapure water, ultrapure water, drying uses scanner pair
Fluorescence signal on unicellular secretory protein capture glass plate is detected, and the secretion situation of unicellular secretory protein is analyzed.
The present invention captures unicellular captured cavity using hydromechanical principle, unicellular capture cavity and its phase
The sense channel of connection forms a hatch unit, and different hatch units is not interfere with each other；The number of increase hatch unit can be passed through
Mesh realizes the detection of unicellular secretory protein high throughput.The unicellular capture rate height of the present invention, secretory protein test chamber volume
Small, secretory protein detection sensitivity is high, unicellular secretory protein capture quickly, reagent can be saved.
Detailed description of the invention
Fig. 1 is unicellular capture and drop incubation layer structure top view
Fig. 2 is unicellular capture and drop incubation layer unit structure top view
Fig. 3 is isolation valve layer structure top view
Fig. 4 is closing valve unit structure top view
Fig. 5 is that secretory protein captures glass plate modification chip structure top view
Fig. 6 is that secretory protein captures glass plate modification chip unit structure top view
Fig. 7 is unicellular capture and drop incubation microfluidic chip structure top view
Fig. 8 is unicellular capture and drop hatch unit structure top view
Fig. 9 is efficiently unicellular capture and quick unicellular secretory protein detection chip structure top view
Figure 10 is efficiently unicellular capture and quick unicellular secretory protein detection chip cellular construction top view
Figure 11 is efficiently unicellular capture and quick unicellular secretory protein detection platform side view
Figure 12 is efficiently unicellular capture and quick unicellular secretory protein detection chip fixture upper plate
Figure 13 is efficiently unicellular capture and quick unicellular secretory protein detection chip fixture lower plate
Figure 14 is that efficient unicellular capture and drop are incubated for micro-fluidic chip fluorescence drop and are incubated for array and generate and scheme.
Figure 15 is the unicellular capture situation of chip.Top is that unicellular capture rate histogram is averaged unicellular capture rate
Up to 80%, the following figure is the unicellular capture figure of chip.
Figure 16 is chip cell survival after 7 hours drops are incubated for.A figure be chip put in be incubated for before light field
Picture；B figure is to check cell survival after dyeing after 7 hours are incubated for using calcein (CA-AM)；C figure is through 7 hours
Cell death situation is checked using propidium iodide (PI) dyeing after incubation.
Figure 17 is cell survival/death condition after 7 hours are incubated for.Its survival rate is up to 94.8%.
Figure 18 is the secretion thermal map of 4 kinds of secretory proteins after being incubated for for macrophage 4 hours.
Numeral mark in figure
1 cell solution entrance, 2 solution inlet, 3 cell solution exports 4 tapholes 5 and 6 cell capture of phase entrance is isolated
7 10 solution channel of straight cell flow channel 8 interface channel, 9 sense channel of cavity, 11 U-shaped or the flowing of arc cell are logical
Valve passage is isolated in 12 isolating valve feeder connection, 13 isolating valve feeder connection, 14 isolating valve channel spacing 15 flared region 16 in road
17 modification chip channel outlet, 18 modification 19 secretory protein of chip channel entrance capture 20 secretory protein of antibody array interval is caught
Obtain 21 fixture upper plate of antibody array band, 22 fixture lower plate
The 23 unicellular captures of isolation valve layer 24 are incubated for 25 hollow iron plate of layer, 26 poly-D-lysine glass plate 27 with drop
Drop is incubated for the unicellular capture of 28 screw of layer channel 29 and fixes with drop incubation layer, 30 screw of diaphragm being isolated between valve layer
Opening
31 cell passages be open 32 solution channels be open 33 cell passages be open 34 solution channels opening 35 and 36 every
From 37 isolation phase entrance opening of valve passage opening.
Specific embodiment
Embodiment 1
Referring to the efficiently unicellular capture of Fig. 1 to Figure 14 and quick unicellular secretory protein detection platform, including unicellular catch
It obtains and is incubated for micro-fluidic chip, secretory protein capture glass plate, chip fixture three parts with drop；Wherein,
1, referring to Figure 11, unicellular capture and drop incubation micro-fluidic chip include: positioned at upper layer be isolated valve layer 23 and
Unicellular capture and drop positioned at lower layer are incubated for layer 24；Unicellular capture is incubated for layer by multiple experimental considerations unit head and the tail phases with drop
It even forms, each experimental considerations unit includes the straight cell flow channel 7 positioned at cell flow channel left and right ends, two straight streams
It is connected between dynamic channel using U-shaped or arc cell flow channel 11, the left end of cell capture cavity 6 and U-shaped or arc cell
Flow channel 11 is connected, and right end is connected by interface channel 8 with the right end of U-shaped or arc cell flow channel 11, cell capture
6 lower section connecting detection channel 9 of cavity, sense channel lower section are connected with solution channel 10；
Wherein, referring to Figure 10, cell capture cavity 6 is connected with 3 channels, and first channel is cell flow channel, even
Wherein one end left end of U-shaped or curved channel 11 is connect, the width in the channel is greater than cell dia, for capturing cell；Article 2
Channel is interface channel 8, and width is less than cell dia, for connecting the right end of U-shaped or curved channel 11；Article 3 channel is inspection
Channel 9 is surveyed, solution channel 10 is led to；There are a solution inlet 2 and a taphole 4 in 10 both ends of solution channel respectively；
Referring to Fig. 3 and Fig. 4, valve layer is isolated and is made of multiple identical structural units, each structural unit is reversed by two
Identical isolation valve passage 16 be spaced a distance 14 compositions, have a flared region 15 in the middle part of structural unit, every
Only one entrance (12 or 13) of valve passage is isolated；Be isolated valve passage 16 above sense channel 9, middle ware alternating floor PDMS every
Film 29, and in 9 range of sense channel, isolating valve flared region 15 is overlapped with sense channel 9；Cell solution entrance 1 with go out
Mouth 3 is at cell flow channel both ends；The entrance 5 that phase channel is isolated connects with cell flowing channel end.
2, secretory protein capture glass plate includes: the glass plate 26 that surface modification has poly-D-lysine, secretory protein capture
Antibody band 20 is modified on poly-D-lysine glass plate 26.
3, fixture includes: fixture upper plate 21 and fixture lower plate 22, and glass plate 26 is in chip fixture upper plate 21, fixture lower plate
Between 22, and one piece of hollow iron plate 25 being placed on fixture upper plate 21, being fixed by screw 28, composition is efficiently unicellular to catch
Obtain with quick unicellular secretory protein detection platform, referring to Figure 12 and Figure 13.
4, the assembling of efficiently unicellular capture and quick unicellular secretory protein detection platform: secretory protein will be modified with and caught
The poly-D-lysine glass plate 26 and unicellular capture for obtaining antibody array 20 are incubated for micro-fluidic chip with drop and calibrate, and secrete egg
White capture antibody array 20 is located in sense channel 9, and between two isolation valve passages 16, antibody array 20 is logical towards detection
Road 9 is simultaneously directly contacted with the solution in sense channel 9.The chip after calibration is fixed using fixture, chip channel entry and exit and
Corresponding chucking opening is corresponding；Platform be respectively from top to bottom hollow iron plate 25, fixture upper plate 21, isolation valve layer 23,
Unicellular capture is incubated for layer 24 with drop, secretory protein captures antibody glass plate 26, fixture lower plate 22, referring to Figure 13.
Preferably, the cell passage (7,11), solution channel 10, isolation 5 width of phase channel are 5-1000 microns, deep
Degree is 5-1000 microns；Sense channel width is 5-1000 microns, and it is highly 5-200 microns that length, which is 5-1000 microns,.
Preferably, a cell capture cavity 6 and a sense channel 9 form a hatch unit, a hatch unit
Along with one group of secretory protein capture antibody band 20 forms a cell secretory protein detection unit；Secretory protein captures antibody
Band 20 is contacted with solution in sense channel 9.
Preferably, the number of secretory protein detection unit is 1-50000 in each secretory protein detection platform.
Preferably, isolation 16 width of valve passage is 5-1000 microns, is highly 5-1000 microns, two channel isolation intervals 14
Distance is 5-1000 microns.
Preferably, the membrane thicknesses being isolated between valve passage 16 and sense channel 9 below are 3-50 microns
Preferably, chip fixture upper plate opening width is 0.1-2 centimetres, and length is 0.1-2 centimetres.Preferably, chip gripper
Have upper plate and be equipped with screw permanent opening 30 and 7 chip channel openings, respectively cell passage opening (31,33), solution leads to
Isolating valve access portal (35,36) and phase entrance opening 37 is isolated in road opening (32,34).
Efficiently unicellular capture and quick unicellular secretory protein detection method, include the following steps:
(1) it uses PDMS to make unicellular capture be incubated for layer with drop, valve layer is isolated, and two layers of calibration is fitted in one
It rises, unicellular capture is incubated for layer together with isolation valve layer thermal bonding with drop by way of thermal bonding；
(2) hydrophobization modification is carried out to the chip made to handle with boiling, handle well rear spare；
(3) channel PDMS chip is modified using modification chip silicon wafer template construct, and will modification chip and poly-D-lysine
Glass plate thermal bonding captures secretory protein on antibody modification to poly-D-lysine glass plate；
(4) fixture that unicellular capture is incubated for micro-fluidic chip with drop is designed and made, is caught using fixture by unicellular
It obtains and is incubated for micro-fluidic chip with drop, the poly-D-lysine glass plate of having modified secretory protein capture antibody is existed by fixture combination
Unicellular capture and quick unicellular secretory protein detection platform are made together；
(5) single celled capture is carried out using platform, isolation is reversely passed through after the completion of capture and mutually generates the liquid for having cell
Drip array；Generation is put into the incubation that a period of time is carried out in cell incubator with the droplet array platform of cell；
(6) the poly-D-lysine glass plate for capturing unicellular secretory protein is subjected to immune enzyme chain reaction processing, output
Fluorescence signal, and fluorescence signal is tested and analyzed.
In preferred embodiments of the present invention, in step (4), it is captured on glass plate in secretory protein and modifies a BSA-
FITC fluorescent bands are caught as being used to calibrate unicellular capture referring to band with incubation micro-fluidic chip and unicellular secretory protein
Obtain glass plate；Unicellular capture is incubated for micro-fluidic chip with drop using fixture later, unicellular secretory protein captures glass
Plate is fixed together, and balancing each screw position point pressure using hollow stainless steel plate can be tested.
In preferred embodiments of the present invention, in step (5), unicellular capture is incubated for the operation of micro-fluidic chip with drop
Time should control within 1.5 hours；Reversely be passed through isolation mutually generate drop when, isolation phase flow velocity should control in 0.8mL/h；Behaviour
The process chip of work should be immersed in ultrapure water.
Embodiment 2
(1) unicellular to capture the thermal bonding for being incubated for layer PDMS chip with drop, valve layer PDMS chip being isolated
First using just setting microscope, by unicellular capture and drop be incubated for layer with valve layer be isolated be aligned, fit to together, general
Then chip is put into 60 degree of baking ovens and heats 20 minutes, then remove it from silicon wafer template, and in chip channel entrance
Place's punching, unicellular capture are incubated for facture of microchip with drop and complete.
(2) unicellular capture is incubated for the modification of micro-fluidic chip with drop
The modification of chip channel is carried out using electronics fluorination liquid 1720, modification ensures molten for 1 hour using baking oven heating after getting well
Agent is volatilized completely, and then chip is placed in PBS and carries out boiling, and after boiling, chip is immersed in PBS, taking-up when testing.
(3) modification of unicellular secretory protein capture glass plate
Production modification channel microfluidic chip, and go out to punch in its entrance；Chip is put into ultrasound 10 in dehydrated alcohol
Minute, chip is then put into ultrasound 10 minutes in ultrapure water, takes out and is dried up surface moisture using nitrogen, chip is placed in dry
In net ware, it is put into 80 degree of baking ovens to heat 30 minutes and dries；Chip channel impurity remained on surface will be modified using adhesive tape
After removing, chip is bonded naturally with poly-D-lysine glass plate, is placed in 80 degree of baking ovens and heats 2 hours progress thermal bondings；To it
After cooling, it is passed through 1.5 microlitres of secretory protein capture antibody and is modified, the modification time is 4 hours.After modification is good, 3% is used
The PBS of BSA carries out closing 1 hour, then successively carries out gradient wash using pure 1X PBS, 50%PBS, ultrapure water, ultrapure water,
Finally secretory protein capture glass plate is spin-dried for, is placed in clean ware and is kept in dark place.
(4) unicellular capture is incubated for the unicellular capture of micro-fluidic chip with drop and droplet array is generated and is incubated for
In the unicellular acquisition procedure of platform, it should be immersed in ultrapure water；It, should be first logical to isolating valve when unicellular capture
Road injects ultrapure water, and downward deformation occurs for the diaphragm being isolated between valve passage and sense channel below, will test channel every
Keep apart from cell capture cavity, flow channel, preventing cell from flowing into sense channel influences single celled capture；Cell flow rate
For 0.01mL/h, cell is captured by cell capture cavity, and is rested in cell capture cavity, and required time is 5 minutes.It catches
5 minutes should be stood after the completion of obtaining, release flow phase pressure is passed through isolation phase convenient for reversed；It is reversely logical from isolation phase entrance and solution
Road outlet is passed through isolation phase, and isolation phase flow velocity is 0.8mL/h, generates and drips one by one with single celled catcher body fluid, array
After generation, air valve is closed；5 minutes are stood after drop formation, the air pressure in channel is drained, then extracts tracheae, will be isolated
Valve passage inner conduit is extracted, pressure release, is restored diaphragm pattern, be will test channel and link together with cell capture cavity, shape
At a hatch unit；Then entrance is closed using adhesive tape, micro-fluidic chip is immersed in ultrapure water, be put into cell training
It supports and is incubated in case；Unicellular to be in cell capture cavity during cell incubation, the secretory protein that cell generates can pass through
The effect of diffusion is diffused into sense channel, and the secretory protein detection antibody array being detected in channel is captured.
The modification of the unicellular secretory protein of embodiment 3 capture glass plate
Production modification channel microfluidic PDMS chip, as shown in figure 5, and going out to punch in its entrance；Chip is put into nothing
Ultrasound 10 minutes in water-ethanol, are then put into ultrasound 10 minutes in ultrapure water for chip, and taking-up is blown surface moisture using nitrogen
It is dry, chip is placed in clean ware, is put into 80 degree of baking ovens to heat 30 minutes and dry；Chip channel table will be modified using adhesive tape
After the remaining impurity removal of face, chip is bonded naturally with poly-D-lysine glass plate, be placed in 80 degree of baking ovens heat 2 hours into
Row thermal bonding；After its cooling, BSA-FITC, IL-8 capture antibody, MCP-1 capture are each led into its 5 modification channels
Antibody, TNF-a capture antibody, MIP-1b capture antibody, each 1.5 microlitres, carry out the modification modification of secretory protein capture antibody, institute
The capture antibody used dilutes one times, and the modification time is 8 hours.After modification is good, closing 1 hour is carried out using the PBS of 3%BSA,
Then gradient wash successively is carried out using the PBS, ultrapure water, ultrapure water of PBS, 50%1X of pure 1X, finally catches secretory protein
It obtains glass plate to be spin-dried for, is placed in clean ware and is kept in dark place.
The single celled capture of embodiment 4 and the viability verifying of cell
Unicellular capture is incubated for micro-fluidic chip with drop and unicellular secretory protein captures glass plate working region pair
Together, it is bonded and is fixed with fixture, can be tested；In experimentation, chip should be immersed in ultrapure water.First give isolation valve layer
Ultrapure water is injected in channel, causes the downward deformation of diaphragm, will test channel and keep apart with cell capture cavity, solution channel；?
Cell passage entrance is passed through cell (cell A549) and is captured, and solution channel is passed through and the consistent cell culture of cell suspending liquid
Liquid, cell concentration should be controlled in 5*106A/mL or so, cell phase and molten liquid phase flow rate are 0.01mL/h, and unicellular capture rate can
Up to 80%, such as Figure 15, stand 5 minutes after the completion of capture, then extract the pipe of cell entry and solution inlet, reversely from every
It is passed through from phase entrance with molten liquid-phase outlet and phase is isolated, isolation phase flow velocity should be 0.8mL/h, generate the drop for having cell one by one
Array is then shut off air pump switch, stands 5min and then extracts tracheae, the entrance of chip is closed using adhesive tape, is put into
It in ultrapure water, then puts it into cell incubator, is incubated for after carrying out incubation a period of time of cell drop.
The entrance for being closed chip after the completion of incubation is opened, and the mixed dye for being passed through CA-AM and PI carries out cell dye
Color judges cells viability, take pictures observation as shown in Figure 16, Figure 17 to the fluorescence signal of cell after twenty minutes, 7 hours
It is incubated for single celled survival rate and has reached 94.8%.
The detection of the unicellular secretory protein of embodiment 5
Unicellular secretory protein after being incubated for is captured into glass plate, is dismantled from chip, and immediately with 3%
The PBS of BSA is closed 30 minutes, is then incubated for 45 minutes with the detection antibody of 4 kinds of secretory proteins with biotin group with it,
It detects antibody and dilutes 200 times；Then it is washed using the PBS of 1%BSA, is caught using the SA-APC for diluting 100 times with secretory protein
Glass plate is obtained to be incubated for 20 minutes；Finally gradient wash, drying, using sweeping are carried out using PBS, 50%PBS, ultrapure water, ultrapure water
It retouches instrument to detect the fluorescence signal on unicellular secretory protein capture glass plate, analyzes the secretion of unicellular secretory protein
Situation, Figure 18 are the secretion thermal map of four kinds of secretory proteins of macrophage.
Claims (10)
1. efficiently unicellular capture and quick unicellular secretory protein detection platform, it is characterised in that: the detection platform packet
It includes cell capture and drop is incubated for micro-fluidic chip and secretory protein capture glass plate；Wherein, unicellular capture is incubated for drop
Micro-fluidic chip includes: that the isolation valve layer positioned at upper layer and the unicellular capture positioned at lower layer and drop are incubated for layer；
It includes multiple units that unicellular capture, which is incubated for layer with drop, and each unit includes being located at cell flow channel left and right ends
Straight cell flow channel (7) is connected using U-shaped or arc cell flow channel between two straight cell flow channels (7),
It further include cell capture cavity (6), the cell capture cavity (6) is connected with 3 channels, and first channel is that cell flowing is logical
Road, connects an arm end of U-shaped or arc cell flow channel, and the width in the channel is greater than cell dia to be captured；Article 2 is logical
Road is interface channel (8), and width is less than cell dia to be captured, for connecting cell capture cavity (6) and U-shaped or arc cell
Another arm end of flow channel；Article 3 channel is sense channel (9), and sense channel (9) leads to solution channel (10)；Solution is logical
There are a solution inlet (2) and a taphole (4) in road (10) both ends respectively；
Be isolated valve layer include the opposite isolation valve passage (16) in two flow directions, two isolation valve passage respectively and detect
Channel (9) intersects；Diaphragm (29) are set between isolation valve passage (16) and sense channel (9)；Cell solution entrance (1) and outlet
(3) at cell flow channel both ends, there are a solution inlet (2) and a taphole (4) in solution channel both ends respectively；Isolation
The entrance (5) in phase channel connects with cell flowing channel end；
Secretory protein capture glass plate includes: the glass plate (26) that surface modification has poly-D-lysine, and secretory protein captures antibody
Array stripe (20) is modified on glass plate (26)；Secretory protein capture antibody array band (20) is located in sense channel (9),
And between two crosspoints that two isolation valve passage (16) and sense channel are formed, secretory protein captures antibody array item
Band (20) is directly contacted towards sense channel (9) and with the solution in sense channel (9).
2. efficiently unicellular capture as described in claim 1 and quick unicellular secretory protein detection platform, it is characterised in that:
Cell flow channel, solution channel (10), isolation phase channel (5) width are 5-1000 microns, and depth is 5-1000 microns；Detection
Channel width is 5-1000 microns, and it is highly 5-200 microns that length, which is 5-1000 microns,.
3. efficiently unicellular capture as described in claim 1 and quick unicellular secretory protein detection platform, it is characterised in that:
One cell capture cavity (6) and a sense channel (9) form a hatch unit, and hatch unit adds one group of secretory protein
It captures antibody band (20) and forms a cell secretory protein detection unit；Secretory protein captures antibody band (20) and detection is logical
The interior solution contact in road (9).
4. efficiently unicellular capture as described in claim 1 and quick unicellular secretory protein detection platform, it is characterised in that:
It is 5-1000 microns that valve passage (16) width, which is isolated, is highly 5-1000 microns, and two channel isolation intervals (14) distance is 5-
1000 microns.
5. efficiently unicellular capture as described in claim 1 and quick unicellular secretory protein detection platform, it is characterised in that:
It further include chip fixture, the chip fixture includes fixture upper plate and fixture lower plate, isolation valve layer, unicellular capture and drop
It is incubated for layer and glass plate is located between fixture upper plate and fixture lower plate.
6. efficiently unicellular capture as claimed in claim 5 and quick unicellular secretory protein detection platform, it is characterised in that:
Fixture upper plate opening width is 0.1-2 centimetres, and length is 0.1-2 centimetres.
7. efficiently unicellular capture as claimed in claim 5 and quick unicellular secretory protein detection platform, it is characterised in that:
Fixture upper plate is equipped with screw permanent opening (30) and 7 chip channel openings, respectively two cell passage openings, two
Phase entrance opening is isolated with one in solution channel opening, two isolating valve access portals.
8. the application of efficient unicellular capture and quick unicellular secretory protein detection platform as described in claim 1-7, is answered
Detection and/or analysis for unicellular secretory protein/intracellular protein.
9. a kind of unicellular secretory protein detection method, includes the following steps:
1) according to the described in any item efficiently unicellular captures of claim 1 to 7 and quick unicellular secretory protein detection platform
Structure, prepare cell capture and drop and be incubated for micro-fluidic chip and secretory protein capture glass plate；
2) it is passed through capture antibody in the modification chip cavity for capturing glass plate thermal bonding with poly-D-lysine, carries out secretory protein
Capture the modification of antibody array；After modification is good, is closed, be washed out using buffer, secretory protein is finally captured into glass
Glass plate is spin-dried for；
3) be aligned unicellular capture with drop incubation micro-fluidic chip with unicellular secretory protein capture glass plate working region,
It posts and fixes；Chip is immersed in ultrapure water；It is captured to unicellular:
A, ultrapure water is first injected in isolation valve passage (16), so that its pressure is become larger, is caused diaphragm (29) downward deformation, will test
Channel (9) is separated with cell capture cavity (6) and solution channel (10)；
B, it is reversely passed through from isolation phase entrance (5) with solution channel outlet (4) and phase is isolated, generated in cell capture cavity (6)
Single celled drop is had one by one；
C, air pump switch should be closed after droplet array generation, stands, then extracts tracheae, by the water of isolating valve feeder connection
Pipe is extracted, and release isolation valve passage (16) pressure, diaphragm (29) pattern restores, there is no isolation effect, make sense channel (9) with
Cell capture cavity (6) is connected to again, forms unicellular hatch unit, is closed by the entrance of chip using adhesive tape；
4) platform is put into cell incubator, carries out the incubation of cell drop, it is unicellular in thin during entire be incubated for
Born of the same parents capture in cavity, and secretory protein is transferred to cell detection channel by diffusion and is caught by secretory protein capture antibody array
It obtains；Unicellular secretory protein capture glass plate after being incubated for is dismantled from chip, and detects slender intracrine egg
Signal on white capture glass plate analyzes the secretion situation of unicellular secretory protein.
10. a kind of unicellular secretory protein detection method as claimed in claim 9, it is characterised in that: step (3) is to unicellular
It is captured, cell concentration is controlled in 5*105-5*107/ mL, cell phase and molten liquid phase flow rate are 0.005-0.05mL/h, reversely
Isolation phase phase flow velocity is 0.5-2.0mL/h when being passed through isolation phase, should close air pump switch after droplet array generation, stand 1-
Then 10min extracts tracheae, the entrance of chip is closed using adhesive tape.
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