CN108627448A - The method of counting micro particles - Google Patents
The method of counting micro particles Download PDFInfo
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- CN108627448A CN108627448A CN201810583361.1A CN201810583361A CN108627448A CN 108627448 A CN108627448 A CN 108627448A CN 201810583361 A CN201810583361 A CN 201810583361A CN 108627448 A CN108627448 A CN 108627448A
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- 239000011859 microparticle Substances 0.000 title claims abstract description 50
- 238000000034 method Methods 0.000 title claims abstract description 43
- 238000001514 detection method Methods 0.000 claims abstract description 106
- 239000012530 fluid Substances 0.000 claims abstract description 97
- 239000002245 particle Substances 0.000 claims abstract description 54
- 239000007788 liquid Substances 0.000 claims description 67
- 239000002699 waste material Substances 0.000 claims description 53
- 238000004140 cleaning Methods 0.000 claims description 19
- 238000005516 engineering process Methods 0.000 claims description 17
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 15
- 230000004899 motility Effects 0.000 claims description 14
- 238000012360 testing method Methods 0.000 claims description 6
- 230000033228 biological regulation Effects 0.000 claims description 5
- 230000005611 electricity Effects 0.000 claims description 4
- 244000005700 microbiome Species 0.000 claims description 4
- 239000000725 suspension Substances 0.000 abstract description 15
- 238000013461 design Methods 0.000 abstract description 14
- 230000003287 optical effect Effects 0.000 abstract description 12
- 238000004458 analytical method Methods 0.000 abstract description 4
- 230000002906 microbiologic effect Effects 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 38
- 239000000243 solution Substances 0.000 description 12
- 238000010586 diagram Methods 0.000 description 8
- 210000000170 cell membrane Anatomy 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000007689 inspection Methods 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 239000003792 electrolyte Substances 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000010191 image analysis Methods 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 241000208340 Araliaceae Species 0.000 description 1
- 241000195493 Cryptophyta Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 244000283207 Indigofera tinctoria Species 0.000 description 1
- 235000005035 Panax pseudoginseng ssp. pseudoginseng Nutrition 0.000 description 1
- 235000003140 Panax quinquefolius Nutrition 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 239000008151 electrolyte solution Substances 0.000 description 1
- 238000007667 floating Methods 0.000 description 1
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- 235000008434 ginseng Nutrition 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 238000003475 lamination Methods 0.000 description 1
- 239000006210 lotion Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/1031—Investigating individual particles by measuring electrical or magnetic effects
- G01N15/12—Investigating individual particles by measuring electrical or magnetic effects by observing changes in resistance or impedance across apertures when traversed by individual particles, e.g. by using the Coulter principle
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- Chemical & Material Sciences (AREA)
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- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
The present invention provides a kind of methods of counting micro particles, it is related to biological particle detection technique field, the method of counting micro particles provided by the invention, using the micro-fluidic chip detection technique based on Coulter principle, analysis cell and/or microbiological specimens flow through the electric impulse signal generated when detection zone on micro-fluidic chip, obtain the grain size and quantity of particle.The error that counting error and cell life or death caused by being layered due to particle when mainly being reduced the differential suspension problem of particulate samples by chip structure design (reducing fluid channel depth) using micro-fluidic chip, and effectively reducing Image Acquisition in conjunction with the depth of field of optical system are differentiated.The optical lens depth of field is more than chip depth, characteristics of image difference caused by avoiding differential suspension.In addition, chip design is but also the quantitative sample being added can all flow through detection zone, to realize full sample count.To sum up so that the result accuracy and consistency of the counting of cell or other biological particle are significantly better than existing detection method.
Description
Technical field
The present invention relates to biological particle detection technique fields, more particularly, to a kind of method of counting micro particles.
Background technology
In a large amount of cell biological research experiment, need (quantitative to cell or other biological particle progress Concentration Testing
Count), concentration of cell suspension (or quantity) is both the monitoring parameters of cell culture, and the necessary ginseng in many experimental projects
Number, the process condition for being very basic but critically important for successfully completing experiment.Existing counting means include mainly using
The artificial counting method of cell counting board, the automated enumeration instrument based on image analysis technology, and utilize electric-resistivity method (Ku Er
Special principle) automated enumeration instrument.
Wherein, artificial counting method is the most universal, and suspension cell sample is injected cell counting board counting chamber by experimenter,
Under the microscope to visually observe and carry out artificial counting by rule.The major defect of the method is：(1) due to counting chamber itself
Depth several times in cell dimensions, differential suspension wherein after cell sample injection is will result in this way, to what is observed
Cellular morphology can difference, cause inaccuracy and the cell activity misjudgment of count results；(2) rule injection cytometer is pressed
The sample of number plates is 10 μ L, but the sample size in microscope viewing area is only sub-fraction, thin in this way less than 1 μ L
Whether born of the same parents' sample is distributed in counting chamber will uniformly cause result prodigious influence；(3) it is according to certain rule when counting
Carry out artificial counting, the difference of operating personnel's level and fatigue strength caused by visually observing just introduce prodigious human error.
Though the self-reacting device based on image analysis technology avoids macroscopic difficulty, there are still it is following not
Foot：(1) use for introducing disposable count slice consumptive material, increases user's testing cost；(2) count slice in structure with cell
Tally is similar, so the problem of leading to result inaccuracy and activity erroneous judgement there is also cell differential suspension on tally；(3) same
Artificial counting is the same, and the instrument for being mostly based on image method has that the few caused result error of detection sample size is big.
The integrated level of traditional coulter counter device entirety is not high, for example wants independent dispensing computer, operates not enough
It is simple and direct.In addition it is exactly the function that traditional coulter counter does not have the judge of cell sample motility rate.
Therefore it provides a kind of microparticle technologies method that error is small, accuracy is high and testing cost is low is particularly important.
In view of this, special propose the present invention.
Invention content
The purpose of the present invention is to provide a kind of methods of counting micro particles, to alleviate detection sample existing in the prior art
Measure the technical problem of result inaccuracy caused by few and cell or other biological particle differential suspension.
The present invention provides a kind of method of counting micro particles, the method includes：
Particulate samples are made to pass through in detection zone using microfluidic chip technology, detection obtains the grain size sum number of the particle
Amount；
The detection zone utilizes Coulter principle, for acquire the particle by when the electric impulse signal that generates；
The particle includes cell and/or microorganism.
Further, the method further includes：
Particulate samples are made to pass through in image acquisition region using microfluidic chip technology, detection obtains the work of the particle
Rate；
Described image pickup area for particle described in continuous acquisition by when image, the particle is cell；
Preferably, particulate samples are made to pass through in image acquisition region using microfluidic chip technology, detection obtains described micro-
The motility rate of grain recycles microfluidic chip technology that particulate samples is made to pass through in detection zone later, and detection obtains the particle
Grain size and quantity.
Further, particle is counted using counting micro particles instrument, the counting micro particles instrument includes being equipped with detection zone
Micro-fluidic chip.
Further, the counting micro particles instrument includes control unit, detection module and processor, described control unit and inspection
Module is surveyed to be electrically connected with the processor respectively；
Described control unit includes the over-pressure control for generating positive pressure for controlling particulate samples disengaging counting micro particles instrument
Unit and vacuum cavitations unit for generating negative pressure；
The detection module includes the micro-fluidic chip equipped with detection zone, and feed liquor is offered on the micro-fluidic chip
Hole, well and waste liquid hole, the micro-fluidic chip are internally provided with fluid channel, the inlet opening, well and waste liquid hole
Between be connected to by the fluid channel；
The processor generates positive pressure for controlling the over-pressure control unit, and it is negative to control the vacuum cavitations unit generation
Pressure so that the particulate samples added from the well enter the fluid channel, and is discharged, the place by the waste liquid hole
The data information that reason device generates the detection module is handled；
The detection zone is set to inside the fluid channel, using being built in the electrode of the micro-fluidic chip to flowing through
The electric impulse signal of the particulate samples of detection zone is acquired；
Preferably, the detection module is detachable.
Further, the counting micro particles instrument further includes waste fluid container and sheath liquid container；
The sheath liquid container is connected with the over-pressure control unit, the waste fluid container and the vacuum cavitations unit phase
Even, the sheath liquid container and the waste fluid container are connected with detection module respectively；
The fluid channel by the inlet opening and the sheath fluid reservoir, the fluid channel by the waste liquid hole with
The waste fluid container connection；
Sheath fluid in the sheath liquid container enters the fluid channel by inlet opening, with the particle sample added from well
Product mix, and carry sample liquids and flow to waste fluid container by waste liquid hole.
Further, the counting micro particles instrument further includes soda liquor container, the over-pressure control unit and the cleaning solution
Container is connected, and the soda liquor container is connected to by the inlet opening with the fluid channel；
Cleaning solution in the soda liquor container enters the fluid channel by the inlet opening, and to the fluid channel
It is cleaned, waste liquid flows to the waste fluid container by the waste liquid hole after cleaning.
Further, the detection module further includes image capture module；
Described image acquisition module is used to acquire multiple images for the particulate samples for flowing through described image acquisition zone, the place
Reason device is for handling the data information of described image acquisition module.
Further, the method includes：
After particulate samples are added to well, by processor regulation unit, particulate samples are made to pass through micro-fluidic
The fluid channel of chip interior flows through image acquisition areas and/or detection zone, and acquired image and/or electricity are analyzed by processor
Pulse signal obtains the motility rate of the particulate samples and/or the grain size and quantity of the particulate samples.
Further, the method includes：
(a) by processor regulation unit, over-pressure control unit is made to generate positive pressure, vacuum cavitations unit generates negative
Pressure, the fluid channel of microcontroller chip is entered by pressure official post sheath fluid by inlet opening；
(b) particulate samples are added to well, sheath fluid carry the particulate samples pass through it is micro- inside micro-fluidic chip
Runner, which flows through, to be discharged from waste liquid hole after image acquisition areas and/or detection zone, and by processor analyze acquired image and/
Or electric impulse signal, obtain the motility rate of the particulate samples and/or the grain size and quantity of the particulate samples.
Further, the method further includes after particulate samples all flow through fluid channel by clear in soda liquor container
The step of washing lotion cleans the detection module.
The method of counting micro particles provided by the invention is divided using the micro-fluidic chip detection technique based on Coulter principle
Analysis cell and/or microbiological specimens flow through the electric impulse signal generated when detection zone on micro-fluidic chip, obtain the grain of particle
Diameter and quantity.It is mainly point that particulate samples are reduced by chip structure design (reducing fluid channel depth) using micro-fluidic chip
Layer suspension problem, and due to counting error caused by particle layering when effectively reducing Image Acquisition in conjunction with the depth of field of optical system
The error differentiated anyway with cell.Such as 60 μm of depths of chip, 80 μm of the optical lens depth of field, avoiding problems caused by differential suspension
Characteristics of image difference.In addition, chip design is but also the quantitative sample being added can all flow through detection zone, to realize bulk sample
Product count.To sum up so that the result accuracy and consistency of the counting of cell or other biological particle are significantly better than existing inspection
Survey method.
Description of the drawings
It, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution in the prior art
Embodiment or attached drawing needed to be used in the description of the prior art are briefly described, it should be apparent that, in being described below
Attached drawing is some embodiments of the present invention, for those of ordinary skill in the art, before not making the creative labor
It puts, other drawings may also be obtained based on these drawings.
Fig. 1 is a kind of connection block diagram of cell counter provided in an embodiment of the present invention；
Fig. 2 is a kind of position view of micro-fluidic chip provided in an embodiment of the present invention；
Fig. 3 is the position view of another micro-fluidic chip provided in an embodiment of the present invention；
Fig. 4 is a kind of structural schematic diagram of micro-fluidic chip provided in an embodiment of the present invention；
Fig. 5 is the structural schematic diagram of another micro-fluidic chip provided in an embodiment of the present invention；
Fig. 6 is the structural schematic diagram of counting micro particles instrument provided in an embodiment of the present invention；
Fig. 7 is the structural schematic diagram of Fig. 6 counting micro particles instrument different angles；
Fig. 8 is the structural schematic diagram of Fig. 6 counting micro particles instrument different angles；
Fig. 9 is the structural schematic diagram of Fig. 6 counting micro particles instrument different angles；
Figure 10 is the structural schematic diagram of detection module.
Icon：110- over-pressure control units；111- positive pressure pressures source；120- vacuum cavitations units；121- negative pressure pressure sources；
130- detection modules；131- micro-fluidic chips；132- inlet openings；133- wells；134- waste liquids hole；135- fluid channels；136-
Electrode；140- sheath liquid containers；150- waste fluid containers；160- soda liquor containers；170- processors；The first solenoid valves of 181-；182-
Second solenoid valve；183- third solenoid valves；The 4th solenoid valves of 184-；The 5th solenoid valves of 185-；The 6th solenoid valves of 186-；187-
Seven solenoid valves；The 8th solenoid valves of 188-；The 9th solenoid valves of 189-；190- fuselages；191- head covers；192- vacuum cups；193- locks
Button；194- display screens；195- power inlets；196- power switches；197-USB mouthfuls；1351- runner branches.
Specific implementation mode
Technical scheme of the present invention is clearly and completely described below in conjunction with embodiment, it is clear that described reality
It is a part of the embodiment of the present invention to apply example, instead of all the embodiments.Based on the embodiments of the present invention, the common skill in this field
The every other embodiment that art personnel are obtained without making creative work belongs to the model that the present invention protects
It encloses.
In the description of the present invention, it should be noted that such as occur term "center", "upper", "lower", "left", "right",
"vertical", "horizontal", "inner", "outside" etc., indicated by orientation or positional relationship be orientation based on ... shown in the drawings or position
Relationship is merely for convenience of description of the present invention and simplification of the description, and not indicating or implying the indicated device or element must have
There is specific orientation, with specific azimuth configuration and operation, therefore is not considered as limiting the invention.In addition, as occurred
Term " first ", " second ", " third " are used for description purposes only, and are not understood to indicate or imply relative importance.
In the description of the present invention, it should be noted that unless otherwise clearly defined and limited, term " peace such as occur
Dress ", " connected ", " connection " shall be understood in a broad sense, for example, it may be being fixedly connected, may be a detachable connection, or integrally
Connection；It can be mechanical connection, can also be electrical connection；Can be directly connected, can also indirectly connected through an intermediary,
It can be the connection inside two elements.For the ordinary skill in the art, it can understand above-mentioned art with concrete condition
The concrete meaning of language in the present invention.
According to an aspect of the invention, there is provided a kind of method of counting micro particles, including：
Particulate samples are made to pass through in detection zone using microfluidic chip technology, detection obtains the grain size and quantity of particle.
The detection zone utilizes Coulter principle, for acquire particle by when the electric impulse signal that generates.
Particle includes cell and/or microorganism.Wherein, microorganism for example can be, but be not limited to virus, bacterium, fungi
And primary algae.
Detection zone in the present invention by acquire particle by when the electric impulse signal that generates, Kurt can be utilized former
Reason (when the particle to suspend in the electrolytic solution passes through small bore tube with electrolyte, replaces the electrolyte of same volume, is designed in constant current
Circuit in cause inside and outside small bore tube two resistance between electrode that transient change occurs, generate potential pulse.The size of pulse signal and
Number is directly proportional to the size and number of particle) it is used for the counting of cell or other biological particle.It is main using micro-fluidic chip
It is the differential suspension problem of particulate samples to be reduced by chip structure design (reducing fluid channel depth), and combine optical system
The error that counting error and cell caused by the depth of field is layered when effectively reducing Image Acquisition due to particle differentiate anyway.Such as core
60 μm of depths of piece, 80 μm of the optical lens depth of field, avoiding problems characteristics of image differences caused by differential suspension.In addition, chip designs
But also the quantitative sample being added can all flow through detection zone, to realize full sample count.To sum up so that cell or other
The result accuracy and consistency of the counting of biological particle are significantly better than existing detection method.Wherein, to being used to acquire particle
By when the electric impulse signal detection zone that generates do not do particular determination, can be used the existing electricity using Coulter principle
Pulse signal detection means can be realized.
As shown in Figures 2 and 3, in one preferred embodiment, the above method further includes：
Particulate samples are made to pass through in image acquisition region using microfluidic chip technology, detection obtains the motility rate of particle；
Above-mentioned image acquisition region for continuous acquisition particle by when image, wherein particle be cell.
Particulate samples are enabled to be continued to flow through in image acquisition region by the control of microfluid on chip, when particle sample
Product flow through during image acquisition region the image acquisition region can automatic collection multiple particle images, which adds total
Particle image pattern amount, and smaller chip runner depth minus has lacked the lamination of particulate samples, substantially reduces existing
Result inaccuracy problem caused by particle differential suspension in picture count method.It, can be accurate by the analysis to above-mentioned particle image
Detect the motility rate information of particulate samples.
Specifically, trypan blue is that most common life or death cell judges that reagent, normal living cells after birth structural integrity, platform are expected
Indigo plant cannot be introduced into cell membrane；And loss of activity or the incomplete cell of cell membrane, the permeability of after birth increase, trypan blue can be into
Enter makes cell color into the cell.It has been generally acknowledged that cell membrane integrity is lost, you can think that cell is dead.Uncoloured work is thin
Born of the same parents and colored dead cell graphic feature have significant difference, as shown, living cells will present bright state after dyeing, and it is dead
Opaque state is presented in cell, is identified by micro- sem observation or image processing software judges, to which tested cell be calculated
The motility rate of sample.
Preferably, particulate samples are made to pass through in image acquisition region using microfluidic chip technology, detection obtains particle
Motility rate recycles microfluidic chip technology that particulate samples is made to pass through in detection zone later, and detection obtains the grain size sum number of particle
Amount.
In one preferred embodiment, particle is counted using counting micro particles instrument, which includes
Micro-fluidic chip equipped with detection zone.
Micro-fluidic chip with detection zone is combined with counting micro particles instrument, enables to entire liquid fluid system more
It is compact and effective, and the absolute counting of full sample size is realized, so that the result accuracy and consistency of counting micro particles
Better than existing apparatus, the integrated level for improving prior art Instrumental entirety is not high, and detects sample size and hanged less with particle layering
Result inaccuracy problem caused by floating.
As shown in Figure 1, in one preferred embodiment, counting micro particles instrument includes control unit, 130 and of detection module
Processor 170, control unit and detection module 130 are electrically connected with the processor 170 respectively；
Control unit includes the over-pressure control unit for generating positive pressure for controlling particulate samples disengaging counting micro particles instrument
110 and the vacuum cavitations unit 120 for generating negative pressure；
Detection module 130 includes the micro-fluidic chip 131 equipped with detection zone, and feed liquor is offered on micro-fluidic chip 131
Hole 132, well 133 and waste liquid hole 134, micro-fluidic chip 131 are internally provided with fluid channel 135, inlet opening 132, sample-adding
It is connected to by fluid channel 135 between hole 133 and waste liquid hole 134；
Processor 170 generates positive pressure for controlling over-pressure control unit 110, and control vacuum cavitations unit 120 generates negative pressure,
So that the particulate samples added from well 133 enter fluid channel 135, and it is discharged by waste liquid hole 134, processor 170 is right
The data information that detection module 130 generates is handled；
In one preferred embodiment, processor 170 further includes the controller of electromechanical component, electric impulse signal and figure
As the acquisition process memory and screen display of signal.
Detection zone is set to inside fluid channel 135, is examined using the electrode 136 for being built in micro-fluidic chip 131 to flowing through
The electric impulse signal for the particulate samples for surveying region is acquired.
The counting micro particles instrument that the present invention uses, is set by the special construction to detection module 130 and micro-fluidic chip 131
Meter so that entire liquid fluid system is compacter and effective, improves the not high problem of prior art Instrumental over all Integration degree.Together
When, the setting of electric impulse signal detection zone can make sample liquids all flow through detection zone, realize full sample size
Absolute counting, that is, be added detecting system sample liquids be all detected so that the result accuracy of counting micro particles and
Consistency is more excellent.
Preferably, detection module 130 is detachable, specific as shown in Figure 10.
Since the size of liquid to be detected sample is different, for example bacterium is small to hundreds of nanometers to a few micrometers, and common
Cell size be then a few micrometers arrive some tens of pm.To meet to the detection demands of different test objects, detection module 130 can
Design form is replaced, it can be different to be adapted to for the different size of micro-fluidic chip 131 for detecting object designs different structure
Detect object.The design for disassembly of above-mentioned detection module 130 considerably increases the flexibility of system, and it is different can be adapted to user
Detection demand.
In one preferred embodiment, counting micro particles instrument further includes waste fluid container 150 and sheath liquid container 140；
Sheath liquid container 140 is connected with over-pressure control unit 110, and waste fluid container 150 is connected with vacuum cavitations unit 120, sheath
Liquid container 140 and waste fluid container 150 are connected with detection module 130 respectively；
Fluid channel 135 is connected to by inlet opening 132 with sheath liquid container 150, and fluid channel 135 passes through waste liquid hole 134 and waste liquid
Container 150 is connected to；
Sheath fluid in sheath liquid container 140 enters fluid channel 135 by inlet opening 132, micro- with being added from well 133
Grain sample mixing, and carry sample liquids and waste fluid container 150 is flowed to by waste liquid hole 134.
Specifically, processor 170 generates positive pressure for controlling over-pressure control unit 110, control vacuum cavitations unit 120 produces
Raw negative pressure so that the sheath fluid in sheath liquid container 140 enters fluid channel 135 by inlet opening 132, and is carried from well 133
The sample liquids of addition flow to waste fluid container 150 by waste liquid hole 134, and processor 170 believes the data that detection module 130 generates
Breath is handled.
In one preferred embodiment, counting micro particles instrument further includes soda liquor container 160, over-pressure control unit 110
It is connected with soda liquor container 160, soda liquor container 160 is connected to by inlet opening 132 with fluid channel 135；
Cleaning solution in soda liquor container 160 enters fluid channel 135 by inlet opening 132, and is carried out to fluid channel 135
Cleaning, waste liquid flows to waste fluid container 150 by waste liquid hole 134 after cleaning.
The cleaning solution contained in soda liquor container 160 can clean the micro-fluidic chip 131 after sample detection, keep away
Exempt from sample carryover to influence instrument detection stability and accuracy.
As a kind of optional embodiment of the present invention, the quantity of inlet opening 132 is one, inlet opening 132 and sample-adding
Fluid channel 135 between hole 133 is set there are two runner branches 1351, specific as shown in Figure 4.Two runner branches 1351 are set respectively
Converge in the both sides of well 133 and with the fluid channel 135 after well 133, the sheath fluid of inlet opening 132 is through runner branches
1351 flow into from the both sides of well 133, and wrap up the sample liquids entered positioned at intermediate slave well 133 and flow to electricity together
Pulse signal detection area, due to the characteristic that fluid dynamics focus, wrapped sample liquids are accumulated into a stable core
Heart streamline flows through electric impulse signal detection zone, to realize cell or counting micro particles；The special construction of above-mentioned runner branches 1351
Design (reducing fluid channel depth) reduces the differential suspension problem of particulate samples, and the depth of field of optical system is combined to effectively reduce
The error that counting error caused by being layered due to particle when Image Acquisition and cell are differentiated anyway, caused by avoiding differential suspension
Characteristics of image difference.In addition, chip design is but also the quantitative sample being added can all flow through detection zone, to realize bulk sample
Product count, and are conducive to the detection of signal, exclude measurement error.
Or, the quantity of inlet opening 132 is no less than two, the both sides of well 133 are respectively arranged on, it is specific as shown in Figure 5.Into
The sheath fluid of fluid apertures 132 flows into from the both sides of well 133 and wraps up the sample liquids one entered positioned at intermediate slave well 133
It rises and flows to electric impulse signal detection zone, due to the characteristic that fluid dynamics focus, wrapped sample liquids are accumulated into stabilization
A core streamline flow through electric impulse signal detection zone, to realize cell or counting micro particles.By at least two inlet openings 132
Be placed in the both sides of well 133, and the sheath fluid and the sample liquids in well 133 for being conducive to inlet opening 132 are sufficiently mixed, i.e.,
Sheath fluid wraps up sample fluid flow, meanwhile, the design of fluid channel depth is reduced in conjunction with the depth of field of optical system, can reduce particle
The differential suspension problem of sample, counting error caused by being layered due to particle when effectively reducing Image Acquisition and cell are sentenced anyway
Other error, characteristics of image difference caused by avoiding differential suspension.In addition, chip design is but also the quantitative sample energy being added
Detection zone all is flowed through, to realize full sample count, is conducive to the detection of signal, excludes measurement error.
In one preferred embodiment, detection module 130 further includes image capture module (not identified in figure)；
Image capture module is used to acquire multiple images for the particulate samples for flowing through image acquisition areas, and processor 170 is used for
The data information of image capture module is handled.
Image capture module can be used existing Image Acquisition means and can be realized, and be not especially limited herein.For example, figure
As acquisition module may include light source, camera and optical lens.Light source and camera are electrically connected with processor respectively, optical lens
It is connect with camera by a standard lens interface.Wherein, optical lens can be the combination of single lens or a plurality of lenses.Its
In, light source is set to the side of micro-fluidic chip, and towards above-mentioned image acquisition areas, and camera is set to the another of micro-fluidic chip
Side, and optical lens faces above-mentioned image acquisition areas.Pass through above-mentioned design so that light source can under the control of a processor upwards
Image acquisition areas transmitted ray is stated, camera can acquire the sample liquids for flowing through above-mentioned image acquisition areas under the control of a processor
Multiple images, and be sent to processor and handled.
Over-pressure control unit 110, vacuum cavitations unit 120 are required to certain pressure source and provide power for it.As this
A kind of optional embodiment of invention, over-pressure control unit 110 are connect with positive pressure pressure source 111；
Vacuum cavitations unit 120 is connect with negative pressure pressure source 121.
According to actual demand, optionally, positive pressure pressure source 111 can be positive pressure gas pump or liquid pump, when positive pressure pressure source
111 be positive pressure air pump when, positive pressure air pump can be diaphragm air pump.When positive pressure pressure source 111 is liquid pump, Ke Yiyou, but it is unlimited
In following to select：Peristaltic pump, diaphragm pump and linear pump.Negative pressure pressure source 121 is negative pressure air pump.
As a kind of optional embodiment of the present invention, counting micro particles instrument further includes several solenoid valves, passes through control
The opening and closing of each solenoid valve combines to realize the flowing of sample liquids, sheath fluid, cleaning solution or waste liquid in fluid channel 135.
Specifically, the first solenoid valve 181 is set between over-pressure control unit 110 and soda liquor container 160, the 4th electromagnetism
Valve 184 is set between soda liquor container 160 and detection module 130, by controlling the first solenoid valve 181 and the 4th solenoid valve
184 opening and closing is flowed to control inlet opening 132 of the cleaning solution on detection module 130；Second solenoid valve 182 is set to just voltage-controlled
Between unit 110 and sheath liquid container 140 processed, the 5th solenoid valve 185 is set between sheath liquid container 140 and detection module 130, is led to
The opening and closing of control second solenoid valve 182 and the 5th solenoid valve 185 is crossed to control inlet opening 132 of the sheath fluid on detection module 130
Flowing；7th solenoid valve 187 is set between vacuum cavitations unit 120 and waste fluid container 150, and the 6th solenoid valve 186 is set to
Between detection module 130 and waste fluid container 150, detection mould is controlled by controlling the 7th solenoid valve 187 and the 6th solenoid valve 186
Waste fluid container 150 is discharged through waste liquid hole 134 in waste liquid in block 130.It is additionally provided between second solenoid valve 182 and waste liquid hole 134
Third solenoid valve 183 is additionally provided with the 8th solenoid valve 188, in well 133 between second solenoid valve 182 and inlet opening 132
The 9th solenoid valve 189 is additionally provided between waste fluid container 150.
According to shown in Fig. 6-9, fluid channel 135 is connected to by well 133 with the external of micro-fluidic chip 131, in well
It is additionally provided with head cover 191 above in the of 133 and on fuselage 190, head cover 191 can be overturn along fuselage.It is additionally provided with vacuum on head cover 191
Suction nozzle 192, vacuum cups 192 are adapted with well 133.Lock 193 is additionally provided on head cover 191.
Be additionally provided with display screen 194 on fuselage 190, user by display screen 194 to counting micro particles instrument operated with
And measurement result inspection.Power inlet 195, power switch 196 and USB port 197 are additionally provided on fuselage 190.
In one preferred embodiment, include to the method that particulate samples are detected using counting micro particles instrument：
After particulate samples are added to well 133, by 170 regulation unit of processor, particulate samples is made to pass through
Fluid channel 135 inside micro-fluidic chip 131 flows through image acquisition areas and/or detection zone, is adopted by the analysis of processor 170
The image and/or electric impulse signal of collection obtain the motility rate of particulate samples and/or the grain size and quantity of the particulate samples.
Specifically, including the following steps：
(1) certain positive pressure pressure is generated by positive pressure pressure source 111, by the output setting of over-pressure control unit 110
Air pressure generates certain negative pressure pressure by negative pressure pressure source 121, by the air pressure of the output setting of vacuum cavitations unit 120, leads to
It crosses and controls the opening and closing combination of each solenoid valve to make sheath fluid be full of the fluid channel 135 of micro-fluidic chip 131.
(2) and then by particulate samples it is added to well 133.After instrument head cover 191 is covered, same instrument passes through tune
Whole different atmospheric pressure and control second solenoid valve 182, the 5th solenoid valve 185, the 6th solenoid valve 186, the 7th solenoid valve
187, it realizes that sample liquids flow through image detection region and electric impulse signal detection zone by certain speed, passes through software algorithm point
Acquired image and electric impulse signal are analysed, it can be achieved that full sample count, obtains the letters such as concentration, grain size, the motility rate of particulate samples
Breath.
In one preferred embodiment, further include passing through cleaning solution after particulate samples all flow through fluid channel 135
The step of cleaning solution in container 160 cleans detection module 130.
Instrument can change whether automatic detection of particles sample all flows through detection zone by certain signal characteristics, at this time
Instrument software can calculate as a result, and from the background automatically into cleaning chip flow.It is all by certain atmospheric pressure and control
The opening and closing of solenoid valve combines that cleaning solution is made to clean the fluid channel 135 of microcontroller chip 131 by certain procedures, and cleaning terminates
It waits for and enters testing process next time.
Finally it should be noted that：The above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent
Present invention has been described in detail with reference to the aforementioned embodiments for pipe, it will be understood by those of ordinary skill in the art that：Its according to
So can with technical scheme described in the above embodiments is modified, either to which part or all technical features into
Row equivalent replacement；And these modifications or replacements, various embodiments of the present invention technology that it does not separate the essence of the corresponding technical solution
The range of scheme.
Claims (10)
1. a kind of method of counting micro particles, which is characterized in that the method includes：
Particulate samples are made to pass through in detection zone using microfluidic chip technology, detection obtains the grain size and quantity of the particle；
The detection zone utilizes Coulter principle, for acquire the particle by when the electric impulse signal that generates；
The particle includes cell and/or microorganism.
2. the method for counting micro particles according to claim 1, which is characterized in that the method further includes：
Particulate samples are made to pass through in image acquisition region using microfluidic chip technology, detection obtains the motility rate of the particle；
Described image pickup area for particle described in continuous acquisition by when image, the particle is cell；
Preferably, particulate samples are made to pass through in image acquisition region using microfluidic chip technology, detection obtains the particle
Motility rate recycles microfluidic chip technology that particulate samples is made to pass through in detection zone later, and detection obtains the grain size of the particle
And quantity.
3. the method for counting micro particles according to claim 1 or 2, which is characterized in that using counting micro particles instrument to particle into
Row counts, and the counting micro particles instrument includes the micro-fluidic chip equipped with detection zone.
4. the method for counting micro particles according to claim 3, which is characterized in that the counting micro particles instrument includes that control is single
Member, detection module and processor, described control unit and detection module are electrically connected with the processor respectively；
Described control unit includes the over-pressure control unit for generating positive pressure for controlling particulate samples disengaging counting micro particles instrument
With the vacuum cavitations unit for generating negative pressure；
The detection module includes the micro-fluidic chip equipped with detection zone, and inlet opening is offered on the micro-fluidic chip, is added
Sample hole and waste liquid hole, the micro-fluidic chip are internally provided with fluid channel, lead between the inlet opening, well and waste liquid hole
The fluid channel is crossed to be connected to；
The processor generates positive pressure for controlling the over-pressure control unit, controls the vacuum cavitations unit and generates negative pressure,
So that the particulate samples added from the well enter the fluid channel, and it is discharged by the waste liquid hole, the processing
The data information that device generates the detection module is handled；
The detection zone is set to inside the fluid channel, using being built in the electrode convection current of the micro-fluidic chip after testing
The electric impulse signal of the particulate samples in region is acquired；
Preferably, the detection module is detachable.
5. the method for counting micro particles according to claim 4, which is characterized in that the counting micro particles instrument further includes that waste liquid holds
Device and sheath liquid container；
The sheath liquid container is connected with the over-pressure control unit, and the waste fluid container is connected with the vacuum cavitations unit, institute
It states sheath liquid container and the waste fluid container is connected with detection module respectively；
The fluid channel by the inlet opening and the sheath fluid reservoir, the fluid channel by the waste liquid hole with it is described
Waste fluid container is connected to；
Sheath fluid in the sheath liquid container enters the fluid channel by inlet opening, is mixed with the particulate samples added from well
It closes, and carries sample liquids and waste fluid container is flowed to by waste liquid hole.
6. the method for counting micro particles according to claim 5, which is characterized in that the counting micro particles instrument further includes cleaning solution
Container, the over-pressure control unit are connected with the soda liquor container, the soda liquor container by the inlet opening with it is described
Fluid channel is connected to；
Cleaning solution in the soda liquor container enters the fluid channel by the inlet opening, and is carried out to the fluid channel
Cleaning, waste liquid flows to the waste fluid container by the waste liquid hole after cleaning.
7. according to the method for claim 4-6 any one of them counting micro particles, which is characterized in that the detection module further includes
Image capture module；
Described image acquisition module is used to acquire multiple images for the particulate samples for flowing through described image acquisition zone, the processor
It is handled for the data information to described image acquisition module.
8. the method for counting micro particles according to claim 7, which is characterized in that the method includes：
After particulate samples are added to well, by processor regulation unit, particulate samples is made to pass through micro-fluidic chip
Internal fluid channel flows through image acquisition areas and/or detection zone, and acquired image and/or electric pulse are analyzed by processor
Signal obtains the motility rate of the particulate samples and/or the grain size and quantity of the particulate samples.
9. the method for counting micro particles according to claim 8, which is characterized in that the method includes：
(a) by processor regulation unit, over-pressure control unit is made to generate positive pressure, vacuum cavitations unit generates negative pressure, leads to
Excess pressure official post sheath fluid enters the fluid channel of microcontroller chip by inlet opening；
(b) particulate samples are added to well, sheath fluid carries the particulate samples and passes through the fluid channel inside micro-fluidic chip
It is discharged from waste liquid hole after flowing through image acquisition areas and/or detection zone, and acquired image and/or electricity is analyzed by processor
Pulse signal obtains the motility rate of the particulate samples and/or the grain size and quantity of the particulate samples.
10. the method for counting micro particles according to claim 9, which is characterized in that the method further includes in particulate samples
All flow through the step of being cleaned to the detection module by the cleaning solution in soda liquor container after fluid channel.
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| CN111398137A (en) * | 2020-04-02 | 2020-07-10 | 华中农业大学 | Detection method based on resistance micron-pore particle counter and application thereof |
| CN112697679A (en) * | 2020-12-04 | 2021-04-23 | 杭州娃哈哈精密机械有限公司 | Device for rapidly detecting number of bacteria in beverage |
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| CN112697679A (en) * | 2020-12-04 | 2021-04-23 | 杭州娃哈哈精密机械有限公司 | Device for rapidly detecting number of bacteria in beverage |
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