CN109596838A - The unicellular interior multiple protein simultaneous quantitative detection system of one kind and method - Google Patents
The unicellular interior multiple protein simultaneous quantitative detection system of one kind and method Download PDFInfo
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- CN109596838A CN109596838A CN201811547637.7A CN201811547637A CN109596838A CN 109596838 A CN109596838 A CN 109596838A CN 201811547637 A CN201811547637 A CN 201811547637A CN 109596838 A CN109596838 A CN 109596838A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
Abstract
The present invention provides a kind of unicellular interior multiple protein simultaneous quantitative detection systems, comprising: micro flow control chip device, including substrate and microchannel, microchannel and substrate form pressure channel, wherein substrate is equipped with the light penetrating slit of light screening material composition；Fluorescent collecting device, including fluorescence microscope, Manifold Light Way detector and data collecting card, wherein, fluorescence microscope is equipped with light source, so that intracellular albumen generates transmitting light after receiving laser, Manifold Light Way detector receives transmitting light, is divided into multiple wave bands for light is emitted according to the wavelength of transmitting light, and multiple wave bands are respectively converted into corresponding multiple electric signal pulses, data collecting card is used to carry out digitized processing to multiple electric signal pulses to obtain multiple digitized pulse signals；Signal acquisition and data processing equipment receive the concentration and quantity for storing multiple digitized pulse signals and calculating multiple albumen.On the other hand the production method of a kind of quantitative detecting method and micro flow control chip device is additionally provided.
Description
Technical field
The present invention relates to detection technique field, more particularly to a kind of unicellular interior multiple protein simultaneous quantitative detection system and
Method.
Background technique
Protein is present in all living things body, is the fundamental of all vital movements, dominates cell and executes physiology
Function participates in the links in cell life, has profound contact with cell function state.Protein amounts are anti-
One of the main indicator of vital movement variation is reflected, the quantification of protein testing result of individual cell level can disclose cell colony
In between single or rare cell protein amounts difference, and then subtly reflect the generating process of life event, thus
Cancer diagnosis and treatment, infection and the fields such as immune, nerve conduction are of great significance.
The technologies such as traditional cell protein quantitative detecting method such as gel electrophoresis, immunoassay, mass spectrum, are directed to mostly
Group's cell collection data, testing result reflect albumen Average expression level.However, even inhereditary material is identical in life entity
Cell between all have differences (i.e. heterogeneous), the result of study in cell colony level may ignore this species diversity,
Cause certain important molecular mechanisms to be hidden, it means that the data that individual cell level measures more can be accurately anti-
The truth of life event is reflected, single cell protein quantitative measurement technology understands that vital movement process has important meaning for deep
Justice, and characterization more can have comprehensive understanding to unicellular while to multiple protein quantitative detection, therefore develop a kind of based on micro-
The unicellular interior multiple protein simultaneous quantitative detection system of fluidics, there is great scientific research and application potential.
For at present, single cell protein detection mode can be divided into conventional method and the new method based on microflow control technique.
Wherein, (one) traditional technology includes fluorescence flow cytometry art, mass spectrum flow cytometry, capillary electrophoresis method, enzyme
Join immunodotting detection method.
Fluorescence flow cytometry art (Florescence Flow Cytometry, FFC) is current most important single cell protein
White detection method, it carries out fast quantitative analysis and sorting to cell of defiled in liquid stream etc. based on fluorescent marker one by one.
However, when fluorescence flow cytometry art is to unicellular internal Protein Detection, brightness scale timing need to use can equivalent inside repair
The standard microballoon for being decorated with fluorescent molecule compares fluorescent brightness therewith, but this quantitative modification technique is current and immature, therefore
Flow cytometry is not used to the absolute quantitation detection of slender intracellular protein.
Mass spectrum flow cytometry (Mass Cytometry) is the combination of Flow Cytometry and mass-spectrometric technique, using a huge sum of money
Belong to specific protein in rubidium marking cell, is detected through mass spectrograph and the data that atomic mass is composed are converted into individual cells testing protein
Relative amount.But this method has the shortcomings that cell apparatus is structurally and operationally complicated, expensive, being unfavorable for promoting makes
With.
Capillary Electrophoresis (Capillary Electrophoresis) is using high-voltage dc (0~30kV) to hair
The technology that tubule (diameter is less than 100 μm) interior sample is separated and detected.Capillary Electrophoresis has using capillary and electrophoretic techniques
Manipulation of single cells and Protein Separation ability have the characteristics that highly sensitive, ultralow sample volume, multicomponent analysis.But it is limited
In input mode, Capillary Electrophoresis is difficult to realize to a large amount of single celled quick detections.
Elisa detection (Enzyme-linked Immunospot Assay, ELISpot) is that a kind of detection is single
The technology of cell exocrine albumen.The porous plate culture cell of specific antibody is modified with using bottom surface and applies particular stimulation,
Spotting out, the number of spot can be presented using chromogenic reaction by antibody capture in the exocrine protein of cell at cell position
Mesh reflects the cell number for secreting the albumen, thus can assess corresponding unicellular ratio.Detection sensitivity is high, but
This method cannot further analyze the protein secretion in individual cells.
It (two) include piece up flow type method, microwell array method, micro channel array method and single cell protein based on microflow control technique
Blotting.
Piece up flow type method is to realize conventional flow cytometer on chip using microflow control technique and divide single cell protein
A kind of method of analysis.Compared with traditional flow cytometry, the advantage of piece up flow type method is (hundreds of to thousands of to a small amount of sample
It is a) detection of such as rare cell, to number of cells demand from 104It is reduced to 102It is a.But with streaming cell art problem
Similar, miniature flow cytometry is equally because scaling method limitation can not absolute quantitation detection intracellular protein.
Microwell array method be it is a kind of using on piece micropore passively capture it is unicellular after the technology cultivating and detect.Its principle is
The microwell array more bigger than unicellular size is produced using micro-nano technology technology, then drips the single-cell suspension of upper low concentration
Liquid, will be fallen into micropore unicellular with certain probability, then cover glass plate and culture that upper surface is modified with specific antibody, carefully
The specific secretory protein of born of the same parents is by antibody capture, to realize that single cell protein detects.This method can one-time detection multiple protein,
And it can be to cell recycling.But it can only detect secretory protein, while be limited to chip size and unicellular formation rate, primary real
The data that can only be obtained lower than 3000~10000 cells are tested, therefore it is lower to detect flux.
Micro- channel array method, also referred to as unicellular bar code chip.Spatially position difference is repaired for microchannel bottom on chip
It is decorated with respective capture antibody, formation " encodes " band one by one and passes through after cell suspending liquid injects sense channel from chip one end
On piece integrated micro-valve forms cellular compartments into thousands of chambers unicellular, and this method not only can detecte cell exocrine egg
It is white, lysate can also be injected from other channels, lytic cell extracts unicellular interior target protein, carries out quantitative analysis.The party
Method can detect unicellular specific protein with absolute quantitation, but due to being integrated with various structures on chip, it is complicated for operation, also limit
Detection flux.
Single cell protein blotting, by microflow control technique, by widely used method of protein detection gel electrophoresis
(Western Blot) has accomplished unicellular level of resolution.First with soft lithography in light-sensitive emulsion (Polyacrylamide
Gel micropore is processed on), using weak solution obtain it is unicellular after, the lysisin situ in hole, then directly carry out on piece gel electrophoresis,
By the Protein Separation of different molecular weight, 12 kinds of albumen can be analyzed simultaneously.But this method is sentenced by comparing shade
The relative amount of disconnected testing protein, can not obtain the absolute quantitation result of albumen.
Summary of the invention
(1) technical problems to be solved
It, can be with by this system the present invention provides a kind of unicellular interior multiple protein simultaneous quantitative detection system and method
The parameters such as concentration, the quantity of quantitative detection intracellular protein.
(2) technical solution
One aspect of the present invention provides a kind of unicellular interior multiple protein simultaneous quantitative detection system, comprising: micro-fluidic core
Sheet devices, including substrate and microchannel, microchannel and substrate form pressure channel, wherein pressure channel sectional area is less than slender
The sectional area of born of the same parents, so that cell is filled up completely pressure channel when passing through pressure channel, substrate is equipped with the saturating of light screening material composition
Light slit；Fluorescent collecting device, including fluorescence microscope, Manifold Light Way detector and data collecting card, wherein fluorescence microscopy
Mirror is equipped with light source, for observing cell by light penetrating slit and emitting laser to cell, so that after intracellular albumen receives laser
Transmitting light is generated, Manifold Light Way detector receives transmitting light, multiple wave bands are divided into for light is emitted according to the wavelength of transmitting light, and
Multiple wave bands are respectively converted into corresponding multiple electric signal pulses, wherein each wave band corresponds to a kind of albumen, data collecting card
Multiple digitized pulse signals are obtained for carrying out digitized processing to multiple electric signal pulses；Signal acquisition and data processing fill
It sets, including signal acquisition module and data processing module, wherein signal acquisition module, for receiving and storing multiple digitlizations
Pulse signal, data processing module, for determining the concentration and quantity of multiple albumen according to multiple digitized pulse signals.
Optionally, it is preset with protein concentration in data processing module and emits the relationship song of light digitization pulse signal strength
Line, comprising:
Y=kx+b
Wherein, x is protein concentration, and y is transmitting light digitization pulse signal strength, known to k and b, wherein different eggs
The different k and b value of white correspondence, brings relation curve by the digitized pulse signal strength that will test and obtains corresponding albumen
Concentration C.
Optionally, the quantity N calculation formula of albumen are as follows:
Wherein, C is protein concentration, and D is the diameter of albumen；
The calculation formula of albumen diameter D are as follows:
Wherein, a is the length and width values of pressure channel, and W is the width of light penetrating slit, trFor the upper of digitized pulse signal
The phase of liter corresponding time span, tsFor the stationary phase corresponding time span of digitized pulse signal, t is digitized pulse signal
Decline phase corresponding time span.
Optionally, further include pressure control device, for for cell in pressure channel by providing driving pressure.
Optionally, the material of microchannel includes dimethyl silicone polymer (polydimethylsiloxane, PDMS).
Optionally, Manifold Light Way detector includes multiple photomultiplier tubes, each photoelectricity times in multiple photomultiplier tubes
Increase pipe for detecting a kind of transmitting light of wave band.
Another aspect of the present invention provides a kind of unicellular interior multiple protein simultaneous quantitative determination, comprising: S1 is used
Panimmunity fluorescent reagent is by the protein staining in cell, cellulation suspension；It is logical to be imported compression by S2 for the cell suspension
Road flows the cell in cell suspension in the pressure channel；S3 emits laser to cell, so that intracellular albumen
Generate transmitting light；S4, acquisition transmitting light, and transmitting light is divided into multiple wave bands, each wave band corresponds to a kind of albumen, by multiple waves
Section is converted into multiple electric signal pulses；S5 calculates the concentration and quantity of multiple protein according to multiple electric signal pulses.
Optionally, further include the relation curve of determining protein concentration and transmitting photosignal intensity, specifically include: will be known
The cell solution of multiple protein concentration imports pressure channel, and the corresponding electrical signal intensity of detection multiple protein concentration obtains albumen
The functional relation of concentration and transmitting photosignal intensity.
Optionally, step S5 specifically: bring the intensity of multiple electric signal pulses into functional relation, obtain multiple telecommunications
It feels the pulse and rushes the concentration of corresponding multiple albumen；The volume that cell is obtained according to the curve of electric signal pulse, according to volume and correspondence
Protein concentration obtain the quantity of the albumen.
Further aspect of the present invention provides a kind of production method of micro flow control chip device, micro-fluidic chip by microchannel and
Substrate composition, microchannel and substrate form pressure channel, and method includes: production microchannel mold；Casting mold, overmolded obtain just
Grade microchannel, punches to obtain microchannel to primary microchannel；The light screening material of preset thickness is set on transparent material, in shading
The light penetrating slit of predetermined width is opened up on material, light penetrating slit is penetrated through to transparent material；PDMS film is set, substrate is generated；It is bonded micro-
Channel and substrate.
(3) beneficial effect
The unicellular interior multiple protein simultaneous quantitative detection system of one kind and method provided through the invention, at least obtain as
It is lower the utility model has the advantages that
1. the present invention is by by microfluidic chip technology and fluorescence antibody technology of preparing, detection technique of fluorescence, signal acquisition
It is combined with data processing technique, realizes unicellular interior multiple proteins simultaneous quantitative detection, be cell biological multifrequency nature
Characterization provides reliable method and detection device simultaneously.
2. the present invention is realized while the transmitting light to different-waveband while being carried out by design multichannel photoelectricity acquisition device
Acquisition, and then realize the purpose of multiple protein simultaneous quantitative detection.
3. the material selection dimethyl silicone polymer of microchannel of the invention, substrate uses silica glass material, on substrate
Light screening material select crome metal, based on micro-nano processing method make micro flow control chip device, have can mass manufacture it is excellent
Gesture.
4. the equipment such as inverted fluorescence microscope used in the present invention, photomultiplier tube, pressure controller, convenient in routine
It realizes in laboratory.
5. sample consumption of the present invention saves sample, cost can be effectively reduced in a microlitre magnitude.
Detailed description of the invention
Fig. 1 diagrammatically illustrates the unicellular interior multiple protein simultaneous quantitative detection system signal in the embodiment of the present disclosure
Figure；
Fig. 2 diagrammatically illustrates micro- in the unicellular interior multiple protein simultaneous quantitative detection system in the embodiment of the present disclosure
The schematic diagram of fluidic chip device 100, wherein Fig. 2A is the top view of micro flow control chip device 100, and Fig. 2 B is micro-fluidic chip
The main view of device 100；
Fig. 3 diagrammatically illustrates the work of the unicellular interior multiple protein simultaneous quantitative detection system in the embodiment of the present disclosure
Flow chart；
Fig. 4 diagrammatically illustrates the unicellular interior multiple protein simultaneous quantitative detection system work in the embodiment of the present disclosure
General flow chart；
Fig. 5 diagrammatically illustrates fluorescence in the unicellular interior multiple protein simultaneous quantitative detection system in the embodiment of the present disclosure
200 work flow diagram of acquisition device；
Fig. 6 diagrammatically illustrates data in the unicellular interior multiple protein simultaneous quantitative detection system in the embodiment of the present disclosure
The work flow diagram of processing module；
Fig. 7 diagrammatically illustrates the unicellular interior multiple protein simultaneous quantitative determination step in the embodiment of the present disclosure
Figure；
Fig. 8 diagrammatically illustrates micro- in the unicellular interior multiple protein simultaneous quantitative detection system in the embodiment of the present disclosure
The production method block diagram of fluidic chip device 100；
Fig. 9 diagrammatically illustrates micro- in the unicellular interior multiple protein simultaneous quantitative detection system in the embodiment of the present disclosure
The schematic diagram of manufacturing method of fluidic chip device 100.
Specific embodiment
To make the objectives, technical solutions, and advantages of the present invention clearer, below in conjunction with specific embodiment, and reference
Attached drawing, the present invention is described in more detail.
The present invention provides a kind of unicellular interior multiple protein simultaneous quantitative detection systems, comprising: micro flow control chip device
100, including substrate 101 and microchannel 102, microchannel 102 and substrate 101 form pressure channel 103, wherein pressure channel 103
Sectional area is less than single celled sectional area, so that cell is by the way that only a cell passes through when pressure channel 103, on substrate 101
Light penetrating slit equipped with light screening material composition；Fluorescent collecting device 200, including fluorescence microscope 201, Manifold Light Way detector
202 and data collecting card 203, wherein fluorescence microscope 201 is equipped with light source, for by light penetrating slit observation cell and to thin
Born of the same parents emit laser, so that intracellular albumen generates transmitting light after receiving laser, Manifold Light Way detector 202 receives the hair
Light is penetrated, multiple wave bands are divided into for light is emitted according to the wavelength of transmitting light, and multiple wave bands are respectively converted into corresponding multiple electricity
Signal pulse, wherein each wave band corresponds to a kind of albumen, and data collecting card 203 is used to carry out the multiple electric signal pulse
Digitized processing obtains multiple digitized pulse signals；Signal acquisition and data processing equipment 300, including signal acquisition module and
Data processing module, wherein signal acquisition module, for receiving and storing multiple digitized pulse signals；Data processing module
For determining the concentration and quantity of multiple albumen, pressure control device 400, for being cell according to multiple digitized pulse signals
By providing driving pressure in the pressure channel, it is specifically described as follows.
Specifically, Fig. 1 diagrammatically illustrates the unicellular interior multiple protein simultaneous quantitative detection system of the embodiment of the present disclosure
Schematic diagram, as shown in Figure 1, the device mainly includes micro flow control chip device 100, fluorescent collecting device 200, signal acquisition and
Data processing equipment 300 and pressure control device 400.
Wherein, micro flow control chip device 100 mainly include substrate 101 and microchannel 102, microchannel 102 with 101 groups of substrate
At pressure channel 103,103 sectional area of pressure channel is less than single celled sectional area, wherein microchannel 102, after it can be poured
Molding material is made, preferably dimethyl silicone polymer, but is not limited to dimethyl silicone polymer, is the flowing and compression of cell
Micro- channel environment is provided, so that cell is compressed when leading to 103 by the compression and guarantees that an only cell passes through；Substrate 101
It is equipped with the light penetrating slit of light screening material composition, the transparent part in substrate 101 is made of clear material, preferably quartz or glass
Glass, but it is not limited to quartz or glass, light screening material is opaque, and for limiting detection zone range, quantization transmitting light emitting receives
Area, material is preferably chromium, but is not limited to chromium, which constitutes detection window 1011, through the detection window 1011
The detection to cell can be achieved in addition, as shown in Figure 2, wherein 2A is the top view of the micro flow control chip device 100, and 2B is should
The main view of micro flow control chip device 100, the micro flow control chip device 100 further include injection hole 104, introduction channel 105, waste liquid
Channel 106 and negative pressure hole 107, wherein injection hole 104 is for injecting cell suspending liquid, and introduction channel 105 is for hanging cell
Supernatant liquid introduces pressure channel 103, and waste fluid channel 106 by the cell that pressure channel 103 detected for that will export, introduction channel
105 height can be higher than pressure channel 103, so that cell suspending liquid flows into pressure channel 103 under gravity, more conducively carefully
The flowing of born of the same parents' suspension, negative pressure hole 107 are connect with pressure control device 400, for being introduction channel 105,106 and of waste fluid channel
Pressure channel provides subnormal ambient, so that cell suspending liquid is under the driving of negative pressure, successively along introduction channel 105, pressure channel
103 and waste fluid channel 106 flow.
Fluorescent collecting device 200, including fluorescence microscope 201, Manifold Light Way detector 202, data collecting card 203 with
And other optical modules etc., wherein light source is equipped in fluorescence microscope 201, which is used for through light penetrating slit namely detection window
Mouth 1011 observes cells and emits laser to cell, which can be mercury lamp, halogen lamp, laser and LED etc., so that carefully
Albumen intracellular generates transmitting light, the preferably several photomultiplier tubes of Manifold Light Way detector 202, for connecing after receiving laser
Light is penetrated in transmitting-receiving, is divided into multiple wave bands for light is emitted according to the wavelength of transmitting light, each photomultiplier tube can receive certain wave band
Emitting light, and multiple wave bands are respectively converted into corresponding multiple electric signal pulses, wherein each wave band corresponds to a kind of albumen,
Multiple electric signal pulse obtains the fluorescent brightness of the individual cells indicated with voltage value by processing of circuit, continuous unicellular
By the way that a series of electric signal pulse can be obtained；Electric signal pulse is carried out number by data collecting card 203 by electric signal pulse
Change handles to obtain multiple digitized pulse signals, and is conducted into signal acquisition and data processing equipment 300, other optical elements
Normal operation for 203 function of secondary fluorescence microscope 201, Manifold Light Way detector 202 and data collecting card.
Signal acquisition and data processing equipment 300 provide visual runnable interface for user comprising signal acquisition mould
Block and data processing module are connect with fluorescent collecting device 200, wherein acquisition module, for receiving, storing and visualize
The multiple digitized pulse signal of display, and the partial devices in quantitative detection system can be controlled；Data processing mould
Block, the interior digitized pulse signal stored of readable signal acquisition module are realized to the filtering of digitlization pulse signal waveform, are determined
The data analysis functions such as position, classification and calculating, can extract the information such as height, time of digitized pulse signal, and cell generates
The brightness of digitized pulse signal the concentration of corresponding different albumen, the digitlization that cell generates can be converted to by relation curve
The time of pulse signal can be converted to the diameter of corresponding cell by mathematical model, and be calculated according to cell equivalent sphere model
The volume of cell, different protein concentrations are multiplied from corresponding cell volume can be obtained the quantity of corresponding intracellular different albumen, lead to
It crosses calculation procedure preset in the data processing module and realizes above-mentioned function, which can believe according to multiple digitized pulses
Number determine the concentration and quantity of multiple albumen, which includes protein concentration and transmitting light digitization pulse signal strength
Relation curve, the corresponding functional expression of the relation curve are as follows:
Y=kx+b (1)
Wherein, x is protein concentration, and y is transmitting light digitization pulse signal strength, wherein different albumen is corresponding not
Same k and b value, brings relation curve by the digitized pulse signal strength that will test and obtains corresponding protein concentration C, has
K the and b value of body determines method are as follows: marks the antibody concentration x gradient having to dilute for same, different antibody concentrations
Corresponding different transmitting brightness, gradually injects pressure channel 103, detects corresponding fluorescent brightness value y, dense by obtaining antibody
Relation curve known to degree and the fitting of multiple points of fluorescent brightness you can get it k and b value, can obtain different eggs in the above manner
The relation curve of white protein concentration and transmitting light digitization pulse signal strength, it is preset in data processing module.
When cell is filled up completely and when uniformly through pressure channel 103, the quantity N calculation formula of albumen are as follows:
Wherein, C is protein concentration, and D is the diameter of albumen, and protein concentration can be calculated by above formula (1)；
The calculation formula of albumen diameter D are as follows:
Wherein, a is the length and width values of pressure channel 103, and W is the width of light penetrating slit, trFor digitized pulse signal
Rising stage corresponding time span, tsFor the stationary phase corresponding time span of digitized pulse signal, t is digitized pulse
The decline phase corresponding time span of signal；
The parameters such as concentration, the quantity of albumen can be calculated by above-mentioned formula.
Pressure control device 400, including pressure controller, negative pressure pump and containment duct, with micro flow control chip device 100
Negative pressure hole 107 connect, for for cell in the channels such as pressure channel by providing driving pressure.
From the foregoing, it will be observed that as shown in figure 3, the workflow of the unicellular interior multiple protein simultaneous quantitative detection system are as follows: adopt
Intracellular multiple protein cellulation suspension is marked with specific antibody；Cell suspending liquid is injected into micro flow control chip device
In 100 injection hole 104, the negative pressure hole 107 of micro flow control chip device 100 and the pressure controller of pressure control device 400 are logical
Cross containment duct connection so that individual cells enter pressure channel 103 driven by pressure, by conduit wall extruding and become
Shape is filled up completely microchannel, and cell is irradiated when flowing through detection window 1011 by the excitation light source of fluorescent collecting device 200, carefully
Different protein emissions intracellular go out the transmitting light of different-waveband, the Manifold Light Way detector being connected on inverted microscope
The a variety of transmitting optical signals being collected into can be converted to multi-channel electric signal pulse by 202, be obtained by processing of circuit with voltage value table
The fluorescent brightness of the individual cells shown, it is continuous unicellular by the way that a series of pulse signal can be obtained；Electric signal pulse passes through
Analog signal digital is imported signal acquisition and data processing equipment 300 and stored, data processing module by data collecting card 203
Openable storing data, by signal waveform filtering, positioning, classification and calculate etc. data analysis it is extractable go out pulse height,
The information such as time, the brightness for the transmitting light pulse that cell generates can be converted to the concentration of corresponding different albumen by calibration curve,
The time for the transmitting light pulse that cell generates can be converted to the diameter of corresponding cell by mathematical model, and according to cell equivalent sphere
Body Model calculates the volume of cell, and corresponding intracellular different eggs can be obtained from corresponding cell volume multiplication in different protein concentrations
White quantity.
Specifically, as shown in figure 4, the workflow that simultaneous quantitative detects unicellular interior multiple protein specifically includes that cell
Preparation, chip prepare, system debug and transmitting light detection, the present embodiment will be carried out specifically for detecting three kinds of albumen
It is bright.
Step 1, prepared by cell.
The cell with fluorescent marker is prepared, cell is made to can produce transmitting light, fluorescent marker master when by laser excitation
It to include fixation, permeable membrane, closing and dyeing, specifically, firstly, paraformaldehyde solution is added in cell suspending liquid, formation
2% fixer is incubated for 15min on 4 DEG C of shaking tables；Then, use Triton X-100 solution as permeable membrane liquid, in 4 DEG C of items
It is incubated for 15min under part, for different cell line, optimal membrane concentration is different；Confining liquid has used 5% BSA, in room temperature
Lower incubation 30min, making into the cell can be capped with the non-specific sites in conjunction with antibody；It is diluted finally, cell is suspended in
The β-of 100 times of the β-Actin antibody for combining FITC coupling, the α-Tubulin antibody of PE coupling and PerCP coupling
(different Staining Protocols can be selected according to detection multiple protein, the present embodiment is with above-mentioned three kinds of Protein Detections in Tubulin antibody
For), make its concentration 1 × 106A/ml is incubated for 4 hours under 37 DEG C of environment, prepares detection brightness after taking out cleaning.
Step 2, chip prepares.
Chip preparation is mainly first just setting microscopically observation to pressure channel 103, it is ensured that pressure channel 103 makes just
Often, and guarantee that foreign blocks in pressure channel 103；Not clean enough quartz glass bottom surface is cleaned again, avoids causing
The variation of window baseline；Then it is used under inverted microscope to bleaching is carried out at 103 detection window 1011 of pressure channel
30mW laser source is directed at 488nm blue light illumination at detection window, at least progress 30min bleaching, can reduce baseline signal value
To stable state.
Step 3, system debug.
When system debug, three PMT are placed into 30min in dark situation, dark noise is reduced, carries out electromagnetism and light screen
It covers, after light source is switched on brightness stability, keeps no sample first on quantitative detection platform, whether detect environment and system noise
It is stable and minimum, it then places micro flow control chip device 100, is directed at detection window 1011, the fluorescence baseline of detection chip, really
It protects at detection chip window and bleaches in place.
Step 4, emit light detection.
Firstly, exhaust bubble, is connected to pressure control by containment duct for the negative pressure hole 107 on micro flow control chip device 100
On device processed, pressure controller output positive pressure or negative pressure, push the liquid in microchannel to flow under software control, and injection hole
104 solution injection is then by syringe or liquid-transfering gun direct injection, about 10~30 μ l of each liquor capacity；Then, make
Two injection holes 104 are filled with buffer, gradually apply the pressure of -1~-12kPa in outlet, using solution by air in channel
It excludes；Then cell is infused, takes the solution of two injection holes 104 away, cell suspending liquid is injected in a hole wherein, greatly containing about cell
1×104It is a, then for the cell line of different-diameter, apply different pressure, pressure range is between -5~-30kPa.
Then acquisition reflection signal, cell can be in three optical path detectors when passing through the detection window 1011 of pressure channel
202 generate a transmitting optical signal pulses simultaneously, wherein the fluorescein of three kinds of albumen couplings generates under 488nm exciting light
The transmitting light of different wave length, the corresponding transmitting light of β-Actin albumen is split, and mouth 534/30, which receives, α-Tubulin albumen is corresponding sends out
It penetrates light to be split the reception of mouth 572/25, the corresponding transmitting light of β-Tubulin albumen is split the reception of mouth 692/40, emits optical signal arteries and veins
It rushes after the amplifier amplification inside photomultiplier tube (PMT) 202, is input to data collecting card 203 with the sample rate number of 80kHz
Word is transferred to signal acquisition and data processing equipment 300.
Wherein, 200 workflow of fluorescent collecting device is as shown in Figure 5.Firstly, initialization photomultiplier tube (PMT) 202,
203 control port of data collecting card (DAQ) and pressure controller interface establish communication connection, and the increasing of photomultiplier tube 202 is arranged
Benefit control voltage, the sample rate of data collecting card 203, buffer size, file storing control parameter etc., then start fluorescence inspection
Ranging sequence, sampled data are continuously transmitted into signal acquisition and data processing equipment 300 after being digitized from data collecting card 203
Signal acquisition module, and draw out real-time waveform, while calculating the statistical information of signal, and simply analyzed.Online
Control platform can also obtain the working condition of PMT and DAQ, and noting abnormalities or testing terminates, then discharge communication port resource, terminate
Program.
Data processing module is as shown in fig. 6, share 4 function divisions, and respectively calibration curve, signal filtering, waveform are fixed
Position and data calculate, using MATLAB software realization, firstly, the calibration curve between specific antibodies concentration and fluorescence intensity is added
It is downloaded to calibration curve subregion on device；Then, the signal filtering point of device will be loaded into after the processing of original transmitted optical signal filtering
Area, and with the impulse waveform in time Series Processing signal；Then, single pulse waveform is positioned and divides trapezoidal temporal
Domain rises domain, stable region and decline domain views in waveform positioning subregion according to trapezoidal be divided into；Finally, according to calculation procedure
Cell dia, protein concentration, unicellular interior protein quantity are shown and calculate subregion in data.
On the other hand, the embodiment of the present disclosure additionally provides a kind of unicellular interior multiple protein simultaneous quantitative determination, ginseng
See Fig. 7, this method specifically includes that S1, using panimmunity fluorescent reagent by the protein staining in cell, cellulation suspension；
Cell suspension is imported pressure channel by S2, so that the cell in cell suspension flows in pressure channel；S3 emits to cell
Laser, so that intracellular albumen generates transmitting light；S4, acquisition transmitting light, and transmitting light is divided into multiple wave bands, each wave band
A kind of corresponding albumen, converts multiple electric signal pulses for multiple wave bands；S5 calculates multiple protein according to multiple electric signal pulses
Concentration and quantity.
Specifically, S1, using panimmunity fluorescent reagent by the protein staining in cell, cellulation suspension；
Intracellular testing protein is dyed using a variety of fluorescent reagents, so that albumen can inspire under the irradiation of laser
The transmitting light of different-waveband, cellulation suspension.
Cell suspension is imported pressure channel by S2, flows the cell in cell suspension in pressure channel；
Cell suspension is imported in pressure channel, so that cell is filled up completely pressure channel, and make the cell in cell suspension
It is flowed in pressure channel.It can control device flowing velocity.
S3 emits laser to cell, so that intracellular albumen generates transmitting light；
Emit laser to cell, intracellular multiple protein generates the transmitting light of different wave length under the excitation of laser.
S4, acquisition transmitting light, and transmitting light is divided into multiple wave bands, each wave band corresponds to a kind of albumen, by multiple wave bands
It is converted into multiple electric signal pulses；
Acquisition transmitting light, and fluorescence compensation is carried out to a variety of transmitting light, exclude spectral cross or the overlapping of different fluoresceins
Caused by influence, and laser is divided by multiple wave bands according to wavelength, and different light is sent into the transmitting light pulse of multiple wave bands
Signal amplification is carried out in the equipment such as electric multiplier tube and is converted into multiple electric signal pulses, and multiple electric signal pulses are subjected to number
Change conversion, can be identified by calculation procedure.
S5 calculates the concentration and quantity of multiple protein according to multiple electric signal pulses.
Firstly the need of determining protein concentration and emitting the reference curve of photosignal intensity, protein concentration and transmitting light are determined
The functional relation of electrical signal intensity.
It brings the intensity of multiple electric signal pulses into functional relation, obtains the corresponding multiple albumen of multiple electric signal pulses
Concentration；The volume that cell is obtained according to the curve of electric signal pulse obtains the egg according to volume and corresponding protein concentration
White quantity.
In another aspect, the embodiment of the present disclosure additionally provides a kind of production method of micro flow control chip device 100, referring to Fig. 8
And Fig. 9, the micro-fluidic chip are made of microchannel 102 and substrate 101, the microchannel 102 is pressed with the substrate 101 composition
Contracting channel 103, which comprises
S801 makes microchannel mold；
Referring to Fig. 9 A, substrate and sputtering layers of chrome are prepared；
Common glass slide is immersed in Piranha Solution natural cooling after ebuillition of heated, is sputtered on substrate
Chromium, 0.1 μm of thickness, as alignment layer.
Referring to Fig. 9 B, positive photoresist spin coating and exposure；
The thickness of the positive photoresist obtained using two step lacquering techniques is about 2.0 μm, as alignment mark exposure mask.
Referring to Fig. 9 C, positive photoresist development；
Positive photoresist development can use 0.3%NaOH or AZ300MIF, and development terminates to toast again, to improve the corrosion resistance of photoresist
Energy.
Referring to Fig. 9 D, chromium corrodes and removes photoresist；
Wet etching is carried out using the strong acid chromium corrosive liquid based on ammonium ceric nitrate.
Referring to Fig. 9 E, seed layer production；
The negtive photoresist that uniform fold a layer thickness is 5 μm on substrate exposes entire substrate, so that flood negtive photoresist retains
As transition zone；Nonvisualization after drying afterwards, is directly warming up to 175 DEG C, seed layer is hardened.
Referring to Fig. 9 F, the spin coating of first layer negtive photoresist and exposure
The production of pressure channel layer: photoresist SU-85 is taken out from refrigerator, is placed in 20~22 DEG C of stabilization room temperature environment to temperature
Degree balance, spin coating, glue are 8.0 μm thick；It puts after substrate to room temperature, mask used in this layer is aligned with the label on substrate, expose
5.5 seconds, do not develop, can see the pattern on surface after cooling.
Referring to Fig. 9 G, the spin coating of second layer negtive photoresist and exposure
Flow channel layer production: using 40 μm of 25 whirl coating thickness of photoresist SU-8, and alignment exposes, and the time 6 seconds, length master
To be judged according to final developing result, if fruit structure has adhesion or distortion, then illustrate under-exposure, if structure lines are wide,
Then illustrate to expose too long, needs the corresponding adjustment time for exposure.
Referring to Fig. 9 H, development and post bake
It puts after substrate to room temperature, develops in SU-8 developer solution, during which tested whether using the mode of sprayed with isopropyl alcohol
Development is clean, if there is white residual then continues to develop；After measuring microchannel width, height, structural parameters, for meeting
It is required that mold can be heated to 175 DEG C on hot plate, toast 2 hours, pattern can be permanently hardened on substrate.
S802 is poured the mold, and overmolded obtains primary microchannel, punches to obtain microchannel 102 to primary microchannel；
It is poured referring to Fig. 9 I, PDMS
According to the mass ratio 15:1 of PDMS performed polymer and curing agent, prepares and be put into mold after being evacuated bubble.
Referring to Fig. 9 J, the molding of the microchannel PDMS
It is put into 80 DEG C of smooth baking ovens and toasts 4 hours, during which cannot move, to prevent uncured PDMS flowing, baking
End separates substrate with PDMS, then cuts the extra part of removal and the PDMS that there is microchannel on surface can be obtained.
Referring to Fig. 9 K, PDMS punching and cleaning
Using the solution injection hole and negative pressure hole of the ordinary straps punch aligned with core on piece of outer diameter 3mm, through entire
PDMS block.Due to the aperture that in the higher situation of the elasticity of PDMS, especially configuration proportion, actually generates 1mm to 1.5mm it
Between.It is impregnated within duration 2min in alcohol when PDMS is cleaned, is then placed on hot plate and dries 1 hour for 80 DEG C, only about shrink
0.5%.
The light screening material of preset thickness is arranged on transparent material, the saturating of predetermined width is opened up on light screening material by S803
Light slit, light penetrating slit are penetrated through to transparent material；
Referring to Fig. 9 L, using quartz substrate, identical as step 9A~9D, details are not described herein again, with pair on mold substrate
Unlike fiducial mark note, minimum thickness is designed as by light transmission experimental verification using shading film made of chromium etc. on substrate
0.15μm.Made chromium slit width degree is 2.5 μm, and control errors are within 10%.
PDMS film is arranged in S804, generates substrate 101；
Referring to Fig. 9 M, n-hexane (n- is added in the prepared PDMS of the mass ratio 15:1 of PDMS performed polymer and curing agent
Hexane the ratio of dilution in), PDMS and n-hexane is 1:16, and 5500rpm revolving speed is run on sol evenning machine after mixing
5min, obtained film thickness are 0.9 μm.After spin coating, substrate is placed on 80 DEG C of hot plates and dries 2h to remove n-hexane.
S805, bonding microchannel 102 and substrate 101.
Referring to Fig. 9 N, using plasma (PDC-32G-2, Harrick Plasma, the U.S.) oxidizing process, in substrate and
PDMS uses watering can to spray a little deionized water as lubricant after air plasma is handled, in substrate surface, while will not shadow
Bond strength is rung, since moisture film is isolated, the two can not be contacted directly, therefore can still be moved freely after PDMS fitting, at this time
It can be aligned under the microscope, place 1~2min and wait for that both water film evaporations are formed, be then carefully moved to smooth hot plate, 80 DEG C
30min is toasted, alignment precision is at 10 μm or so.
Above step S801~S802 makes for making microchannel, step S803~S804 for making substrate
Sequencing is not limited by the embodiment of the present invention, and all methods that can obtain above-mentioned micro flow control chip device are in the present invention
Protection scope in.
Particular embodiments described above has carried out further in detail the purpose of the present invention, technical scheme and beneficial effects
It describes in detail bright, it should be understood that the above is only a specific embodiment of the present invention, is not intended to restrict the invention, it is all
Within the spirit and principles in the present invention, any modification, equivalent substitution, improvement and etc. done should be included in guarantor of the invention
Within the scope of shield.
Claims (10)
1. a kind of unicellular interior multiple protein simultaneous quantitative detection system, comprising:
Micro flow control chip device, including substrate and microchannel, the microchannel and the substrate form pressure channel, wherein institute
Pressure channel sectional area is stated less than the single celled sectional area, so that the cell when pressure channel by being filled up completely
The pressure channel, the substrate are equipped with the light penetrating slit of light screening material composition；
Fluorescent collecting device, including fluorescence microscope, Manifold Light Way detector and data collecting card, wherein the fluorescence
Microscope is equipped with light source, for observing cell by the light penetrating slit and emitting laser to the cell, so that described intracellular
Albumen receive the laser after generate transmitting light, the Manifold Light Way detector receives the transmitting light, according to the hair
The transmitting light is divided into multiple wave bands by the wavelength for penetrating light, and the multiple wave band is respectively converted into corresponding multiple electric signals
Pulse, wherein each wave band corresponds to a kind of albumen, and the data collecting card is used to carry out number to the multiple electric signal pulse
Change handles to obtain multiple digitized pulse signals；
Signal acquisition and data processing equipment, including signal acquisition module and data processing module, wherein signal acquisition module,
For receiving and storing the multiple digitized pulse signal, data processing module, for according to the multiple digitized pulse
Signal determines the concentration and quantity of the multiple albumen.
2. multiple protein simultaneous quantitative detection system according to claim 1, which is characterized in that the data processing module
In be preset with protein concentration and emit light digitization pulse signal strength relation curve, comprising:
Y=kx+b
Wherein, x is protein concentration, and y is transmitting light digitization pulse signal strength, known to k and b, wherein different albumen pair
Answer different k and b values, by the digitized pulse signal strength that will test bring into the relation curve obtain it is corresponding
Protein concentration C.
3. multiple protein simultaneous quantitative detection system according to claim 1, which is characterized in that the quantity N of the albumen
Calculation formula are as follows:
Wherein, C is the protein concentration, and D is the diameter of the albumen；
The calculation formula of the albumen diameter are as follows:
Wherein, a is the length and width values of the pressure channel, and W is the width of the light penetrating slit, is believed for the digitized pulse
Number rising stage corresponding time span,sIt is the number for the stationary phase corresponding time span of the digitized pulse signal
The decline phase corresponding time span of word pulse signal.
4. multiple protein simultaneous quantitative detection system according to claim 1, further includes pressure control device, it is used for as institute
Cell is stated in the pressure channel by providing driving pressure.
5. multiple protein simultaneous quantitative detection system according to claim 1, the material of the microchannel includes poly- diformazan
Radical siloxane.
6. multiple protein simultaneous quantitative detection system according to claim 1, the Manifold Light Way detector includes more
A photomultiplier tube, each photomultiplier tube is used to detect a kind of transmitting light of wave band in the multiple photomultiplier tube.
7. a kind of unicellular interior multiple protein simultaneous quantitative determination, comprising:
S1, using panimmunity fluorescent reagent by the protein staining in cell, cellulation suspension；
The cell suspension is imported pressure channel, flows the cell in the cell suspension in the pressure channel by S2；
S3, Xiang Suoshu cell emit laser, so that the intracellular albumen generates transmitting light；
S4 acquires the transmitting light, and transmitting light is divided into multiple wave bands, and each wave band corresponds to a kind of albumen, will be the multiple
Wave band is converted into multiple electric signal pulses；
S5 calculates the concentration and quantity of the multiple protein according to the multiple electric signal pulse.
8. multiple protein simultaneous quantitative determination according to claim 7, which is characterized in that further include the determining egg
The relation curve of white concentration and transmitting photosignal intensity, specifically includes:
The cell solution of known multiple protein concentration is imported into pressure channel, detects the corresponding electric signal of the multiple protein concentration
Intensity obtains the protein concentration and emits the functional relation of photosignal intensity.
9. multiple protein simultaneous quantitative determination according to claim 8, which is characterized in that step S5 specifically:
It brings the intensity of the multiple electric signal pulse into the functional relation, show that the multiple electric signal pulse is corresponding
The concentration of multiple albumen；
The curve of pulse obtains the volume of the cell according to the electric signal, is obtained according to the volume and corresponding protein concentration
The quantity of the albumen out.
10. a kind of production method of micro flow control chip device, the micro-fluidic chip are made of microchannel and substrate, described micro- logical
Road and the substrate form pressure channel, which comprises
Make microchannel mold；
It is poured the mold, overmolded obtains primary microchannel, punches to obtain the microchannel to the primary microchannel；
The light screening material of preset thickness is set on transparent material, the light penetrating slit of predetermined width is opened up on the light screening material,
The light penetrating slit is penetrated through to the transparent material；
PDMS film is set, substrate is generated；
It is bonded the microchannel and the substrate.
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