CN104928163A - Micro-fluidic chip used for raw milk body cell counting - Google Patents
Micro-fluidic chip used for raw milk body cell counting Download PDFInfo
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- CN104928163A CN104928163A CN201510288102.2A CN201510288102A CN104928163A CN 104928163 A CN104928163 A CN 104928163A CN 201510288102 A CN201510288102 A CN 201510288102A CN 104928163 A CN104928163 A CN 104928163A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
- C12M23/16—Microfluidic devices; Capillary tubes
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
- C12M41/36—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of biomass, e.g. colony counters or by turbidity measurements
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Abstract
The invention discloses a micro-fluidic chip used for raw milk body cell counting. The micro-fluidic chip comprises a micro-fluidic channel unit plate arranged on a substrate, wherein a group of micro-fluidic channels for bearing a measured raw milk sample fluid is formed on the micro-fluidic channel unit. Each micro-fluidic channel comprises an inlet for injecting a pre-treated to-be-measured raw milk sample fluid, 2-6 channels which are connected on the inlet at certain ends and are communicated with the inlet, wherein each of the other ends of the 2-6 channels is connected with a fluid collecting cavity for collecting a detected waste liquid; 2-6 micro cavities which are communicated with the channel for placing the to-be-measured raw milk sample fluid are formed in each channel at equal intervals. The distribution, in the micro cavities, of the body cells is limited by the height of the micro cavities, so that the body cells within the field of view are guaranteed to be almost in a focal plane at the same height, and thus, focusing in an imaging process is facilitated to form a clear microscopic image, and accurate counting is realized. The micro-fluidic chip can be directly applied to counting raw milk body cells by a microscopic method, and can be used for remarkably improving the detection precision.
Description
Technical field
The present invention relates to a kind of micro-fluidic chip.Particularly relate to a kind of micro-fluidic chip for raw milk Somatic Cell Count being applied to direct Microscopical Method For Detection.
Background technology
Milk somatic cell number (somatic cell count, SCC) refers to the total cellular score in every ml milk, and majority is white corpuscle, accounts for 95% of somatic number, and somatic number reflects the healthy state of milk qualities and milk cow.Under normal circumstances, in milk somatic number generally at 20,000 ~ 200,000/mL.If its content is more than 500,000/mL, then milk cow presents the clinical symptom of mazoitis.Somatic number is when 20 ~ 500,000/mL, and milk cow suffers from stealthy mazoitis more, and symptom is not easily discovered, but has a strong impact on quality and the mouthfeel of fresh milk.Existing raw milk Somatic Cell Count method has the traditional detection means such as direct Microscopical Method For Detection, electrical conductivity method, fluorescence colour, flow cytometric methods.Often kind of detection method all be unable to do without the carrying platform of milk sample to be detected.
Microfluidic chip technology can be integrated on the chip of one piece of micro-meter scale the basic operation unit such as sample preparation, pre-treatment, reaction, separation, detection that is biological, chemical analysis processes, the operation such as transport, mixing, sorting, enrichment, logical operation of fluid, micro-nano particle or drop can be realized, the means of micro-nano technology can be adopted to prepare, there is advantages such as consuming sample few (only microliter quantity level), testing cost is low, detection speed is fast.
China's application 200710067168.4 (being applied to the slide glass of automatic counting instrument for body cell of milk/bacteria through direct microscopic) give a kind of slide configurations scheme being applied to the automatic counter for counting milk somatic cell/bacteria of direct microscopy, utilize the relative engagement of the peripheral frame of slide and slide, form shallow disk container, ensure that the abundant dyeing of abundant milk sample.But in this kind of design, the height of tray container is difficult to ensure the contour distribution of somatocyte to be detected in short transverse, is unfavorable for the accurate focusing in direct microscopy process, thus have impact on the accuracy of detected result.China's application 201220232862.3 (milk somatic cell 4 hole Test papers) give the paper substrate Test paper of sandwich structure, detect for milk somatic cell, but the depth of this method colour developing depends on reaction substrate on test paper and corresponding reaction enzymes, and must could estimate somatic content by means of spectrophotometer, for indirectly testing method, have impact on the reliability of detected result.
In order to realize the accurate focusing in direct microscopy process, be convenient to form steady and audible image under the microscope, it is good that general requirement Microscopical Method For Detection slide used has light transmission, and somatocyte is evenly distributed and is in same focussing plane as far as possible.
Summary of the invention
Technical problem to be solved by this invention is, provides a kind of micro-fluidic chip for raw milk Somatic Cell Count that can meet raw milk Somatic Cell Count and mammitis of cow rapid detection.
The technical solution adopted in the present invention is: a kind of micro-fluidic chip for raw milk Somatic Cell Count, including substrate and be arranged on described suprabasil microchannel cell board, described microchannel cell board being formed with one group for carrying the microchannel of tested fresh milk sample fluid.
Described microchannel includes: for injecting the entrance through pretreated fresh milk sample fluid to be measured, one end is connected to 2 ~ 6 passages be connected on described entrance and with described entrance, the other end of described 2 ~ 6 passages respectively each connection one for collect detect after the liquid collecting cavity of waste liquid, all equally spacedly on each passage be provided with 2 ~ 6 microcavitys for placing fresh milk sample fluid to be measured be connected with described passage.
Described microcavity is circular microcavity, and the diameter of described circular microcavity is 0.1 ~ 5mm, is highly 10 ~ 100um.
Described microcavity is square microcavity, and the length of side of described square microcavity is 0.1 ~ 5mm, is highly 10 ~ 100um.
Described entrance and liquid collecting cavity are the communicating pores of up/down perforation, and the aperture of described communicating pores is 1 ~ 10mm, is highly the thickness of microchannel cell board, is 0.1 ~ 2cm.
A kind of micro-fluidic chip for raw milk Somatic Cell Count, include substrate and be arranged on described suprabasil microchannel cell board, described microchannel cell board is formed with the microchannel for carrying tested fresh milk sample fluid that M × N group structure is identical, wherein M is the integer of more than 5, and N is the integer of more than 1.
Described substrate is that length is 1 ~ 10cm, the wide 1 ~ 10cm of being and height are the slab construction of 0.1 ~ 2cm.
Described microchannel includes: for injecting the entrance through pretreated fresh milk sample fluid to be measured, one end is connected to 2 ~ 6 passages be connected on described entrance and with described entrance, the other end of described 2 ~ 6 passages respectively each connection one for collect detect after the liquid collecting cavity of waste liquid, all equally spacedly on each passage be provided with 2 ~ 6 microcavitys for placing fresh milk sample fluid to be measured be connected with described passage.
Described microcavity is circular microcavity or is square microcavity, and when described microcavity is circular microcavity, the diameter of described circular microcavity is 0.1 ~ 5mm, is highly 10 ~ 100um; When described microcavity is square microcavity, the length of side of described square microcavity is 0.1 ~ 5mm, is highly 10 ~ 100um.
Described entrance and liquid collecting cavity are the communicating pores of up/down perforation, and the aperture of described communicating pores is 1 ~ 10mm, is highly the thickness of microchannel cell board, is 0.1 ~ 2cm.
A kind of micro-fluidic chip for raw milk Somatic Cell Count of the present invention, because the height of the distribution of somatocyte in microcavity by microcavity limited, ensure that somatocyte within the vision is in the focal plane of sustained height substantially, be conducive to the focusing in imaging process, obtain micro-image clearly, and then realize accurate counting.The somatic counting of Microscopical Method For Detection raw milk can be directly applied to, traditional slide glass can be replaced, and significantly improve the tolerance range of detection.
Accompanying drawing explanation
Fig. 1 is the one-piece construction schematic diagram of first embodiment of the invention;
Fig. 2 is the one-piece construction schematic diagram of second embodiment of the invention;
Fig. 3 is the one-piece construction schematic diagram of third embodiment of the invention;
Fig. 4 is the structural representation of microchannel in third embodiment of the invention;
Fig. 5 is the structural representation of microchannel in third embodiment of the invention.
In figure
1: substrate 2: microchannel cell board
3: microchannel 31: entrance
32: passage 33: microcavity
34: liquid collecting cavity 11: substrate
12: microchannel cell board 13: microchannel
131: entrance 132: passage
133: microcavity 134: liquid collecting cavity
Embodiment
Below in conjunction with embodiment and accompanying drawing, a kind of micro-fluidic chip for raw milk Somatic Cell Count of the present invention is described in detail.
As shown in Figure 1, a kind of micro-fluidic chip for raw milk Somatic Cell Count of the present invention, the microchannel cell board 2 including substrate 1 and be arranged in described substrate 1, described microchannel cell board 2 is formed with one group for carrying the microchannel 3 of tested fresh milk sample fluid.The material of substrate 1 and microchannel cell board 2 can be the material such as glass, quartz, acrylic, ABS, PDMS, size with reference to slide glass, substrate 1 and microchannel cell board 2 by thermal bonding, pressure key and or the mode of plasma bonding complete encapsulation.
Described microchannel 3 includes: for injecting the entrance 31 through pretreated fresh milk sample fluid to be measured, one end is connected to 2 ~ 6 passages 32 be connected on described entrance 31 and with described entrance 31, the other end of described 2 ~ 6 passages 32 respectively each connection one for collect detect after the liquid collecting cavity 34 of waste liquid, all equally spacedly on each passage 32 be provided with 2 ~ 6 microcavitys 33 for placing fresh milk sample fluid to be measured be connected with described passage 32.Described entrance 31 and liquid collecting cavity 34 are the communicating pores of up/down perforation, and the aperture of described communicating pores is 1 ~ 10mm, is highly the thickness of microchannel cell board 2, is 0.1 ~ 2cm.Described microcavity 33 is circular microcavity, and the diameter of described circular microcavity is 0.1 ~ 5mm, is highly 10 ~ 100um.Somatocyte flows through the circular microcavity 33 of 9 shown in Fig. 1 with fluid, distributes respectively in 9 microcavitys, and completes microscopy counting by microscope.By asking for the mean value of Somatic Cell Count in 9 microcavitys, and according to microcavity volume, can realize the estimation of somatocyte concentration level in milk sample and the diagnosis of mammitis of cow.
As shown in Figure 2, described microcavity 33 can also be square microcavity, and the length of side of described square microcavity is 0.1 ~ 5mm, is highly 10 ~ 100um.
In actual applications, can according to the microchannel structural design and processing micro-fluidic chip shown in Fig. 1 and Fig. 2, the single completing milk sample detects.
Also pattern array can be obtained M × N number of microchannel structure, same micro-fluidic chip realize the detection to multiple milk sample, the structure shown in Fig. 3 as described below.Except above-mentioned bonding pattern can obtain except micro-fluidic chip, this structure also obtains by injection moulding or the once shaped of 3D printing technique.
As shown in Figure 3, a kind of micro-fluidic chip for raw milk Somatic Cell Count of the present invention, it can also be the microchannel cell board 12 including substrate 11 and be arranged in described substrate 11, described microchannel cell board 12 is formed with the microchannel 13 for carrying tested fresh milk sample fluid that M × N group structure is identical, wherein M is the integer of more than 5, and N is the integer of more than 1.Described substrate 11 is that length is 1 ~ 10cm, the wide 1 ~ 10cm of being and height are the slab construction of 0.1 ~ 2cm.
As shown in Figure 4, described microchannel 13 includes: for injecting the entrance 131 through pretreated fresh milk sample fluid to be measured, one end is connected to 2 ~ 6 passages 132 be connected on described entrance 131 and with described entrance 131, the other end of described 2 ~ 6 passages 132 respectively each connection one for collect detect after the liquid collecting cavity 134 of waste liquid, described entrance 131 and liquid collecting cavity 134 are the communicating pores of up/down perforation, the aperture of described communicating pores is 1 ~ 10mm, highly being the thickness of microchannel cell board 12, is 0.1 ~ 2cm.All equally spacedly on each passage 132 be provided with 2 ~ 6 microcavitys 133 for placing fresh milk sample fluid to be measured be connected with described passage 132.
As shown in Figure 4, Figure 5, described microcavity 133 is circular microcavity or is square microcavity, and when described microcavity 133 is circular microcavity, the diameter of described circular microcavity is 0.1 ~ 5mm, is highly 10 ~ 100um; When described microcavity 133 is square microcavity, the length of side of described square microcavity is 0.1 ~ 5mm, is highly 10 ~ 100um.
Illustrate that the making processes of a kind of micro-fluidic chip for raw milk Somatic Cell Count of the present invention is as follows with two examples below:
Embodiment 1:
Make light with reference to Fig. 3 and paint mask egative film, mask dimensions is 70 × 20mm, is distributed with 14 detecting units be made up of microchannel.Cleaning slide glass, completes the plastic packaging of photosensory membrane and slide glass, is placed in above photosensory membrane by mask egative film, complete the microstructure after uv-exposure, developing process as Mold Making microchannel structure.Be cast in above-mentioned microchannel after being mixed according to mass ratio 10:1 with fluidizer by PDMS prepolymer, keep 2 hours at 80 DEG C in thermostatic drying chamber, complete solidification, obtain microchannel structure.Utilize punch tool in injection port place and the punching of waste liquid outlet place, then microchannel and clean slide glass substrate are placed in plasma bonder and plasma treatment is carried out to it, complete the bonding of microchannel and substrate of glass, finally obtain the micro-fluidic chip needed for raw milk Somatic Cell Count.
Embodiment 2:
Make light with reference to Fig. 3 and paint mask egative film, mask dimensions is 70 × 20mm, is distributed with 14 detecting units be made up of microchannel.Cleaning slide glass, utilizes photoresist spinner on slide glass, complete the gluing process of SU8 photoresist material, and controlling photoresist layer thickness is 30um.Mask egative film is placed in above photosensory membrane, completes the microstructure after uv-exposure, developing process as Mold Making microchannel structure.Be cast in above-mentioned microchannel after being mixed according to mass ratio 10:1 with fluidizer by PDMS prepolymer, keep 2 hours at 80 DEG C in thermostatic drying chamber, complete solidification, obtain microchannel structure.Utilize punch tool in injection port place and the punching of waste liquid outlet place, then microchannel and clean acrylic substrate of glass are placed in plasma bonder and plasma treatment is carried out to it, complete the bonding of microchannel and acrylic substrate, obtain the micro-fluidic chip needed for raw milk Somatic Cell Count.
Claims (10)
1. the micro-fluidic chip for raw milk Somatic Cell Count, it is characterized in that, including substrate (1) and be arranged on the microchannel cell board (2) in described substrate (1), described microchannel cell board (2) being formed with one group for carrying the microchannel (3) of tested fresh milk sample fluid.
2. a kind of micro-fluidic chip for raw milk Somatic Cell Count according to claim 1, it is characterized in that, described microchannel (3) includes: for injecting the entrance (31) through pretreated fresh milk sample fluid to be measured, one end is connected to described entrance (31) and goes up and 2 ~ 6 passages (32) be connected with described entrance (31), the other end of described 2 ~ 6 passages (32) respectively each connection one for collect detect after the liquid collecting cavity (34) of waste liquid, all equally spacedly on each passage (32) be provided with 2 ~ 6 microcavitys (33) for placing fresh milk sample fluid to be measured be connected with described passage (32).
3. a kind of micro-fluidic chip for raw milk Somatic Cell Count according to claim 2, is characterized in that, described microcavity (33) is circular microcavity, and the diameter of described circular microcavity is 0.1 ~ 5mm, is highly 10 ~ 100um.
4. a kind of micro-fluidic chip for raw milk Somatic Cell Count according to claim 2, is characterized in that, described microcavity (33) is square microcavity, and the length of side of described square microcavity is 0.1 ~ 5mm, is highly 10 ~ 100um.
5. a kind of micro-fluidic chip for raw milk Somatic Cell Count according to claim 2, it is characterized in that, described entrance (31) and liquid collecting cavity (34) are the communicating pores of up/down perforation, the aperture of described communicating pores is 1 ~ 10mm, highly being the thickness of microchannel cell board (2), is 0.1 ~ 2cm.
6. the micro-fluidic chip for raw milk Somatic Cell Count, it is characterized in that, include substrate (11) and be arranged on the microchannel cell board (12) in described substrate (11), described microchannel cell board (12) is formed with the microchannel (13) for carrying tested fresh milk sample fluid that M × N group structure is identical, wherein M is the integer of more than 5, and N is the integer of more than 1.
7. a kind of micro-fluidic chip for raw milk Somatic Cell Count according to claim 6, is characterized in that, described substrate (11) is that length is 1 ~ 10cm, the wide 1 ~ 10cm of being and height are the slab construction of 0.1 ~ 2cm.
8. a kind of micro-fluidic chip for raw milk Somatic Cell Count according to claim 6, it is characterized in that, described microchannel (13) includes: for injecting the entrance (131) through pretreated fresh milk sample fluid to be measured, one end is connected to described entrance (131) and goes up and 2 ~ 6 passages (132) be connected with described entrance (131), the other end of described 2 ~ 6 passages (132) respectively each connection one for collect detect after the liquid collecting cavity (134) of waste liquid, all equally spacedly on each passage (132) be provided with 2 ~ 6 microcavitys (133) for placing fresh milk sample fluid to be measured be connected with described passage (132).
9. a kind of micro-fluidic chip for raw milk Somatic Cell Count according to claim 6, it is characterized in that, described microcavity (133) is for circular microcavity or be square microcavity, when described microcavity (133) is for circular microcavity, the diameter of described circular microcavity is 0.1 ~ 5mm, is highly 10 ~ 100um; When described microcavity (133) is for square microcavity, the length of side of described square microcavity is 0.1 ~ 5mm, is highly 10 ~ 100um.
10. a kind of micro-fluidic chip for raw milk Somatic Cell Count according to claim 6, it is characterized in that, described entrance (131) and liquid collecting cavity (134) are the communicating pores of up/down perforation, the aperture of described communicating pores is 1 ~ 10mm, highly being the thickness of microchannel cell board (12), is 0.1 ~ 2cm.
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Cited By (1)
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CN108845001A (en) * | 2018-05-29 | 2018-11-20 | 哈尔滨工业大学 | The online electric detection means of milk somatic cell and method based on microflow control technique |
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