CN110787851B - Multi-channel liquid drop quantitative measuring device and method based on pressure driving - Google Patents
Multi-channel liquid drop quantitative measuring device and method based on pressure driving Download PDFInfo
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- CN110787851B CN110787851B CN201911025326.9A CN201911025326A CN110787851B CN 110787851 B CN110787851 B CN 110787851B CN 201911025326 A CN201911025326 A CN 201911025326A CN 110787851 B CN110787851 B CN 110787851B
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Abstract
The invention discloses a pressure-driven multichannel quantitative liquid drop measuring device, which comprises: a spotting probe for performing spotting operation on the sample solution; a working medium reservoir for containing a working medium; the pressure control system is used for controlling the pressure in the working medium liquid storage tank; the fluid damper is provided with a micro-channel structure and has a damping effect on fluid in the micro-channel structure, the liquid inlet end of the fluid damper is communicated with the medium liquid storage tank, and the liquid outlet end of the fluid damper is communicated with the liquid inlet end of the sample application probe in a sealing way. The invention has the capability of quantitatively measuring the liquid drops from pico-liter to micro-upgrade and the precision of quantitatively measuring the liquid drops from pico-upgrade. The invention can measure various solutions with different viscosities in parallel, which is an essential capability for large-scale high-throughput screening applications such as drug screening, protein crystallization condition screening and the like which need to process a large amount of solutions with different properties. The device is easy to construct, convenient to operate and good in reliability.
Description
Technical Field
The invention relates to the field of micro-fluidic of analytical chemistry, in particular to a pressure-driven multi-channel liquid drop quantitative measuring device and method.
Background
The liquid drop refers to a discontinuous fluid formed by separating one phase of liquid from another phase or a plurality of phases of liquid which are not mutually soluble. Where the droplets are formed, the discontinuous phase is referred to as the dispersed phase and the liquid separating the droplets is referred to as the continuous phase. In the field of microfluidics, the most common droplet systems use aqueous solutions as the dispersed phase and mineral or fluoro oils as the continuous phase, where the droplet volumes are typically on the order of picoliters to nanoliters.
In the previous research, the inventor's research group developed a quantitative liquid drop measuring and operating method which can measure liquid with pico-liter precision by matching a precision syringe pump, a micro-sampler and a tip-pulling capillary, sequentially mix a plurality of solutions to assemble a composite liquid drop, and linearly store the liquid drop in the capillary to perform reaction or measurement. On this basis, the SODA technology has been produced. The SODA technology does not store liquid drops in a capillary linearly, but stores the liquid drops on an open chip, and completes generation, addressing, sampling, transferring, splitting and fusion of liquid drops from picoliter to nanoliter by three primitive operations of sucking, spotting and moving. The SODA system adopting the SODA technology has been applied in the fields of drug screening, protein crystallization condition screening and the like. However, in actual use, the deficiency of insufficient flux of existing SODA systems is revealed. The best way to increase the throughput of a SODA system is to develop a multi-channel SODA system. However, since the conventional SODA technology employs a micro-fluid driving system with a precision syringe pump and a micro-injector as a core, the system structure is complicated, and initialization is inconvenient, so that it is difficult to realize a reliable and easy-to-use multi-channel SODA system under the conventional SODA technology.
The pressure driving is a common fluid driving mode in the field of microfluidics, and has the characteristics of simple device and convenient control. For a specific microfluidic channel and specific liquid, due to the fact that the fluid resistance is constant, the volume of the liquid flowing through the microfluidic channel can be effectively controlled by controlling the pressure and the application time. The method has application in the aspect of quantitative distribution of trace liquid and shows good parallel operation capability. However, as can be seen from the principle of the above method, the precondition for quantitatively measuring a small amount of liquid by using the method is to know the viscosity of the liquid to be measured first, so although the above method is simple in device, convenient to control, and wide in controllable liquid volume range, the method is mainly used for large-scale distribution of one or a few kinds of liquid with known viscosity, but cannot be used for quantitatively measuring a liquid with unknown viscosity, and even cannot be used for quantitatively measuring a plurality of kinds of liquid with unknown viscosity in a parallelization manner.
Good controllability of the fluid resistance is another key element of pressure-driven multichannel micro-liquid quantitative metering. In the practice of quantitative measurement of trace liquid, the tip-drawing capillary tube can effectively improve the manipulation capability of the trace liquid. However, the problem that the fluid resistance of the tip capillary is difficult to control due to the preparation process causes that the overall fluid resistance of the pipeline is difficult to accurately control. This makes it difficult to apply the tapered capillary to a multi-channel micro-liquid quantitative measuring device based on pressure driving.
Disclosure of Invention
The invention provides a pressure-driven multichannel quantitative liquid drop measuring device and an operation method, which can be used for picoliters (10)-12L) to microliter (10)-6Liter) quantitative measurement and operation of the stage droplets.
By utilizing the characteristics of multiphase liquid drops, the invention overcomes the defect that the traditional pressure-driven liquid quantitative measuring method cannot be used for quantitatively measuring liquid with unknown viscosity. By introducing a fluid damper to increase the total fluid resistance by several orders of magnitude, the present invention ensures that the accuracy of the fluid resistance of each channel in a multi-channel device is not reduced even if a tipped capillary is used. The device for quantitatively measuring the liquid drops has the advantages of simple structure, convenient operation and easy large-scale parallelization, and is very suitable for massively generating the liquid drop arrays consisting of a large amount of different solutions and performing parallelization operation on the liquid drop arrays by combining the operation method. The invention has high flux characteristic, so the invention is especially suitable for drug screening, protein crystallization condition screening and other applications.
The scheme provided by the invention is as follows:
a pressure-driven multichannel quantitative liquid drop measuring device comprises:
a spotting probe for performing spotting operation on the sample solution;
a working medium reservoir for containing a working medium;
the pressure control system is used for controlling the pressure in the working medium liquid storage tank;
the fluid damper is provided with a micro-channel structure and has a damping effect on fluid in the micro-channel structure, the liquid inlet end of the fluid damper is communicated with the medium liquid storage tank, and the liquid outlet end of the fluid damper is communicated with the liquid inlet end of the sample application probe in a sealing way.
The pressure control system of the present invention may be a system for realizing pressure control only, and a matched pressure source device is required to be arranged at this time, or a pressure source and a pressure control system comprising a pressure source may be adopted. The pressure control system can provide positive and negative pressure sources and control the magnitude and timing of the pressure application. The control of the pressure and the application time can be realized by an industrial computer or a control chip or a control circuit. The pressure control system or the pressure source and the pressure control system can be directly connected with the working medium liquid storage tank through a pipeline.
The pressure source and pressure control system can provide positive and negative pressure sources, wherein the pressure source can be, but is not limited to, a pneumatic source (such as a high pressure air tank or other gas source), a hydraulic source, or a syringe pump, and the pressure is generated by providing a flow. The pressure source in the pressure source and pressure control system has three pressure options of positive pressure source, negative pressure source and atmosphere, and can be composed of one or more positive pressure sources and one or more negative pressure sources. The three pressure options of the positive pressure source, the negative pressure source and the atmosphere can be switched by a pressure control system consisting of a series of electromagnetic valves, and the control of the pressure application time is completed. Preferably, the pressure source and pressure control system comprises an adjustable pressure source capable of providing a continuously adjustable pressure. The pressure control system of the invention can also be a simple hydraulic bottle device with a control valve, the control valve can be a manual valve or an electromagnetic valve, and when the electromagnetic valve is adopted, the automatic control can be realized by a control unit. The control unit can be a computer, a control board or a control chip programmed with set functions, or a control circuit with equivalent functions, and the like, and can be selected from existing products or programmed by adopting an existing conventional method or designed by parameters of computer software. Alternatively, the pressure source and pressure control system employs a servo pressure control device capable of directly providing positive and negative pressures and controlling the pressure application time.
Preferably, two or all of the medium reservoir, the deposition probe and the fluid damper are of an integrated construction. That is, the deposition probe, the fluid damper, the working medium reservoir and the fixture (i.e. the connector or fixture for achieving a previous fixation of the deposition probe, the fluid damper or/and for achieving an overall fixation of the deposition probe, the fluid damper) may be manufactured separately and finally assembled. Or the spotting probe and the fluid damper can be integrally manufactured (at the moment, a connecting piece between the spotting probe and the fluid damper can be omitted), and finally the spotting probe and the fluid damper are assembled with the working medium reservoir. Or the fluid damper and the working medium liquid storage tank can be integrally manufactured and finally assembled with the sample application probe. And the sample application probe, the fluid damper and the working medium liquid storage tank can be completely integrated, so that the assembly process is avoided. In an integrated fabrication scheme, the array of deposition probes and fluidic dampers may or may not require fixings for positioning and fixing. The device adopts a partially integrated or fully integrated structure, so that the device is more compact in structure, simpler and more convenient to process, and more easy to realize standardized manufacture and use.
Preferably, the drop dosing channel of the deposition probe and the fluid dampener can be a single channel or multiple parallel channels. Preferably, the spotting probe and the fluid damper form a liquid drop quantitative measuring channel to form an array, the whole positioning and fixing relative to the whole device are realized through a fixing piece, and the spotting probe and the fluid damper can be fixed through a connecting piece in front.
Preferably, the spotting probe and the fluid damper form a quantitative drop tapping channel that can be multiplexed and parallel, and the number of parallel channels can be, but is not limited to, 8 channels, 12 channels, 96 channels, 384 channels, and 1536 channels.
Preferably, the deposition probe of the invention is made using a capillary, such as a tipped capillary. For water-soluble samples, the surface of the sample application probe is subjected to low surface energy treatment or the surface energy of the material is low, so that the sample is not adhered to the wall of the probe, and the sample can be accurately measured.
In the present invention, the fluid damper is mainly used for providing a specified (set amount or determined amount) of fluid resistance, and may be constituted by a long and thin pipe, a minute hole, or a combination of both. The cross-section of the conduits and holes may be, but not limited to, circular, polygonal, or other one or more contoured shapes. The cross-sectional shape of the conduit may be constant or may vary.
Preferably, the fluid damper may be cut out of a circular capillary tube having a thin inner diameter. According to different actual requirements, the inner diameter of the capillary tube used as the fluid damper can be 0.1-1000 μm, preferably 0.5-500 μm, and more preferably 1-250 μm; the length of the capillary tube as the fluid damper may be 0.1 to 1000mm, preferably 0.5 to 500mm, and more preferably 1 to 500 mm. The capillary tube may be made of, but not limited to, non-metal materials such as quartz, glass, and PEEK, or metal materials such as stainless steel and brass. The fluid damper with required precision can be directly cut by the capillary tube, and the precision of the fluid damper can be further improved by measurement and finish machining.
Preferably, the fluid damper can be obtained by processing the microchannel by a microfluidic chip processing technology. By utilizing the micro-fluidic chip processing technology, one fluid damper or a plurality of fluid dampers or an array formed by the fluid dampers can be obtained at one time.
Further, the fluid resistance of the fluid damper accounts for a majority of the fluid resistance of the entire flow path at the rated flow rate, preferably, 90% or more, more preferably, 95% or more, more preferably, 99% or more, more preferably, 99.9% or more. The higher the fluid resistance ratio of the fluid damper, the higher the fluid resistance control accuracy of the entire flow path.
Further, the fluidic resistance of the fluidic damper is much greater than the deposition probe, so that the fluidic resistance error of the deposition probe does not have a significant impact on the overall flow path fluidic resistance accuracy. Preferably, the fluidic resistance of the fluidic damper is more than 10 times, more preferably more than 20 times, more preferably more than 50 times, more preferably more than 100 times, more preferably more than 500 times, more preferably more than 1000 times the fluidic resistance of the deposition probe.
Furthermore, the whole device composed of the sample application probe, the working medium reservoir, the pressure control system and the fluid damper can be combined into a larger-scale composite device with the same specification or different specifications, and each device in the composite device can be independently controlled.
Preferably, the fluid resistance of the fluid damper accounts for more than 90% of the fluid resistance of the entire flow path at the rated flow rate, and the entire flow path consists of the micro flow channel structure in the fluid damper and the internal channel of the spotting probe; or the fluidic resistance of the fluidic damper is more than 10 times the fluidic resistance of the deposition probe. More preferably, the fluid resistance of the fluid damper accounts for 95% or more of the fluid resistance of the entire flow path at the rated flow rate; or the fluidic resistance of the fluidic damper is more than 20 times the fluidic resistance of the deposition probe.
Preferably, the present invention further comprises:
a temperature sensor for detecting a temperature of a working environment;
and the controller receives the temperature information of the temperature sensor and determines the operating pressure and the operating time of the pressure control system by combining the viscosity information of the working medium.
According to the working medium (called as the working medium I, namely the working medium I is placed in the working medium liquid storage tank and the fluid damper), liquid with known viscosity can be selected, the relation between the viscosity and the temperature can be detected in advance, the temperature of a working environment can be obtained through the temperature sensor, the viscosity of the working medium I in the working environment state can be further obtained, further, appropriate operating pressure can be selected according to the volume of the sample liquid to be operated, the fluid damping (which can be detected in advance) of the fluid damper and the viscosity of the working medium, the operating time is calculated, and further manual or automatic control can be achieved.
The device of the invention can be produced and sold separately, and can also be provided with a matched porous plate or chip. The multi-hole plate is mainly used for containing sample solution, and the multi-hole plate with the appropriate number of holes can be selected according to needs. The chip is mainly used for generating liquid drops, and the chips with the appropriate number of micro pits can be arranged according to the requirement.
Preferably, the method further comprises the following steps:
a multi-well plate for holding a sample solution;
the chip is used for storing liquid drops, and a micro-pit array formed by micro-pits with large openings and small bottoms is arranged on the chip.
Preferably, the well of the multi-well plate corresponds to the position of the spotting probe array (the spotting probe adopts an array channel structure at this time) and the position of the micro-pits of the chip, so as to facilitate the realization of automatic operation.
According to the invention, the micro-pits on the chip have a structure with a large top and a small bottom, are used for storing liquid drops, and are filled with a working medium II. The key point of the chip design is that the side wall of the micro-pit always keeps a larger taper and extends to the bottom surface of the micro-pit. The size of the bottom of the micro-pit is selected in relation to the size of the droplet finally contained by the micro-pit, and the size of the bottom of the micro-pit should be not more than 3 times, preferably 1 time, the diameter of the droplet finally contained (which can be determined by experience or preliminary experiment) so as to ensure effective fusion between different droplets after centrifugation.
The micro-pits on the chip can be in a round table or square table structure with a large upper part and a small lower part, and the included angle formed by the side walls of the micro-pits and the vertical line of the chip is not more than 60 degrees, more preferably not more than 45 degrees, more preferably not more than 30 degrees. The smaller included angle is beneficial to the subsequent fusion operation of the liquid drops. The array formed by the micro pits on the chip can correspond to a standard orifice plate, and the number of the orifices and the intervals of the orifices can be designed according to requirements. Preferably, the array of micro-wells on the chip can be a standard 96-well plate array, a standard 384-well plate array, a standard 1536-well plate array, or a higher density array.
For a common aqueous solution system, the chip is made of a low surface energy material, or the surface of the micro-pit in the chip is subjected to low surface energy treatment, so that the phenomenon that liquid drops are adhered to the surface of the chip and cannot fall into the bottom surface of the micro-pit is avoided. Preferably, the diameter of the bottom surface of the micro-pits should be no more than 3 times the diameter of the finally contained droplets; the included angle formed by the side wall of the micro pit and the vertical line of the chip is not more than 60 degrees.
The invention provides a pressure-driven multichannel quantitative liquid drop measuring device, which has the following principle:
in a complete flow path formed by the point sample probe, the fluid damper, the working medium liquid storage tank and the pressure control system (the pressure source and the pressure control system), for the flow rate of the order of picoliters/minute to microliter/second, the pressure drop on the fluid damper can be mostly caused by design, and the pressure difference between two ends of the fluid damper is approximately considered to be the pressure provided by the pressure source and the pressure control system. According to the fluid dynamics principle, under the condition that the fluid damping of the fluid damper and the viscosity of the working medium are known, the pressure source and the pressure control system can be used for controlling the pressure and the application time at two ends of the fluid damper, and further controlling the volume of the working medium flowing through the fluid damper. Because the fluid damper is connected with the spotting probe, the working medium flowing through the fluid damper will produce a displacement effect in the spotting probe, thereby causing the spotting probe to inject or aspirate a specified volume of sample.
The invention also provides a method for quantitatively measuring the liquid drops by the pressure-driven multi-channel quantitative liquid drop measuring device, which comprises the following steps:
(1-1) the pressure control system applies a set pressure to make the working medium I fill the fluid damper and the sample application probe;
(1-2) inserting the spotting end (or tip) of the spotting probe into the sample solution;
(1-3) under the control of a pressure control system (executing set pressure and operation time), injecting the sample solution with a target volume out of the sample application probe or sucking the sample solution into the sample application probe to finish the suction or dripping of the sample solution;
in the present invention, the sample solution may be placed in a multi-well plate to be used.
The working medium I is immiscible with the sample solution.
Before the step (1-1), enough working medium I needs to be injected into the working medium storage tank so as to meet the requirement of the step (1-1).
When the droplet generation operation is performed, a contact type spotting, a semi-contact type spotting or a non-contact type spotting method can be adopted, so that the sample solution liquid injected out of the spotting probe is separated from the spotting probe to form an independent droplet.
As a preferable scheme, the pressure-driven multi-channel quantitative liquid drop measuring method comprises the following steps:
(2-1) injecting a working medium II into the micro-pits of the chip in advance; the operations of step (1-1) may be performed simultaneously or separately;
(2-2) injecting a target volume of sample solution or reagent solution into the working medium II in the corresponding micro-pit by using the operation spotting probe in the step (1-2) and the step (1-3) to generate 2 or more droplets to be fused;
(2-3) centrifuging the whole chip to promote the liquid drops to sink to the bottom surface of the micro-pit, and realizing the fusion of the liquid drops under the limitation of the wall of the micro-pit to form new liquid drops;
the working medium II is immiscible with the sample solution.
In the present invention, preferably, the working medium I is a liquid that is immiscible with the sample solution and has a suitable viscosity. The working medium II is a liquid which is not mutually soluble with the sample solution and has a density smaller than that of the sample solution. The working medium I may be the same as or different from the working medium II.
More preferably, for common aqueous systems, working medium I and working medium II may be oily liquids, including but not limited to hydrocarbons, esters, silicone oils, fluoropolyether oils, and combinations thereof.
As a preferable scheme, the ambient temperature can be measured or controlled by a temperature sensor, and the viscosity of the working medium in the current working environment is obtained through the viscosity-temperature relationship of the working medium;
then, selecting proper operation pressure according to the volume of the liquid to be operated, the fluid damping of the fluid damper and the viscosity of the working medium, and calculating operation time; then, the operations of the steps (1-1) to (1-3) or the operations of the steps (2-1) to (2-3) are carried out.
The invention also provides an operating method for fusing two or more droplets, comprising:
I. the chip is placed under an ion fan to blow ion wind so as to eliminate the static electricity on the surface of the chip.
And II, injecting a working medium into the chip, and centrifuging to fill all the micro pits with the working medium.
And III, blowing the spotting probe by using ion wind generated by an ion fan to eliminate the static electricity on the surface of the spotting probe.
And IV, transferring a specified volume of sample solution from the multi-hole plate to a specified micro-pit of the chip through the operation cycles (1-2) to (1-3) to generate a droplet to be fused.
And V, centrifuging the whole chip to promote the liquid drops to settle to the bottom of the micro-pit and to be fused with other liquid drops to form new liquid drops.
The invention adopts the ion wind generated by the ion fan to eliminate the static electricity on the surfaces of the chip and the sample application probe, thereby improving the success rate of the liquid drop fusion. And the step I and the step III are carried out under the ionic wind generated by the ionic wind machine, so that the success rate of liquid drop fusion is further improved.
Further, the initialization steps of the multi-channel liquid drop quantitative measuring device based on pressure driving of the invention are as follows: and injecting enough working medium into the working medium liquid storage tank, and then applying proper pressure to ensure that the working medium is filled with the fluid damper and the spotting probe.
Since the liquid level of the spotting probe tip is not equal to the liquid level of the working medium reservoir, the liquid flow resulting from the difference in the liquid levels of the two during long waiting times can be significant. The liquid flow can be eliminated by applying a certain compensation pressure by the pressure source and the pressure control system during waiting, thereby improving the reliability of quantitative measurement of the liquid drops. Preferably, when the liquid level of the spotting probe tip is not equal to the liquid level of the working medium reservoir, a compensating pressure of a set magnitude for eliminating the differential pressure flow is applied to the spotting probe by the pressure control system during the waiting period.
The multichannel liquid drop quantitative measuring device based on pressure driving can be manually operated or automatically completed under the control of a computer.
Preferably, the multi-channel liquid drop quantitative measuring device based on pressure driving can be arranged on an automatic translation stage, and the operation cycle of sampling and sample application can be automatically completed under the control of a computer.
The invention has the advantages that:
A. the invention has the capability of quantitatively measuring the liquid drops from picoliter to micro-upgrade and has the precision of quantitatively measuring the liquid drops from picoliter to picoliter. For the applications of drug screening, protein crystallization condition screening and the like, the micro-liquid manipulation capability can greatly reduce the reagent consumption and save the cost.
B. The quantitative measuring and parallel channel number of the liquid drops can be conveniently expanded, and the liquid drop measuring and parallel channel number measuring device has high parallel capability. For the applications of drug screening, protein crystallization condition screening and the like, the capability can greatly improve the screening flux and accelerate the screening speed.
C. The invention can measure various solutions with different viscosities in parallel, which is an essential capability for large-scale high-throughput screening applications such as drug screening, protein crystallization condition screening and the like which need to process a large amount of solutions with different properties.
D. The device is easy to construct, convenient to operate and good in reliability.
Based on the advantages, the invention has wide application prospect in high-throughput screening fields such as drug screening, protein crystallization condition screening and the like.
Drawings
Fig. 1 is a schematic diagram of a multi-channel liquid drop quantitative measuring device based on pressure driving.
FIG. 2 is a schematic diagram of a droplet fusion operation.
FIG. 3 is an explanatory view of the operation of example 2.
FIG. 4 is a diagram illustrating the structure of embodiment 3.
FIG. 5 is a diagram illustrating the structure of example 4.
Detailed Description
The invention will now be illustrated by way of example.
Example 1
This example applies the invention to protein crystallization condition screening. As shown in fig. 1 and 2, a multi-channel quantitative liquid drop measuring device based on pressure driving includes:
a pressure source and pressure control system 1 capable of providing positive and negative pressure sources and controlling the magnitude and application time of the pressure;
the working medium liquid storage tank 3 is used for containing a working medium 2 with a known viscosity-temperature relation;
a temperature sensor 8 capable of measuring a working environment temperature;
the liquid inlet end of the fluid damper 4 is connected with the working medium liquid storage tank 3 and communicated with the medium inner cavity of the fluid damper;
the liquid inlet end of the spotting probe 7 is hermetically connected with the liquid outlet end of the fluid damper 4 through a connecting piece 23;
a fixture 6 for positioning and fixing the array of deposition probes 7.
The pressure source and the pressure control system 1 are connected with the working medium liquid storage tank 3 and used for controlling the pressure in the working medium liquid storage tank 3, and the point sample probe 7 is connected with the working medium liquid storage tank 3 through the fluid damper 4. The connector 23 is a length of rubber tubing for sealingly connecting the fluid damper 4 and the sampling probe 7. The fixing piece 6 is of a mounting plate structure with a positioning hole, and the positioning hole is used for fixing the array of the sample application probes 7;
The chip 11 has a micro-pit array of micro-pits with large top and small bottom for storing liquid droplets, and is filled with a working medium 12. The dimples 13 on the chip 11 may be in the form of truncated cones or square mesas with larger top and smaller bottom, and the sidewalls 14 of the dimples may form an angle with the vertical of the chip of no more than 60 °, more preferably no more than 45 °, and still more preferably no more than 30 °. The smaller included angle is beneficial to the subsequent fusion operation of the liquid drops.
In this embodiment, the pressure source and pressure control system 1 is composed of an air compressor, a vacuum pump, a pressure regulating valve, a two-position three-way solenoid valve, a pipeline and an electronic control system. The high-pressure air provided by the air compressor is subjected to pressure reduction and pressure stabilization by the pressure regulating valve to form high-low two paths of positive pressure output; the vacuum pump forms strong and weak negative pressure output after the pressure is reduced and stabilized by the pressure regulating valve. The two positive pressures, the two negative pressures and the atmosphere are gated by an electromagnetic valve pipeline controlled by an electronic control system. The electronic control system is ultimately controlled by a computer program.
In the embodiment, high-low positive pressure is used for quantitative injection at two speeds, namely high speed and low speed, strong negative pressure is used for quantitative suction, and weak negative pressure is used for balancing additional pressure caused by liquid level difference during operation waiting.
In this example, the working environment temperature is 25 ℃ and the working medium 2 is a mineral oil having a viscosity of 40 mPas at 25 ℃. The fluid damper 4 was a capillary tube having an inner diameter of 50 μm and a length of 20cm, which was tested and finished to have a fluid damping of 2nL/s/bar for a mineral oil having a viscosity of 40 mPas.
In this example the deposition probe 7 is a 2cm long, 250 μm inner diameter, tipped capillary. The surface of the capillary tube is subjected to low-energy treatment, so that the water-soluble sample is ensured not to remain on the tube wall.
In this embodiment, the fluid damper 4 and the spotting probe 7 form a droplet quantitative measuring channel sharing 12 channels, and the 12 channels share one working medium reservoir 3.
In this example, the multi-well plate 9 is a standard 96-well plate and the sample solution 10 is a solution of various water-soluble precipitants used in protein crystallization condition screening. The chip 11 comprises a plurality of micro pits 13, wherein each micro pit 13 is of an inverted frustum structure, the diameter of the upper surface of each micro pit is 1.2mm, the diameter of the bottom surface (namely the bottom 15 of each micro pit) of each micro pit is 0.3mm, and the height of each micro pit is 2 mm. The working medium 12 is mineral oil. The chip 11 is made of low surface energy plastic (or made of other materials, and the side wall 14 of the micro-pit is subjected to hydrophobic surface treatment), is oleophilic but not hydrophilic, and ensures that liquid drops cannot remain on the side wall 14 of the micro-pit.
In this embodiment, the multi-channel liquid droplet quantitative measuring device is mounted on an automatic translation stage, which is also controlled by the computer program. Working medium 12 mineral oil is added into the chip 11 in advance, and the multichannel quantitative liquid drop measuring device firstly quantitatively sucks 10nL of precipitator in the porous plate 9 and then quantitatively injects 15nL into the chip 11 to ensure that the liquid drops 16 fall into the designated micro-pits 13 on the chip 11. The above operation is repeated so that each crater 13 on the chip contains one droplet of precipitant solution. Then 10nL droplets of the solution containing the protein to be screened were added to each of the micro-wells. After the addition of the protein solution droplets is completed, the entire chip 11 is centrifuged 18 to fuse the precipitant solution droplets with the protein solution droplets to obtain fused droplets 17. After the droplet fusion is completed, incubation is performed for a period of time, and the protein crystallization condition screening result can be obtained by observing the crystallization condition of the protein in the droplet 17 in the chip 11, as shown in fig. 2.
In this embodiment, before injecting the working medium 12, the chip may be placed under an ion blower to blow an ion wind to eliminate the static electricity on the surface of the chip. Before sampling by using the sampling probe, the ion wind generated by the ion fan can be used for blowing the spotting probe to eliminate the static electricity on the surface of the spotting probe. The ion blower is used according to the description of the invention, and the processing parameters are adjusted according to different environments.
In this embodiment, the data of the relationship between the viscosity and the temperature of the working medium 2 may be stored in the computer in advance, and the operating time and the operating pressure may be obtained by software or a preset program, thereby realizing automatic control of sampling and spotting.
Example 2
This example first generates an array of droplets of different drug solutions and performs a quantitative dilution operation on the droplets therein.
In this embodiment, the multi-channel quantitative droplet measuring device is similar to that of embodiment 1, but the number of channels is 6, as shown in fig. 3. The multi-channel quantitative liquid drop measuring device is also arranged on an automatic translation table, and the automatic translation table is controlled by a computer program.
In this example, the multi-well plate 9 is a 24-well plate, the sample solution 10 is an aqueous solution of 24 different drugs, the chip 11 is a glass chip with micro-pits, and the working medium 12 is mineral oil. A reservoir 19 is provided with pure water 20 for dilution.
In this embodiment, the multichannel quantitative droplet measuring device first quantitatively aspirates 10nL of the drug solution from the porous plate 9, then moves to the chip 11 to inject 5nL of the drug solution, and transfers the drug solution to the micro-wells of the chip 11 to generate a droplet array. This process was repeated 4 times to construct a droplet array consisting of 4 columns of 24 different drug solutions. Subsequently, 60nL of pure water 20 was aspirated from the reservoir 19, and then 45nL of pure water 20 was injected into each column of droplets on the chip, thereby completing the 10-fold dilution operation of the entire drug droplet array. And subsequently, target enzyme and a substrate thereof can be added into the drug liquid drop by adopting a similar operation method to carry out enzyme inhibitor screening. The target enzyme and the substrate thereof can be added by adopting the multichannel quantitative measuring device for liquid drops, and the target enzyme and the substrate thereof can be added into each medicine liquid drop in sequence by adopting a single-channel liquid adding device.
This example demonstrates that the invention can be used directly for droplet metering and manipulation.
Example 3
This embodiment is a highly integrated and modified version of the present invention.
In this embodiment, the pressure source and control system 1 is comprised of an air compressor, a vacuum pump and a servo air pressure controller, as shown in fig. 4. The compressed air and the vacuum required by the servo air pressure controller are respectively provided by an air compressor and a vacuum pump. By adopting the servo air pressure controller, the pressure source and pressure control system 1 can generate continuously adjustable air pressure of-0.1 MPa to 0.6 MPa. The pressure source and the pressure control system 1 are finally controlled by a computer program.
In this embodiment the working medium reservoir 3, the fluid damper 4, the deposition probe 7 are integrated into one microfluidic chip 21. The working medium reservoir 3 is connected to a pressure source and a pressure control system via a pneumatic connection 22. The height of the micro-channel cavity at the working medium liquid storage tank 3 is 1 mm. The channel height at the fluid damper 4 was about 5 μm, the width was 50 μm, and the length was 60mm, and the fluid damper 4 had a fluid damping of 100pL/s/bar when the working medium 2 and the working environment were at the same temperature as in example 1. The height of the channel at the deposition probe 7 was 50 μm. The tip required for the deposition probe (7) is obtained by laser or machining.
This embodiment illustrates that the pressure source and pressure control system 1, the working medium reservoir 3, the fluid damper 4 and the spotting probe 7 are the core features of the multi-channel quantitative liquid drop measuring device based on pressure driving in the present invention.
Example 4
As shown in fig. 5, this embodiment illustrates a multi-channel drop dosing device where the pressure source and pressure control system 1 is a syringe pump.
In this embodiment, the multi-channel quantitative droplet measuring device is the same as that of embodiment 1 except for the pressure source and the pressure control system 1.
In this embodiment, the pressure source and pressure control system 1 is served by a syringe pump. Since the fluid resistance of each fluid damper 4 is known, the flow rate generated by the syringe pump will first build up a certain pressure in the working medium reservoir 3, which in turn drives the working medium 2 through the fluid damper 4, which plays a role in quantitative metering.
In this embodiment, there may be bubbles in the lines between the pressure source and the pressure controller 1 to the fluid damper 4 or there may be elasticity in the lines, but the presence of bubbles or elasticity in the lines greatly affects the response speed of the system and thus the accuracy of the quantitative measurement of the droplets.
Claims (10)
1. The utility model provides a multichannel liquid drop ration volume gets device based on pressure drive which characterized in that includes:
a spotting probe for performing spotting operation on the sample solution;
a working medium reservoir for containing a working medium;
the pressure control system is used for controlling the pressure in the working medium liquid storage tank;
the fluid damper is provided with a micro-channel structure and has a damping effect on fluid in the micro-channel structure, the liquid inlet end of the fluid damper is communicated with the medium liquid storage tank, and the liquid outlet end of the fluid damper is communicated with the liquid inlet end of the sample application probe in a sealing way.
2. The pressure-driven multichannel quantitative droplet measuring device according to claim 1, wherein two or all of the medium reservoir, the spotting probe, and the fluid damper are integrated into a single structure.
3. The pressure-driven multi-channel quantitative droplet measuring device according to claim 1, further comprising:
a temperature sensor for detecting a temperature of a working environment;
and the controller receives the temperature information of the temperature sensor and determines the operating pressure and the operating time of the pressure control system by combining the viscosity information of the working medium.
4. The pressure-driven multichannel quantitative droplet tapping device according to claim 1, wherein the fluid resistance of the fluid damper is 90% or more of the fluid resistance of the entire flow path composed of the micro flow channel structure and the internal channels of the spotting probe in the fluid damper at a rated flow rate; or the fluidic resistance of the fluidic damper is more than 10 times the fluidic resistance of the deposition probe.
5. The pressure-driven multi-channel quantitative droplet measuring device as claimed in claim 1, wherein the fluid damper is formed of an elongated pipe or a minute hole, or a combination of the two; when a long and thin pipeline is adopted, the inner diameter of the pipeline is 0.1-1000 mu m, and the length of the pipeline is 0.1-1000 mm.
6. The pressure-driven multi-channel quantitative droplet measuring device according to claim 1, further comprising:
a multi-well plate for holding a sample solution;
the chip is used for storing liquid drops, and a micro-pit array formed by micro-pits with large openings and small bottoms is arranged on the chip.
7. The multi-channel quantitative liquid drop measuring device based on pressure driving of claim 6, wherein the diameter of the bottom surface of the micro-pit is not more than 3 times of the diameter of the finally contained liquid drop; the included angle formed by the side wall of the micro pit and the vertical line of the chip is not more than 60 degrees.
8. The method for quantitatively measuring the liquid drops by using the pressure-driven multi-channel quantitative liquid drop measuring device according to any one of claims 1 to 7, comprises the following steps:
the pressure control system applies set pressure to ensure that the working medium I is filled with the fluid damper and the spotting probe;
inserting the sample application end of the sample application probe into the sample solution;
under the control of a pressure control system, injecting a sample solution with a target volume out of the sample application probe or sucking the sample solution into the sample application probe;
the working medium I is immiscible with the sample solution.
9. The method for quantitatively taking the liquid drops by using the pressure-driven multi-channel quantitative liquid drop taking device according to claim 8, wherein the method comprises the following steps:
injecting a working medium II into the micro-pits of the chip in advance;
injecting a sample solution or a reagent solution with a target volume into a working medium II in the corresponding micro-pit by using a sample application probe to generate a liquid drop to be fused;
after 2 or more than 2 liquid drops to be fused are generated, the chip is wholly centrifuged to promote the liquid drops to be settled to the bottom surface of the micro pit, and the fusion of the liquid drops is realized under the limitation of the wall of the micro pit to form new liquid drops;
the working medium II is immiscible with the sample solution.
10. The method for quantitatively taking the liquid drops by using the pressure-driven multi-channel quantitative liquid drop taking device according to claim 8, wherein the method comprises the following steps:
when the liquid level of the point sampling probe tip is not equal to the liquid level of the working medium liquid storage tank, a pressure control system applies a set compensation pressure for eliminating pressure difference flowing to the point sampling probe in a waiting process.
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| CN113495085B (en) * | 2020-04-03 | 2023-07-07 | 深圳市帝迈生物技术有限公司 | POCT blood cell analyzer and liquid flow driving device of impedance detection cell |
| CN111665111A (en) * | 2020-07-03 | 2020-09-15 | 上海百傲科技股份有限公司 | Sample applicator |
| CN112191189A (en) * | 2020-09-26 | 2021-01-08 | 宁波大学 | Method and device for generating picoliter single liquid drop |
| CN113368918B (en) * | 2021-06-21 | 2022-04-26 | 合肥瀚海星点生物科技有限公司 | Multi-channel liquid separation device and method based on microfluidic printing |
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