CN113600250B - Chip for micro-channel assisted high-throughput reagent quantitative distribution and analysis - Google Patents
Chip for micro-channel assisted high-throughput reagent quantitative distribution and analysis Download PDFInfo
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- CN113600250B CN113600250B CN202110825892.9A CN202110825892A CN113600250B CN 113600250 B CN113600250 B CN 113600250B CN 202110825892 A CN202110825892 A CN 202110825892A CN 113600250 B CN113600250 B CN 113600250B
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/50273—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the means or forces applied to move the fluids
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/10—Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0887—Laminated structure
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2400/00—Moving or stopping fluids
- B01L2400/04—Moving fluids with specific forces or mechanical means
- B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
- B01L2400/0409—Moving fluids with specific forces or mechanical means specific forces centrifugal forces
Abstract
The invention belongs to the technical field of microfluidics, and discloses a microchannel-assisted chip for quantitative reagent distribution and analysis, wherein a detection unit in the chip comprises at least 1 quantitative reagent distribution channel arranged from inside to outside relative to a rotation center; the outer wall of the liquid distribution drainage channel (3, 23) is provided with a plurality of quantitative chambers (4, 24) for quantitative distribution; the adjacent 2 reagent quantitative distribution channels are connected through hydrophobic micro-channels (8, 28); the hydrophobic microchannel can generate resistance effect on a liquid reagent or a sample, so that the centrifugal force is controlled through the rotating speed, and the relation between the centrifugal force and the resistance is regulated, namely whether the hydrophobic microchannel conducts the reagent or the sample or not can be controlled, so that quantitative distribution and the function of mixing the reagent or the sample after quantitative distribution are realized. According to the invention, by arranging the hydrophobic micro-channel, high-flux quantitative distribution and mixing of the reagent or the sample can be completed without integrating a large amount of valve control on a chip.
Description
Technical Field
The invention belongs to the technical field of microfluidics, and particularly relates to a chip for assisting high-flux reagent quantitative distribution and analysis through a microchannel, which is applicable to high-flux quantitative distribution and analysis of liquid reagents or liquid samples.
Background
The microfluidic technology is a new scientific technology, is applied to various fields such as chemistry, biology, engineering, physics and the like, has strong subject crossability, makes a breakthrough in the precise control of time, space and an analysis object, and can solve many key problems of life analysis. The microfluidic technology can integrate the detection experiment which can only be completed in a laboratory originally on a small chip, so that the material cost and the time cost are saved, more importantly, various detection technologies can be integrated into a whole, and the detection efficiency is improved.
With the continuous development of the microfluidic technology, the advantages of the microfluidic technology in the field of analysis and detection are more and more obvious, so that the loss of reagents can be reduced, and the micro-reactor under the micro-scale is more favorable for various biochemical reactions. Firstly, no matter whether the normal operation of various reactions is ensured or the detection and analysis of the concentration of substances are carried out, quantitative addition and mixing of various required reaction reagents are required firstly, and the quantitative distribution and mixing of liquid are realized manually in a laboratory through tools such as a pipette, a dropper, a measuring cylinder, a pipette and the like, but the flux is not high, the human error is large, and the operation is complicated, so that a plurality of micro-fluidic chips capable of automatically and quantitatively distributing the liquid are designed by many researchers by virtue of the advantages of the micro-fluidic chip technology. However, most of the prior microfluidic chips can only complete the quantification of single liquid, or the chip structure is relatively complex, or the design of the utilized principle is difficult to realize. For example, the granted patent CN101486004B in 2012 skillfully utilizes the advantage of the fluid shear force of the incompatible liquid at the micro scale, but only can complete the quantitative distribution of a single liquid sample, and the patent application No. CN109806920A published in 2019 is similar to the principle and concept of its design, but also can complete the quantitative distribution of a single sample; in order to improve the flux of quantitative distribution in the chip, the patent CN108855254B granted in 2020 provides a disc type chip, the chip has two layers, and the two layers of chips need to be moved relatively when quantitative mixing is performed, although high-flux quantitative distribution can be performed, the relative movement causes the influence of the processing technology and the sealing of liquid, and at the same time, only the quantitative distribution of a single sample can be performed. Meanwhile, in patent CN102989533B granted in 2015, a disc-type chip is also designed, which ingeniously uses PDMS material with vacuum negative pressure retention as fluid driving force to provide negative pressure adsorption, so as to complete quantitative distribution of two samples, but it is necessary to precisely control air pressure and control valves, and at the same time, it is necessary to perform advanced treatment on the PDMS chip layer. However, with the continuous development of science, the types of reaction reagents involved in each biochemical reaction and substance concentration analysis are increased, so that a microfluidic chip which is convenient to process, simple in structure and applicable to high-throughput quantitative distribution analysis of various reagents is urgently needed in the analysis field.
Disclosure of Invention
In view of the above defects or improvement requirements of the prior art, an object of the present invention is to provide a chip for quantitatively distributing and analyzing a high-throughput reagent with the assistance of a microchannel, wherein a hydrophobic microchannel is arranged, and the resistance effect of the hydrophobic microchannel on the reagent or a sample is utilized, and whether the action mechanism of the resistance effect can be overcome by centrifugal forces corresponding to different rotation speeds, so that whether the hydrophobic microchannel conducts the reagent or the sample can be controlled by rotating the rotation speed, and the high-throughput quantitative distribution and mixing of the reagent (or the sample) can be completed without integrating a large number of valve controls on the chip in cooperation with the design of a quantitative chamber and a reaction chamber which are preset along the volume size on the outer side wall of a liquid distribution drainage channel, so that the problems of difficulty in chip bonding, poor stability and the like caused by the complexity of chip processing technology and the excessive number of chip layers due to the excessive number of integrated valves and the difficulty in the simultaneous quantitative distribution of multiple reagents (or samples) in the prior art can be effectively solved.
To achieve the above objects, there is provided a microchannel-assisted chip for reagent quantitative distribution and analysis according to the present invention, wherein the chip is provided with a rotation center and has one or more detection units therein; the detection unit comprises at least 1 reagent quantitative distribution channel which is arranged from inside to outside relative to the rotation center;
each reagent quantitative distribution channel comprises 1 arc-shaped liquid distribution drainage channel (3, 23) for quantitatively distributing the reagent or the sample to be quantitatively distributed;
along a preset rotation direction, marking two ends of the liquid distribution diversion channel (3, 23) as a head end and a tail end respectively, from the head end to the tail end, keeping the distance from the liquid distribution diversion channel (3, 23) to the rotation center unchanged or gradually increasing, connecting the head end with a reagent storage chamber (1, 21), and using the reagent storage chamber (1, 21) to store liquid reagents or liquid samples to be quantitatively distributed;
the liquid distribution drainage channel (3, 23) has a first side wall and a second side wall which are oppositely arranged, wherein the first side wall is positioned at the inner side close to the rotation center, and the second side wall is positioned at the outer side far away from the rotation center; a plurality of quantitative chambers (4, 24) are arranged on the second side wall, and the volume of the quantitative chambers (4, 24) is preset for quantitative distribution;
any one of the detection units: for 2 adjacent said reagent dosing channels, the dosing chamber (4, 24) adjacent to the inner reagent dosing channel is connected to the liquid dispensing drainage channel (3, 23) adjacent to the outer reagent dosing channel by a hydrophobic microchannel (2, 28); such that all reagent dosing channels are connected in series, the dosing chamber (4, 24) of the outermost reagent dosing channel is also connected to the more outer reaction chamber (7, 27) by a hydrophobic microchannel (2, 28); the inner wall of the hydrophobic microchannel (2, 28) has hydrophobicity and can generate resistance effect on a liquid reagent or a liquid sample, so that the centrifugal force is controlled through the rotating speed, the magnitude relation between the centrifugal force and the resistance is regulated and controlled, and whether the hydrophobic microchannel (2, 28) conducts the reagent or the sample or not can be controlled; the reaction chamber (7, 27) is used for collecting the quantified reagent or sample, and the reagent or sample is mixed in the reaction chamber (7, 27);
the analysis chip is capable of quantitatively dispensing a reagent or a sample when the analysis chip is rotated about the rotation center and the hydrophobic microchannel (2, 28) is in a non-conductive state with respect to the reagent or the sample;
when the analysis chip rotates around the rotation center and the hydrophobic micro-channel (2, 28) is in a conduction state for a reagent or a sample, the analysis chip can mix the reagent or the sample after quantitative distribution.
As a further preference of the present invention, any one of the reagent quantitative distribution channels includes at least 2 quantitative chambers (4, 24); wherein the dosing chamber (4, 24) closest to the first end is a sacrificial chamber for receiving excess reagent or excess sample generated during spin centrifugation.
As a further preferred aspect of the present invention, the reagent quantitative distribution channel located outside the sacrificial chamber has a uniform or gradually increasing volume from the head end to the tail end of the quantitative chamber (4, 24);
for the reagent dosing channel located inside, the volume of the dosing chamber (4, 24) is kept uniform or gradually decreases from the head end to the tail end, except for the sacrificial chamber.
As a further preference of the present invention, the hydrophobic microchannel (2, 28) preferably has a height of 10 to 500 μm and a width of 10 to 500 μm; correspondingly:
when the rotating speed is 500-1000 rpm, the analysis chip can quantitatively distribute the liquid reagent or the liquid sample;
when the rotating speed is 2000-3000 rpm, the analysis chip can mix the liquid reagent or the liquid sample after the quantitative distribution.
As a further preference of the invention, the inner wall of the hydrophobic microchannel (2, 28) is provided with a hydrophobic coating, and the hydrophobic coating is specifically made of wax, silanization reagent, teflon AF, CYTOP or electronic fluorination liquid; the electronic fluorinated liquid is preferably ECG1700.
As a further preference of the present invention, the hydrophobic microchannel (2, 28) has a bent structure for increasing its resistance to a liquid reagent or a liquid sample;
preferably, any one of the hydrophobic microchannels (2, 28) corresponding to the reagent dosing channel positioned at the outer side has a more bent structure, and any one of the hydrophobic microchannels (2, 28) corresponding to the reagent dosing channel positioned at the inner side has a less bent structure.
As a further preferable aspect of the present invention, the reagent storage chamber (1, 21) is further provided with a reagent sample injection port (2, 22) for replenishing a liquid reagent or a liquid sample to be quantitatively dispensed into the reagent storage chamber (1, 21);
the reaction chamber (7, 27) is also connected with an air hole (6, 26) for balancing the air pressure in the reaction chamber (7, 27);
the tail end of any one of the liquid distribution drainage channels (3, 23) is also communicated with a waste liquid chamber (5, 25);
preferably, the liquid distribution drainage channels (3, 23) in any one of the detection units are communicated with the same waste liquid chamber (5, 25).
As a further preference of the present invention, the chip has a 2-layer structure in which the hydrophobic micro-channels (2, 28) are located in a 1-layer structure, and the reagent storage chamber (1, 21), the reagent addition port (2, 22), the liquid distribution drainage channel (3, 23), the quantification chamber (4, 24), the waste liquid chamber (5, 25), and the reaction chamber (7, 27) are all located in another 1-layer structure;
preferably, the chip is a disc chip.
Compared with the prior art, the chip for assisting high-flux reagent quantitative distribution and analysis by the hydrophobic microchannel has the advantages that the chip for assisting high-flux reagent quantitative distribution and analysis is realized by the aid of the hydrophobic microchannel, a rotary micro-fluidic chip structure is adopted, centrifugal force is used as driving force of fluid, the simple hydrophobic microchannel is used as a flow resistance valve, high-flux quantitative distribution of multiple reagents (or samples) in a single reaction is completed, and the chip is suitable for high-flux quantitative analysis of various reactions; meanwhile, the chip has a simple structure (particularly, the chip can be designed into a double-layer chip structure, the hydrophobic microchannel is positioned on the microchannel layer, and other structures can be positioned on the reagent layer), so that the processing technology of the chip is simplified, and the stability of chip detection is improved; meanwhile, the whole complex quantitative distribution process is finished by the chip and the centrifugal instrument, the operation of professional detection personnel is not needed, and the characteristics of micro-fluidic miniaturization are combined, so that the reagent consumption is saved, the detection speed is greatly increased, and the detection cost is reduced.
The centrifugal chip is provided with a plurality of detection units to complete high-throughput processing of a plurality of samples, each detection unit can complete rapid quantitative mixing of a plurality of reagents or samples, each detection unit is provided with a plurality of reaction chambers, and each reaction chamber is an independent reaction chamber and can complete a plurality of biochemical reactions. The centrifugal chip can accurately and quantitatively distribute liquid in a low-speed centrifugation or simple manual sample introduction mode, and finally, the mixing of different liquids is completed at a high speed. Specifically, when a low-speed centrifugation or simple manual sample introduction mode is adopted, liquid can enter the quantitative chamber, but the hydrophobic micro-channel cannot be broken through, and redundant liquid enters the waste liquid pool along with the distribution channel; adopt high-speed centrifugation after the ration is accomplished, quantitative liquid can get into the reaction chamber in proper order according to the order by the outside ration cavity in the centre of a circle and mix, because during low-speed centrifugation, some liquid can be remained to the reagent sample application port, and this partial unnecessary liquid can get into the sacrifice cavity under high-speed centrifugation's the condition, and can not influence the liquid ration of reaction chamber. According to the invention, any one detection unit can comprise a plurality of quantitative chambers, and can realize the simultaneous quantitative distribution and mixing of a plurality of reaction reagents (or samples). In addition, the mixing mode can be various, for example, preliminary mixing can be achieved by controlling the sequential entering of the reagents or samples, and mixing can also be achieved by controlling the acceleration of the centrifugal rotation speed and the continuous change of the centrifugal direction.
Specifically, the present invention can achieve the following advantageous effects:
(1) The chip has simple structure, simple and efficient processing technology, does not need complex valve control, and increases the stability of the chip; the whole structure of the chip can only have an upper cover and a lower cover (namely, the micro-channel layer and the reagent layer), the chip structure is relatively simple, the chip processing technology is greatly simplified, the processing and the manufacturing are simple, and the later-stage mass production development is facilitated.
(2) The chip can realize simultaneous proceeding of various reactions and simultaneously complete simultaneous accurate quantification of various reagents in one reaction, thereby realizing high-throughput quantitative distribution and reaction analysis of various reagents.
(3) The chip drives liquid to flow by adopting centrifugal force, and the magnitude of fluid driving force can be accurately controlled by controlling the centrifugal speed.
In conclusion, the invention provides the automatic microfluid quantitative distribution chip which is cheap to process, mass-produced, simple, rapid and high-flux, and can be applied to rapid detection scenes of immunity, nucleic acid, pathogenic bacteria and the like.
Drawings
FIG. 1 is a schematic diagram of the overall structure of a first type of assay chip for high throughput reagent quantification according to the present invention.
FIG. 2 is a schematic diagram of the structure of the detection unit of the first type of analysis chip for high throughput reagent quantitative distribution according to the present invention.
FIG. 3 is a schematic diagram of the overall structure of a second type of analysis chip for high throughput reagent quantitative distribution according to the present invention.
FIG. 4 is a schematic diagram of the second type of analysis chip detection unit for high throughput reagent quantitative distribution according to the present invention.
FIG. 5 shows the results of the pigment test of the first type of assay chip with high throughput reagent dosing according to the present invention; in fig. 5, a indicates a low-speed quantitative detection result (specifically, 800 rpm), and B indicates a high-speed mixing detection result (specifically, 2500 rpm).
FIG. 6 shows the result of the pigment test of the second type of analysis chip with high-throughput reagent quantitative distribution according to the present invention; in fig. 6, a indicates a low-speed quantitative detection result (specifically, 800 rpm), and B indicates a high-speed mixing detection result (specifically, 2500 rpm).
The meanings of the reference symbols in the figures are as follows: 1-reagent storage chamber, 2-reagent sample loading port, 3-liquid distribution drainage channel, 4-quantification chamber, 5-waste liquid chamber, 6-air hole, 7-reaction chamber, 8-hydrophobic microchannel, 21-reagent storage chamber, 22-reagent sample loading port, 23-liquid distribution drainage channel, 24-quantification chamber, 25-waste liquid chamber, 26-air hole, 27-reaction chamber, 28-hydrophobic microchannel, 100-first class analysis chip and 200-second class analysis chip.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
In general, the chip for the microchannel assisted high-throughput reagent quantitative distribution and analysis of the present invention may be a disk-type analysis chip, one disk-type analysis chip is designed with one or more detection units, the single detection unit includes a reagent storage chamber, a quantitative distribution unit and an auxiliary microchannel unit, the reagent storage chamber is connected with the quantitative distribution unit; the quantitative distribution unit is used for quantitatively distributing liquid during low-speed centrifugation or manual sample introduction, and one or more quantitative distribution units can be arranged on one detection unit; the quantitative distribution unit comprises a liquid distribution drainage channel and a quantitative cavity; the microchannel unit is a microchannel connecting the quantitative cavity and the reaction cavity and connecting the quantitative cavity and the quantitative cavity.
The volume of the dosing chamber is determined by the size of the chamber it is designed for, and is not limited to a fixed format.
The micro-channel is a hydrophobic micro-channel, has hydrophobic property, can be made of hydrophobic materials, and can also be caused by hydrophobic modification. The hydrophobic material may be Polydimethylsiloxane (PDMS), carbon powder, wax film, or the like; the hydrophobic modification agent is one of wax, silanization agent, teflon AF, CYTOP and ECG1700 coating on the surface of the micro-channel.
Taking the reagents which are all the reagents taking water as a solvent as an example, due to the resistance effect of the hydrophobic microchannels on the reagents, under the condition of low rotating speed, due to the small centrifugal force, the resistance of the hydrophobic microchannels cannot be overcome, the hydrophobic microchannels are in a non-conducting state (at the moment, the hydrophobic microchannels are in a non-conducting state on the reagents), and the analysis chip can quantitatively distribute the reagents; at high rotational speeds, the hydrophobic microchannel will be in a conducting state (at this time, the hydrophobic microchannel will be in a conducting state to the reagent) due to the greater centrifugal force, which exceeds the resistance of the hydrophobic microchannel. Certainly, for a reagent which is not water as a solvent (for example, ethanol as a solvent), because the resistance of the hydrophobic microchannel to the reagent still objectively exists, the analysis chip of the invention still has similar technical effects of high-flux reagent quantitative distribution, but only the low-speed condition of quantitative distribution and the high-speed condition of reagent mixing occur at the moment, the specific rotating speed value intervals of the low-speed condition and the high-speed condition change, and meanwhile, the material modified by the microchannel hydrophobicity can be changed to increase the resistance to the ethanol solution. Further, in order to increase the resistance of the hydrophobic microchannel to the reagent, the hydrophobic microchannel may be configured to be bent, and the more the bending times, the greater the resistance (of course, if the linear hydrophobic microchannel can meet the resistance requirement, the linear hydrophobic microchannel may also be designed).
When a manual sample feeding mode is adopted (such as a syringe pump is utilized), after the liquid is continuously injected, a section of air is injected, similar to a low-speed centrifugation mode, the liquid can be injected into the quantitative chamber, and the redundant liquid is pushed by the air to enter the sacrificial chamber (of course, the stability may not be as good as that of the low-speed centrifugation mode).
In addition, the liquid distribution drainage channel extends from the reagent storage chamber to the waste liquid chamber, and can be designed to be continuously away from the circle center (namely, continuously more away from the circle center before from the sample adding port to the waste liquid chamber), so that the liquid flow during centrifugation is facilitated (of course, the distance from the circle center can also be kept unchanged).
The liquid distribution drainage channel is communicated with the waste liquid pool, the size of the quantitative cavity can be preset and adjusted at will, the size of the quantitative cavity determines the volume of the quantitative liquid, and the liquid can be particularly quantified in an equal or gradient manner. Further, accurate quantitative distribution of the sample concentration gradient in the reaction chamber can be achieved. The height of the dosing chamber may be, for example, but is not limited to, 0.5 to 3 mm.
Analysis chips can be classified into two types, which are respectively referred to as a first type analysis chip and a second type analysis chip, according to whether the quantification is equivalent or the quantification is gradient.
These two types of analysis chips are described in detail below with reference to specific examples:
example 1 (gradient quantitation):
the chip for the quantitative distribution and analysis of the microchannel-assisted high-flux reagent is a disc-type analysis chip, the structure of the chip comprises a microfluidic channel layer and a reagent layer (the 2 layers are arranged one above the other, and the upper layer and the lower layer are not limited), and fig. 1 is a schematic diagram of the overall structure of a first type of analysis chip for the quantitative distribution of the hydrophobic microchannel-assisted high-flux reagent. The disc-type assay chip comprises one or more detection units (2 detection units are shown in FIG. 1; the number of the detection units can be flexibly adjusted, for example, the number of the detection units can be selected according to the quantity of the quantitative chambers, and if the quantity of the quantitative chambers is less, the detection units of the chip can be correspondingly increased), and FIG. 2 is a schematic structural diagram of the detection units of the first type of assay chip for the hydrophobic microchannel assisted high-throughput reagent quantitative distribution. The detection unit comprises a reagent storage chamber 1, a reagent sample adding port 2, a liquid distribution drainage channel 3, a quantification chamber 4, a waste liquid chamber 5, an air hole 6, a reaction chamber 7 and a hydrophobic microchannel 8 (wherein the hydrophobic microchannel 8 can be independently arranged on a microfluidic channel layer, and other structures can be arranged on a reagent layer); the agent storage chamber 1 is provided with a sample adding port 2 for adding a sample; the liquid distribution drainage channel 3 is communicated with the reagent storage chamber 1, the quantitative chamber 4 and the waste liquid chamber 5 and is used for accurately and quantitatively distributing the reagent during low-speed centrifugation; the hydrophobic microchannel 8 is communicated with the quantitative chamber 4 and the reaction chamber 7 and is used for draining liquid in the quantitative chamber 4 during high-speed centrifugation, so that accurately quantitative reagents enter the final reaction chamber 7 in sequence and impact mixing and diffusion effects are generated; the waste liquid chamber 5 communicates with the liquid distribution tapping channel 3 for collecting waste liquid.
The disc-type analysis chip has a rotation center, and the liquid distribution drainage channel 3 is far away from the rotation center relative to the reagent storage chamber 1 and the reagent sample adding port 2; the dosing chamber 4 is further away from the centre of rotation with respect to the liquid dispensing tapping channel 3 (i.e. the dosing chamber 4 is provided on a side wall of the liquid dispensing tapping channel 3 remote from the centre of rotation); the waste liquid chamber 5 and the reaction chamber are further away from the center of rotation with respect to the hydrophobic microchannel 8.
As shown in fig. 1 and 2, in the present embodiment, for any one of the liquid distribution drainage channels 3, from the head end to the tail end thereof, there are several dosing chambers 4, wherein the 1 st dosing chamber 4 appears as a sacrificial chamber (the volume may be set larger in advance); and starting from the 2 nd dosing chamber 4:
(a) For the reagent dosing channel located on the outside, the volume of the dosing chamber 4 increases gradually;
(b) For the reagent dosing channel located inside, the volume of the dosing chamber 4 gradually decreases.
Any one of the detection units shown in fig. 1 and 2 includes 3 reagent quantitative distribution channels, which is only an example, and the number of the reagent quantitative distribution channels can be flexibly adjusted according to the actual situation as long as at least 1 reagent quantitative distribution channel is included; for example, in an extreme case, a detection unit may include only 1 reagent quantitative distribution channel (where several quantitative chambers 4 are connected to the reaction chamber 7 through the hydrophobic micro-channel 2), and still perform the functions of quantitative distribution and mixing (at this time, some reagents, such as powder, may be pre-buried in the reaction chamber 7, and mixing of the liquid reagent and the powder after quantitative distribution may be achieved).
In this embodiment, as shown in fig. 5, the size of the chip is a disc-shaped chip with a diameter of 120mm, and three dyeing dye solutions of red, blue and green are sequentially added from the center of the circle to the outside of the three chambers.
The chip was first subjected to quantification at a low speed and then to mixing at a high speed, and the results of the detection are shown in FIG. 5. As can be seen from FIG. 5, under low-speed centrifugation, the three dyed dyes can achieve good accurate quantification, and the volume of the quantification is determined by the volume of the quantification chamber; under high-speed centrifugation, all liquid is smoothly drained into the reaction chamber, the final reaction volume is basically kept consistent, the quantification has higher accuracy, and the mixing of various reagents according to the gradient proportion can be realized.
Example 2 (equivalent quantification):
the invention relates to a chip for high-flux reagent quantitative distribution and analysis assisted by a hydrophobic microchannel, which is a disc type analysis chip, the structure of the chip comprises a microfluidic channel layer and a reagent layer, and figure 3 is a schematic diagram of the overall structure of a second type of analysis chip for high-flux reagent quantitative distribution assisted by the hydrophobic microchannel. The disc-type assay chip comprises one or more detection units (3 detection units are shown in fig. 3, the number of the detection units can be flexibly adjusted), and fig. 4 is a schematic structural diagram of the detection units of a second type of assay chip for hydrophobic microchannel assisted high-throughput reagent quantitative distribution. The detection unit comprises a reagent storage chamber 21, a reagent loading port 22, a liquid distribution drainage channel 23, a quantification chamber 24, a waste liquid chamber 25, an air hole 26, a reaction chamber 27 and a hydrophobic microchannel 28; the agent storage chamber 21 is provided with a sample adding port 22 for adding sample; the liquid distribution drainage channel 23 is communicated with the reagent storage chamber 21, the quantitative chamber 24 and the waste liquid chamber 25, and is used for accurately and quantitatively distributing the reagent during low-speed centrifugation; the hydrophobic micro-channel 28 is communicated with the quantitative chamber 24 and the reaction chamber 27 and is used for draining liquid in the quantitative chamber 24 during high-speed centrifugation, so that accurately quantitative reagents enter the final reaction chamber 27 in sequence and impact mixing and diffusion effects are generated; the waste liquid chamber 25 communicates with the liquid distribution tapping channel 23 for collecting waste liquid.
The disc-type assay chip of the present invention has a rotation center, and the liquid distribution drainage channel 23 is located farther from the rotation center than the reagent storage chamber 21 and the reagent addition port 22; the dosing chamber 24 is further from the centre of rotation with respect to the liquid dispensing drainage channel 23; the waste liquid chamber 25 and the reaction chamber are further away from the center of rotation with respect to the hydrophobic microchannel 28.
As shown in fig. 3 and 4, in the present embodiment, for any one of the liquid distribution drainage channels 23, from the head end to the tail end thereof, there are several dosing chambers 24, wherein the 1 st dosing chamber 24 appears as a sacrificial chamber (the volume may be set larger in advance); whereas starting from the 2 nd dosing chamber 24 the volume of the dosing chamber 24 remains fixed.
Since the disc-type chip drives the fluid to move by means of centrifugal force, the magnitude of the centrifugal force is related to the distance between the quantitative chamber 24 and the circle center, the farther away from the circle center, the larger the centrifugal force at the same speed, and therefore, the more resistance the hydrophobic microchannel needs to be farther away from the circle center, the more channels are bent.
Any one of the detecting units shown in fig. 3 and 4 includes 6 reagent quantitative distribution channels, which is only an example, and the number of the reagent quantitative distribution channels can be flexibly adjusted according to actual situations as long as at least 1 reagent quantitative distribution channel is included; for example, in an extreme case, a detection unit may include only 1 reagent quantitative distribution channel (the quantitative chambers 24 on the reagent quantitative distribution channel are connected to the reaction chamber 27 through the hydrophobic micro-channels 22), and still perform the functions of quantitative distribution and mixing (at this time, some reagents, such as powder, may be pre-filled in the reaction chamber 27, and mixing of the liquid reagent after quantitative distribution and the powder may be achieved).
In this embodiment, as shown in fig. 6, the size of the chip is a disk-type chip with a diameter of 240mm, six reagent sample inlets are included on one detection unit, so that accurate quantification of six reagents can be completed, and six staining dye solutions with different colors of red, green, blue, purple, light red, and black are sequentially added into six reagent chambers from the center of the circle to the outside, so as to represent six different reagents.
The chip was first subjected to quantification at a low speed and then to mixing at a high speed, and the results of the detection are shown in FIG. 6. As can be seen from FIG. 6, under low-speed centrifugation, six dyes with different colors can be well and accurately quantified, the quantitative volume is determined by the volume of the quantitative chamber, under high-speed centrifugation, all liquid is smoothly drained into the reaction chamber, the final reaction volume is basically kept consistent, and the quantification has higher accuracy, so that the precise quantification and mixing of various reagents can be realized.
In addition, the processing material of the chip for the hydrophobic microchannel assisted high-flux reagent quantitative distribution and analysis in the invention can be PMMA, PC, PP, PDMS, etc., and the processing method includes CNC numerical control processing, laser engraving, soft lithography, 3D printing and injection molding to form a mold, but is not limited thereto. For example, because the main structure of the disc-type chip is a microfluidic channel layer and a reagent layer, when the chip is processed by using the CNC technology, the chip is only attached in a double-layer structure; when the laser engraving technology is used for processing, the chip only needs to be provided with the upper cover plate and the lower cover plate. Of course, other shapes than a disk are also applicable as long as the chip has a rotation center and the distribution of the liquid distribution channels satisfies an arc distribution with reference to the rotation center.
It will be understood by those skilled in the art that the foregoing is only an exemplary embodiment of the present invention, and is not intended to limit the invention to the particular forms disclosed, since various modifications, substitutions and improvements within the spirit and scope of the invention are possible and within the scope of the appended claims.
Claims (11)
1. A micro-channel assisted chip for reagent quantitative distribution and analysis, characterized in that the chip is provided with a rotation center and one or more detection units inside; the detection unit comprises at least 1 reagent quantitative distribution channel which is arranged from inside to outside relative to the rotation center;
each reagent quantitative distribution channel comprises 1 arc-shaped liquid distribution drainage channel (3, 23) for quantitatively distributing the reagent or the sample to be quantitatively distributed;
along a preset rotation direction, marking two ends of the liquid distribution drainage channel (3, 23) as a head end and a tail end respectively, from the head end to the tail end, keeping the distance from the liquid distribution drainage channel (3, 23) to the rotation center unchanged or gradually increasing, connecting the head end with a reagent storage chamber (1, 21), and storing a liquid reagent or a liquid sample to be quantitatively distributed in the reagent storage chamber (1, 21);
the liquid distribution drainage channel (3, 23) has a first side wall and a second side wall which are oppositely arranged, wherein the first side wall is positioned at the inner side close to the rotation center, and the second side wall is positioned at the outer side far away from the rotation center; a plurality of quantitative chambers (4, 24) are arranged on the second side wall, and the volume of the quantitative chambers (4, 24) is preset for quantitative distribution;
any one of the detection units: for 2 adjacent reagent dosing channels, the dosing chamber (4, 24) adjacent the inner reagent dosing channel is connected to the liquid dispensing drainage channel (3, 23) adjacent the outer reagent dosing channel by a hydrophobic microchannel (2, 28), and the hydrophobic microchannel (2, 28) extends into the liquid dispensing drainage channel (3, 23) adjacent the outer reagent dosing channel to connect the dosing chambers between adjacent reagent dosing channels; such that all reagent dosing channels are connected in series, the dosing chamber (4, 24) of the outermost reagent dosing channel is further connected to the further outer reaction chamber (7, 27) by a hydrophobic microchannel (2, 28), and the hydrophobic microchannel (2, 28) extends into the reaction chamber (7, 27); the inner wall of the hydrophobic microchannel (2, 28) has hydrophobicity and can generate resistance effect on a liquid reagent or a liquid sample, so that the centrifugal force is controlled through the rotation speed, and the relation between the centrifugal force and the resistance is regulated and controlled, namely whether the hydrophobic microchannel (2, 28) conducts the reagent or the sample can be controlled; the reaction chamber (7, 27) is used for collecting the quantified reagent or sample, and the reagent or sample is mixed in the reaction chamber (7, 27);
and, the chip has a 2-layer structure, 2 layers being arranged one on top of the other, wherein the hydrophobic microchannel (2, 28) is located in a 1-layer structure thereof, and the reagent storage chamber (1, 21), the liquid distribution drainage channel (3, 23), the quantifying chamber (4, 24), and the reaction chamber (7, 27) are located in another 1-layer structure thereof;
the analysis chip is capable of quantitatively dispensing a reagent or a sample when the analysis chip is rotated about the rotation center and the hydrophobic microchannel (2, 28) is in a non-conductive state with respect to the reagent or the sample;
when the analysis chip rotates around the rotation center and the hydrophobic micro-channel (2, 28) is in a conducting state for a reagent or a sample, the analysis chip can drain and mix the quantitatively distributed reagent or sample;
at least 2 quantitative chambers (4, 24) are arranged in any reagent quantitative distribution channel; wherein the dosing chamber (4, 24) closest to the first end is a sacrificial chamber for receiving excess reagent or excess sample generated during spin centrifugation.
2. The microchannel-assisted chip for reagent dosing and analysis according to claim 1, wherein the volumes of the dosing chambers (4, 24) are kept uniform or gradually increased from the head end to the tail end except for the sacrificial chamber for the reagent dosing channel located outside;
for the reagent dosing channel located inside, the volume of the dosing chamber (4, 24) is kept uniform or gradually decreases from the head end to the tail end, except for the sacrificial chamber.
3. The microchannel-assisted chip for reagent quantitative distribution and analysis according to claim 1, wherein the hydrophobic microchannel (2, 28) has a height of 10 to 500 μm and a width of 10 to 500 μm; correspondingly:
when the rotating speed is 500-1000 rpm, the analysis chip can quantitatively distribute the liquid reagent or the liquid sample;
when the rotating speed is 2000-3000 rpm, the analysis chip can drain and mix the liquid reagent or the liquid sample after quantitative distribution.
4. Micro-channel assisted chip for the quantitative dispensing and analysis of reagents according to claim 1, characterized in that the hydrophobic micro-channels (2, 28) have a hydrophobic coating on their inner walls, in particular with wax, silanization reagents, teflon AF, CYTOP or electron-fluorinated liquids.
5. The microchannel-assisted chip for reagent dosing and analysis of claim 4, wherein the electronic fluoridating fluid is ECG1700.
6. The microchannel-assisted chip for reagent dosing and analysis according to claim 1, wherein the hydrophobic microchannel (2, 28) has a meandering structure for increasing its resistance to liquid reagents or liquid samples.
7. The micro-channel-assisted chip for reagent dosing and analysis according to claim 6, wherein any one of the hydrophobic micro-channels (2, 28) corresponding to the reagent dosing channel located at the outer side has a more bent structure, and any one of the hydrophobic micro-channels (2, 28) corresponding to the reagent dosing channel located at the inner side has a less bent structure.
8. The microchannel-assisted chip for reagent metering and analysis according to claim 1, wherein the reagent storage chamber (1, 21) is further provided with a reagent loading port (2, 22) for replenishing a liquid reagent or a liquid sample to be metered into the reagent storage chamber (1, 21);
the reaction chamber (7, 27) is also connected with an air hole (6, 26) for balancing the air pressure in the reaction chamber (7, 27);
the tail end of any one of the liquid distribution drainage channels (3, 23) is also communicated with a waste liquid chamber (5, 25).
9. The microchannel-assisted chip for quantitatively dispensing and analyzing a reagent as set forth in claim 8, wherein the liquid-dispensing drainage channels (3, 23) in any one of the detecting units communicate with the same waste liquid chamber (5, 25).
10. The microchannel-assisted chip for reagent dosing and analysis according to claim 8, wherein the reagent storage chamber (1, 21), the reagent loading port (2, 22), the liquid distribution drainage channel (3, 23), the dosing chamber (4, 24), the waste chamber (5, 25) and the reaction chamber (7, 27) are located in the same layer of a 2-layer structure of the chip.
11. The microchannel-assisted chip for reagent dosing and analysis of claim 10, wherein the chip is a disk-type chip.
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| CN115350733B (en) * | 2022-07-13 | 2023-12-22 | 中国科学院上海微系统与信息技术研究所 | Microfluidic chip with single-layer inclined structure liquid drop storage cavity and preparation method thereof |
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