CN113528504A - Device for purifying nucleic acid in low-load pathogen - Google Patents
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Abstract
The invention provides a device for purifying nucleic acid in low-load pathogens, which comprises a micro-fluidic chip, a gas-liquid supply mechanism, a liquid distribution mechanism, a magnetic field generation mechanism and an acquisition mechanism, wherein the micro-fluidic chip is connected with the gas-liquid supply mechanism; the gas-liquid supply mechanism comprises a liquid supply driver, a composite sample storage and a plurality of liquid storages, wherein the composite sample storage and the liquid storages are independently arranged; or directly conveying the composite sample liquid in the composite sample storage to the microfluidic chip; the magnetic field generating mechanism generates a magnetic field, and uniformly mixes, fixes or separates magnetic substances in the microfluidic chip; the inlet of the collecting mechanism is communicated with the outlet of the microfluidic chip to output gas or/and liquid. The magnetic beads required by nucleic acid purification are transplanted into the microfluidic chip, and the automatic nucleic acid treatment and purification of the low-load pathogen sample are realized through the peripheral mechanical structure, so that the nucleic acid extraction efficiency is improved, the stability is good, and the cross contamination can be avoided.
Description
Technical Field
The invention relates to the field of in-vitro detection, in particular to a device for purifying nucleic acid in low-load pathogens.
Background
For a long time, infectious diseases caused by pathogens are extremely easy to threaten human health and cause social panic, common pathogens which cause infectious diseases mainly comprise viruses, bacteria, fungi and parasites, wherein the viruses are one of important factors which cause the infectious diseases, and the viruses have the characteristics of high mutation probability, high traceability difficulty, multiple types of infected animals, multiple infection routes (such as contact transmission, air transmission and the like), and the like, so that the viruses are extremely easy to spread, and further develop into the infectious diseases and even sudden epidemic situations.
In the face of sudden epidemic caused by pathogen infection and regional transmission, timely and accurate diagnosis is needed in the control and treatment process, so that a reliable and effective diagnosis method is an important means for preventing and treating epidemic transmission.
When nucleic acid detection is adopted, nucleic acid purification of pathogens is mainly realized by a Trizol method, a centrifugal column method, a magnetic bead method and the like, the method needs manual operation of corresponding reagents to complete, the purification process is complex, the time is long, the nucleic acid extraction efficiency is low, the stability is poor, meanwhile, for application scenes with low pathogen content, after the extraction by the method, nucleic acid samples with effective detection amount are difficult to obtain, in order to efficiently and stably extract nucleic acid stock solution, a plurality of automatic nucleic acid extraction instruments are available, and the purification principle of the nucleic acids is usually based on a magnetic rod method and a rotary centrifugation method.
For example, the invention patent with application number 201810404724.0 discloses a magnetic bar type automatic nucleic acid extraction method, which comprises the steps of adsorbing magnetic beads with nucleic acids adsorbed on the surfaces on a magnetic bar, mechanically moving the magnetic bar to participate in different reaction processes, and simultaneously driving the magnetic bar to perform vertical high-frequency reciprocating vibration to rapidly mix and stir reagent liquid so as to complete complex nucleic acid extraction processes such as cracking, adsorption, washing, elution and the like, but the mixed nucleic acid extraction method with high-frequency reciprocating vibration is easy to generate liquid splashing and has the risk of sample cross contamination; meanwhile, the high-frequency reciprocating vibration can generate strong vibration to nucleic acid, and the risk of damaging the integrity of DNA chains and RNA chains exists.
The invention patent with application number 201710435931.8 discloses a rotary automatic nucleic acid extraction device and a control method thereof, which adopts a rotary mode to uniformly mix a sample and a reagent to be extracted, realizes the functions of stirring, magnetic attraction, heating and the like through high-speed rotation, and simultaneously realizes the whole flow of nucleic acid extraction such as cracking, adsorption, washing, elution and the like by matching with vertical and/or horizontal movement. Although the device reduces the cross contamination rate between extracted samples, the device is formed by staggered arrangement of a plurality of transmission gears and motors, the structure is complex, and the failure rate of the device is high.
Therefore, how to find a nucleic acid extraction method with high extraction efficiency, good stability and no cross contamination between samples is a problem that needs to be solved at present.
Disclosure of Invention
The invention aims to provide a device for purifying nucleic acid in low-load pathogens, which realizes the functions of rapid and automatic nucleic acid treatment and purification and improves the stability of sample collection.
In order to achieve the aim, the invention provides a device for purifying nucleic acid in low-load pathogens, which comprises a microfluidic chip, a gas-liquid supply mechanism, a liquid distribution mechanism, a magnetic field generation mechanism, a collection mechanism and a drive controller arranged on the periphery of the microfluidic chip, wherein the gas-liquid supply mechanism is connected with the liquid distribution mechanism through a pipeline; the gas-liquid supply mechanism comprises a liquid feeding driver, an independently arranged composite sample storage and a plurality of independently arranged liquid storages, and reaction gas/liquid in the liquid storages is conveyed to the microfluidic chip through a liquid distribution mechanism at a certain flow rate under the control of the liquid feeding driver; or the composite sample liquid in the composite sample storage is directly conveyed to the microfluidic chip under the control of the liquid feeding driver; the magnetic field generating mechanism is configured to generate a magnetic field to uniformly mix, fix or separate a magnetic substance in the microfluidic chip and a nucleic acid sample; the inlet of the collecting mechanism is communicated with the outlet of the microfluidic chip and is configured to output gas and/or liquid.
According to the invention, the micro-fluidic chip is provided with a sample inlet, a sample outlet and a plurality of functional cavities, the functional cavities are communicated through a flow channel, each sample inlet is correspondingly communicated with one gas/liquid inlet, the gas/liquid inlets are used for introducing gas/liquid into the corresponding functional cavities or flow channels, and the gas/liquid flows among different functional cavities through the flow channels.
Preferably, the microfluidic chip is provided with a bubble capturing mechanism communicated with the functional cavity, the bubble capturing mechanism is composed of a group of circular chambers with the diameter larger than the width of the flow channel, and the bubble capturing mechanism can be arranged on the flow channel between the liquid inlet and the functional cavity.
According to the present invention, the liquid dispensing mechanism includes a liquid dispenser and a dispensing controller for controlling the liquid dispenser; the liquid distributor is a multi-interface passage with a solenoid valve gating function.
According to the invention, the liquid storage comprises a composite sample storage, and a flushing liquid storage, a magnetic particle storage, a washing liquid storage and an eluent storage which are independently arranged and respectively communicated with the liquid distributor; the flushing liquid storage, the magnetic particle storage, the washing liquid storage and the eluent storage respectively convey the stored reaction liquid to the liquid distributor under the control of the liquid feeding driver, and realize the one-way circulation of the corresponding reaction liquid to the solution input cavity under the control of the distribution controller; the outlet of the compound sample storage is communicated with the other inlet of the solution input cavity, and the stored compound sample solution is directly conveyed into the solution input cavity under the control of the liquid feeding driver.
According to the invention, a solution input cavity, a bubble capturing mechanism and an enrichment purification cavity which are sequentially communicated are arranged on the micro-fluidic chip, and an inlet of the solution input cavity is respectively communicated with an outlet of the composite sample storage and an outlet of the liquid distributor; the outlet of the enrichment and purification cavity is communicated with the acquisition mechanism.
According to the invention, the collection mechanism comprises an enrichment sample collector, a waste liquid collector and a collection controller, the nucleic acid solution enriched and purified in the enrichment and purification cavity is guided into the enrichment sample collector under the control of the collection controller, and the magnetic bead waste liquid, the sample waste liquid, the washing waste liquid and the flushing waste liquid of the system pipeline are sequentially guided into the waste liquid collector.
According to the invention, the magnetic field generating mechanism is arranged at the position on the microfluidic chip where the magnetic substances are required to be mixed, fixed or separated, preferably above and below the enrichment and purification cavity.
Preferably, the magnetic field generating mechanism comprises a magnet driver and two sets of magnets, and the two sets of magnets are symmetrically distributed above and below the enrichment and purification cavity.
According to the invention, the flow channel comprises a straight line structure and a curved line structure. Preferably, the flow channel is a micro flow channel or a capillary flow channel. Preferably, the width of the same flow channel is the same or different at different positions.
The invention has the beneficial effects that:
1) the invention provides a method for purifying nucleic acid in a low-load pathogen, which ensures that a micro-fluidic chip has excellent comprehensive treatment function and good control capability through module function combination of the micro-fluidic chip and a peripheral functional module, improves the extraction efficiency of the nucleic acid in the low-load pathogen, solves the problems of low nucleic acid and superparamagnetic nano magnetic beads combination, low elution efficiency, poor stability, large consumption of purification reagents, high cost, cross contamination among samples and the like in the prior art, and effectively improves the automatic nucleic acid treatment and purification capability of the low-load pathogen sample.
2) The magnetic beads required by nucleic acid purification are transplanted into the microfluidic chip, and meanwhile, the automatic nucleic acid treatment and purification of the low-load pathogen sample are realized through the peripheral mechanical structure, so that the nucleic acid extraction efficiency is improved, the stability is good, and the cross contamination can be avoided.
3) According to the invention, the magnetic beads and nucleic acid molecules are subjected to specific adsorption and desorption processes under different environments (the change of the salt content and the pH value of the solution) so as to realize the rapid and automatic nucleic acid treatment and purification functions and improve the stability of sample collection.
4) According to the invention, the processes of binding nucleic acid and magnetic beads, impurity washing and nucleic acid elution are integrated on one microfluidic chip, so that on one hand, the loss of reagents used in the process of extracting nucleic acid is reduced through a micro flow channel, on the other hand, the problems of cross contamination among samples and the like are avoided in the totally-enclosed extraction process, and the bubble in the flow channel can be captured by arranging a bubble capturing mechanism in the microfluidic chip, so that the binding, washing and elution of nucleic acid and magnetic beads are prevented from being influenced by the bubble, and the efficiency of releasing and extracting nucleic acid in low-load pathogens is effectively improved.
Drawings
FIG. 1 is a schematic diagram of the structure of an apparatus for purifying nucleic acid from a low load of pathogens according to an embodiment of the present invention.
FIG. 2 is a schematic diagram of a process for enriching binding of magnetic particles and nucleic acids in a purification chamber according to an embodiment of the present invention.
Fig. 3 shows a state where dynamic magnetic bead plugs are formed in the microfluidic channel when the upper and lower magnets move at a constant speed to a certain state.
Reference numerals: 1-microfluidic chip, 11-solution input cavity, 12-bubble capture mechanism, 13-enrichment purification cavity, 2-gas-liquid supply mechanism, 21-liquid supply driver, 22-liquid storage, 221-flushing liquid storage, 222-magnetic particle storage, 223-eluent storage, 224-washing liquid storage, 225-composite sample storage, 3-liquid distribution mechanism, 31-liquid distributor, 32-distribution controller, 4-magnetic field generation mechanism, 41-magnet driver, 42, 43-magnet, 5-collection mechanism, 51-enrichment sample collector, 52-waste liquid collector and 53-collection controller.
Detailed Description
The construction and use of the invention will be further explained with reference to the drawings and specific embodiments. It is to be understood that the following examples are only illustrative and explanatory of the present invention and should not be construed as limiting the scope of the present invention. All the technologies realized based on the above-mentioned contents of the present invention are covered in the protection scope of the present invention.
Referring to fig. 1, the present invention provides an apparatus for purifying low-load pathogen nucleic acid, including a microfluidic chip 1, a gas-liquid supply mechanism 2, a liquid distribution mechanism 3, a magnetic field generation mechanism 4, a collection mechanism 5, and a drive controller disposed at the periphery of the microfluidic chip 1.
The gas-liquid supply mechanism 2 is used as a power source for driving gas-liquid two-phase substances, and provides driving force for three types of reaction reagents and composite pathogen samples to enter the microfluidic chip 1 to participate in reaction, and driving force for flushing liquid to carry out self-cleaning on a system pipeline. The gas-liquid supply mechanism 2 comprises a liquid supply driver 21, an independently arranged composite sample storage 225 and a plurality of independently arranged liquid storages 22, and the reaction gas/liquid in the liquid storages 22 is conveyed to the microfluidic chip 1 through the liquid distribution mechanism 3 at a certain flow rate under the control of the liquid supply driver 21; or the composite sample liquid in the composite sample storage 225 is directly conveyed to the microfluidic chip 1 under the control of the liquid feeding driver 21. The magnetic field generating mechanism 4 is configured to generate a magnetic field to mix, fix or separate the magnetic substance in the microfluidic chip 1 and the accounting sample. The inlet of the acquisition mechanism 5 is communicated with the outlet of the microfluidic chip 1 and is configured to output gas or/and liquid.
The liquid reservoir 22 can store liquid solution or solid powder therein, and the number of the liquid reservoirs 22 can be expanded or reduced according to actual situations.
In the invention, a pathogen solution is placed in a composite sample storage 225 for cracking and then is introduced into a microfluidic chip 1; magnetic particles required by nucleic acid purification are transplanted or guided into the microfluidic chip 1, and meanwhile, automatic nucleic acid treatment and purification of low-load pathogen samples are achieved through a peripheral mechanical structure, so that nucleic acid extraction efficiency is improved, stability is good, and cross contamination can be avoided.
According to the invention, the micro-fluidic chip 1 is provided with a sample inlet, a sample outlet and a plurality of functional cavities, the functional cavities are communicated through a flow channel, each sample inlet is correspondingly communicated with a gas/liquid inlet, the gas/liquid inlets are used for introducing gas/liquid into the corresponding functional cavities or flow channels, and the gas/liquid flows among different functional cavities through the flow channels. The number and the size of the functional cavities are set according to actual requirements, and the functional cavities can be one or more of sample adding, reaction, filtration, detection, mixing and the like. The functional cavity and the flow channel can be prepared by photoetching, numerical control, hot pressing, injection molding and other methods on a chip material.
Preferably, the micro-fluidic chip 1 is further provided with a bubble capturing mechanism 12 communicated with the functional cavity, and the bubble capturing mechanism 12 is arranged in the micro-fluidic chip 1, so that bubbles in a flow channel can be captured, further, the influence on the combination, washing and elution of nucleic acid and magnetic beads due to the bubbles is avoided, and further, the efficiency of releasing and extracting nucleic acid in low-load pathogens is effectively improved. The position of the bubble capturing mechanism is set according to actual requirements, and the bubble capturing mechanism can be arranged on a flow channel between different functional cavities, or at the intersection of the flow channel and the functional cavity, or at the intersection of the liquid inlet and the functional cavity.
As shown in fig. 1, the liquid dispensing mechanism 3 includes a liquid dispenser 31 and a dispensing controller 32 for controlling the liquid dispenser 31; the liquid distributor 31 is a multi-port passage with a solenoid valve having a selective function.
The liquid storage 22 comprises a composite sample storage 225, and a washing liquid storage 221, a magnetic particle storage 222, a washing liquid storage 223 and an eluent storage 224 which are independently arranged and respectively communicated with the liquid distributor 31, wherein the washing liquid storage 221, the magnetic particle storage 222, the washing liquid storage 223 and the eluent storage 224 respectively convey the stored reaction liquid into the liquid distributor 31 under the control of the liquid feeding driver 21, and realize the unidirectional circulation of the corresponding reaction liquid to the solution input cavity 11 under the control of the distribution controller 32. The outlet of the composite sample reservoir 225 communicates with the other inlet of the solution input chamber 11, and the stored composite sample solution is directly delivered into the solution input chamber 11 under the control of the feeding actuator 21.
As shown in fig. 1, in an embodiment of the present invention, a solution input chamber 11, a bubble capturing mechanism 12 and an enrichment and purification chamber 13 are disposed on a microfluidic chip 1 and sequentially connected to each other, wherein the solution input chamber 11 has two independent inlets respectively connected to an outlet of a composite sample storage 225 and an outlet of a liquid distributor 31 for allowing a pathogen sample and a corresponding reagent to enter the microfluidic chip 1.
The outlet of the enrichment and purification cavity 13 is communicated with the acquisition mechanism 5. Wherein, the collecting mechanism 5 can be a part of the microfluidic chip 1 or independent from the microfluidic chip 1.
As shown in fig. 1, the collecting mechanism 5 includes an enrichment sample collector 51, a waste liquid collector 52 and a collecting controller 53, under the control of the collecting controller 53, the nucleic acid solution enriched and purified in the enrichment and purification chamber 13 is introduced into the enrichment sample collector 51, and the magnetic bead waste liquid, the sample waste liquid, the washing waste liquid and the flushing waste liquid of the system pipeline are sequentially introduced into the waste liquid collector 52.
The magnetic field generating mechanism 4 is arranged at the position on the microfluidic chip 1 where the magnetic substances are required to be mixed, fixed or separated, and is preferably arranged above and below the enrichment and purification cavity 13. Preferably, the magnetic field generating mechanism 4 comprises a magnet driver 41 and two sets of magnets 42, 43, the two sets of magnets 42, 43 are symmetrically distributed above and below the enrichment and purification chamber 13.
The microfluidic chip 1 is further connected with a chip clamping device, the clamping device comprises interfaces between the solution input cavity 11 and the liquid distributor 31, between the solution input cavity 11 and the composite sample storage 225, and interfaces between the chip enrichment and purification cavity 13 and the acquisition controller 53.
In the present invention, the flow path includes a straight line structure and a curved line structure. Preferably, the flow channel is a micro flow channel or a capillary flow channel.
The channel structure in the device is realized in the form of three channels, namely a main channel, a reaction system supply branch channel and a waste liquid collection branch channel, and specifically is a main channel which is connected with a solution input cavity 11, a bubble capturing mechanism 12 and an enrichment and purification cavity 13; the reaction system supply branch channel comprises two types of medium supply branch channels, specifically: one type of the reagent supplying branch channel consists of a solution input cavity 11 and four reaction reagent supplying branch channels, wherein the reagent supplying branch channels comprise 4 mutually independent reaction reagent branch channels, namely a flushing liquid storage 221, a magnetic particle storage 222, a washing liquid storage 223 and an eluent storage 224 which are sequentially connected with corresponding valve ports in a liquid distributor 31, and a multiplexing reaction reagent transmission branch channel, wherein the liquid distributor 31 is connected with the solution input cavity 11. Another type of independent feeding composite pathogen feeding branch channel is connected with the composite sample storage 225 by the solution input cavity 11; the waste liquid collecting branch channel is composed of branch channels between the waste liquid collector 52 and the collection controller 53, and is used for transporting magnetic bead waste liquid, sample waste liquid, washing waste liquid generated by reaction and washing waste liquid generated by a washing system.
According to another aspect of the present invention, there is also provided a method for purifying nucleic acids from low-load pathogens using the apparatus for purifying low-load pathogen nucleic acids, comprising:
According to the invention, the processes of nucleic acid and magnetic bead combination, impurity washing and nucleic acid elution are integrated on one microfluidic chip, so that on one hand, the loss of reagents used in the nucleic acid extraction process is reduced through a micro flow channel, and on the other hand, the problems of cross contamination among samples and the like are avoided in the totally-enclosed extraction process. Meanwhile, immunomagnetic beads and nucleic acid molecules are subjected to specific adsorption and desorption processes under different environments (the change of the salt content and the pH value of the solution) so as to realize the rapid and automatic nucleic acid treatment and purification functions and improve the stability of sample collection.
In a preferred embodiment of the present invention, the corresponding reaction liquid stored in the liquid reservoir 22 is pumped into the solution input chamber 11 of the microfluidic chip 1 in an intermittent pumping mode, which is a gas-liquid supply mode of "liquid a-gas-liquid B" by pumping a segment of gas between two types of reaction liquid.
Taking 100 μ L of pathogen sample as an example, the method of the invention is adopted to purify nucleic acid in low-load pathogen:
the step 1 comprises the following steps: the lysis mixed solution and the sample stock solution required for pathogen lysis are sequentially introduced into the composite sample reservoir 225 according to a certain volume ratio (for example, according to 3:1-5:1), and the second switch component 102 is closed to prevent the insufficiently reacted composite sample from entering the microfluidic chip 1. In the process of lysing the pathogen sample, the lysis of the pathogen sample can be promoted by externally connecting a vibrator and applying a heat source to assist in heating the composite sample reservoir 225.
The step 2 comprises the following steps: step 2-1, the liquid distributor 31 opens the first switch part 101 on the reagent feeding branch channel by mechanically gating the corresponding channel of the magnetic particle storage 222, and inputs the fully and uniformly mixed magnetic bead liquid into the microfluidic chip solution input cavity 11 through the first switch part 101 under the action of the liquid feeding driver 21.
And 2-2, closing the second switch component 102 to prevent the magnetic bead liquid from flowing back to the composite sample storage 225 connected to the second switch component 102, and supplying the magnetic bead liquid to the enrichment and purification cavity 13 at a flow rate lower than 300 μ L/min under the control of the liquid supply driver 21.
Step 2-3, as shown in fig. 2-3, the magnetic bead particles stay in the enrichment and purification chamber 13 to form a magnetic bead plug under the control of the first magnet 42 and the second magnet 43 at the periphery of the enrichment and purification chamber 13.
And 2-4, under the control of the acquisition controller 53, gating a waste liquid collecting branch channel at an outlet of the microfluidic chip 1, and after the enrichment of the peripheral magnetic field, allowing the residual magnetic bead waste liquid to enter the waste liquid collector 52 through the waste liquid collecting branch channel.
The step 3 comprises the following steps: opening the second switch component 102 of the reagent feeding branch channel, and inputting the fully cracked composite sample into the solution input cavity 11 of the microfluidic chip 1 through the second switch component 12 under the action of the feeding driver 21; the first switch part 101 is closed to prevent the composite sample liquid from flowing back to the liquid distributor 31 connected to the first switch part 101, and the composite sample liquid is driven by the liquid feeding driver 21 to flow through the bubble trap mechanism 12 and then enter the enrichment and purification chamber 13. As shown in fig. 2-3, the magnets 42 and 43 at the periphery of the enrichment and purification chamber 13 rotate at a constant speed under the control of the magnet driver 41, and the static magnetic bead plugs start to move regularly in the enrichment and purification chamber 13 under the action of the constantly changing magnetic field at the periphery, so as to form the dynamic magnetic bead plugs. In the process, the flow rate of the composite sample liquid needs to be controlled within 150 mu L/min. The outlet of the microfluidic chip is gated with a waste liquid collecting branch channel, so that the residual waste liquid after nucleic acid-magnetic bead combination enters the waste liquid collector 52 through the waste liquid collecting branch channel.
The combination of nucleic acid and magnetic beads can be completed on the microfluidic chip 1 or realized in the composite sample storage 225, so that the process is accompanied with the fragmentation of a pathogen sample under the action of a lysis solution, the released nucleic acid is combined with the magnetic beads in the composite sample storage 225, and when the sample is completely cracked, the released nucleic acid molecules are also in bonding energy connection with the surfaces of the magnetic bead particles.
In the above process, the pathogen sample lysis in step 1 and the nucleic acid-magnetic bead binding in step 3 occur simultaneously, that is, step 1 and step 3 are combined, the nucleic acid-magnetic bead complex generated in the composite sample reservoir 225 is processed through the above process to complete step 2, and a nucleic acid-magnetic bead complex plug is formed in the microfluidic chip enrichment and purification chamber 13.
Step 4 comprises the following steps: the liquid distributor 31 gates the corresponding channel of the washing liquid storage 223, opens the first switch part 101 of the reagent feeding branch channel, and feeds the washing liquid into the solution input cavity 11 of the microfluidic chip 1 through the first switch part 101 under the action of the liquid feeding driver 21. The second switch component 102 is closed, the washing liquid is prevented from flowing back to the composite sample storage 225 connected with the second switch component 102, the washing liquid is driven by the liquid supply driver 21 to flow through the bubble capturing mechanism 12 and the enrichment and purification cavity 13 in sequence, and contacts with the nucleic acid-magnetic bead complex in the cavity, so that the washing effect of impurity components brought by pathogen lysis is realized. The outlet of the microfluidic chip 1 is gated with a waste liquid collecting branch channel, so that the washed waste liquid containing impurity components such as protein and high-salt ions enters the waste liquid collector 52 through the waste liquid collecting branch channel.
Wherein the washing process requires the immobilization of the nucleic acid-magnetic bead complex, it is preferable to immobilize the first magnet 42 and the second magnet 43 above and below the enrichment and purification chamber 13 to form a static magnetic bead plug for the washing operation. More preferably, the magnet driver 41 controls the upper first magnet 42 and the lower second magnet 43 to rotate at a constant speed, so as to form a dynamic magnetic bead plug to make the whole washing process more complete.
The washing process can involve a washing solution, and washing reagents with various components can be sequentially introduced to effectively wash and purify original protein, high-salt ions, phenol, EDTA and other impurity components in the nucleic acid-magnetic bead environment.
The step 5 comprises the following steps: the liquid distributor 31 gates a channel corresponding to the eluent reservoir 224, under the action of the liquid supply driver 21, the eluent flows through the solution input cavity 11, the bubble capturing mechanism 12 and the enrichment purification cavity 13 of the microfluidic chip 1 in sequence, contacts with the nucleic acid-magnetic bead complex purified in the cavity, and performs a specific desorption process with the nucleic acid molecules by using the silicon hydroxyl nano-magnetic beads under different environments (the change of the salt content and the pH value of the solution) to realize the elution and enrichment work of the purified nucleic acid sample. Under the control of the acquisition controller 53, the outlet of the microfluidic chip is connected with the main enrichment sample acquisition channel, so that after nucleic acid elution and enrichment, the eluent enters the enrichment sample acquisition device 51 through the main enrichment sample acquisition channel.
Wherein, the elution process needs to fix the nucleic acid-magnetic bead complex, and preferably the first magnet 42 and the second magnet 43 are fixed above and below the enrichment and purification cavity 13 to form a static magnetic bead plug for the elution operation. Preferably, the first magnet 42 and the second magnet 43 are controlled by the magnet driver 41 to rotate at a constant speed, so as to form a dynamic magnetic bead plug, thereby making the whole elution process more complete. The enrichment concentration of the enrichment sample is related to the amount of the eluent, when the amount of the eluent is less than the input amount of the pathogen sample, the concentration of the nucleic acid enrichment solution is high, and when the amount of the eluent is far more than the input amount of the pathogen sample, the concentration of the nucleic acid enrichment solution is lower.
Wherein, the magnet components in the magnetic field generating module comprise a first magnet 42 arranged on the upper surface of the microfluidic chip channel of the enrichment and purification chamber 13 and a second magnet 43 arranged on the lower surface of the microfluidic chip channel of the enrichment and purification chamber 13. The distance H1 between the lower surface of the first magnet 42 and the upper surface of the microfluidic chip channel of the enrichment and purification chamber 13 is equal to the distance H2 between the upper surface of the second magnet 43 and the lower surface of the microfluidic chip channel of the enrichment and purification chamber 13.
Preferably, the widths of the first magnet 42 and the second magnet 43 are 4-5 times of the width W of the microfluidic chip channel of the enrichment and purification chamber 13.
The first magnet 42 and the second magnet 43 are fixed above and below the enrichment and purification cavity 13, at this time, static magnetic bead plugs are formed in the microfluidic channel in the cavity, and the corresponding liquid flow rate needs to be lower than 300 muL/min; the first magnet 42 and the second magnet 43 move at a constant speed above and below the enrichment and purification chamber 13 at a stable rotation speed, and at this time, a dynamic magnetic bead plug moving regularly is formed in the microfluidic channel in the chamber, and the corresponding liquid flow rate needs to be lower than 150 μ L/min.
Preferably, a process of self-cleaning the system pipeline by using a flushing liquid is further included before the step 1 and after the step 5.
Preferably, the reaction liquid in the liquid storage 22 is a washing liquid, a reaction sample solution, a washing solution, a lysis solution, an elution solution or other types of solutions.
Preferably, the rinsing solution, the washing solution and the elution solution relate to enzyme-free pure water, ethanol, isopropanol, a magnetic particle solution and a high salt solution.
Preferably, the reaction sample is purified or unpurified saliva, urine, semen, a cell solution, a protein solution, a nucleic acid solution, a bacterial solution, or a virus solution.
Preferably, the magnetic particle solution is obtained by performing targeted modification on the surface of magnetic beads.
Preferably, the magnetic beads are superparamagnetic magnetic beads, the surfaces of the magnetic beads are wrapped with high molecular compounds, the high molecular compounds are polystyrene compounds, polyvinyl fluoride or polyfiber compounds, the surfaces of the high molecular compounds are modified with functional ligand groups, and the functional ligand groups comprise amino groups, carboxyl groups, silicon groups, streptomycin, avidin, aldehyde groups or sulfydryl groups. More preferably, the ligand group is further modified with an antibody, a DNA fragment and/or an RNA fragment. Most preferably, the surface of the magnetic bead is modified with a poly fiber compound, and the surface of the poly fiber compound is modified with a silicon hydroxyl group.
The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. The device for purifying the low-load pathogen nucleic acid is characterized by comprising a microfluidic chip (1), a gas-liquid supply mechanism (2), a liquid distribution mechanism (3), a magnetic field generation mechanism (4), a collection mechanism (5) and a drive controller arranged on the periphery of the microfluidic chip (1);
the gas-liquid supply mechanism (2) comprises a liquid supply driver (21), an independently arranged composite sample storage (225) and a plurality of independently arranged liquid storages (22), and reaction gas/liquid in the liquid storages (22) is conveyed to the microfluidic chip (1) at a certain flow rate through a liquid distribution mechanism (3) under the control of the liquid supply driver (21); and/or the composite sample liquid in the composite sample storage (225) is directly conveyed to the microfluidic chip (1) under the control of the liquid feeding driver (21);
the magnetic field generating mechanism (4) is configured to generate a magnetic field to uniformly mix, fix or separate magnetic substances in the microfluidic chip (1);
and the inlet of the acquisition mechanism (5) is communicated with the outlet of the microfluidic chip (1) and is configured to acquire and output gas and/or liquid.
2. The device for purifying low-load pathogen nucleic acid according to claim 1, wherein the microfluidic chip (1) is provided with a sample inlet, a sample outlet, and a plurality of functional cavities, wherein the functional cavities are communicated with each other through a flow channel, each sample inlet is correspondingly communicated with one gas/liquid inlet, the gas/liquid inlets are used for introducing gas/liquid into the corresponding functional cavities or flow channels, and gas/liquid circulation is performed between the different functional cavities through the flow channels.
3. The device for purifying low-load pathogen nucleic acid according to claim 2, wherein the microfluidic chip (1) is further provided with a bubble capture mechanism (12) communicated with the functional cavity, and the bubble capture mechanism (12) is arranged on the flow channel between the liquid inlet and the functional cavity.
4. The apparatus for purifying a low load of pathogen nucleic acid according to claim 1, wherein the liquid dispensing mechanism (3) comprises a liquid dispenser (31) and a dispensing controller (32) for controlling the liquid dispenser (31); the liquid distributor (31) is a multi-interface passage with the function of selecting the electromagnetic valve.
5. The apparatus for purifying a low load of pathogen nucleic acid according to claim 4,
the liquid storage (22) comprises a composite sample storage (225), and a flushing liquid storage (221), a magnetic particle storage (222), a washing liquid storage (223) and an eluent storage (224) which are independently arranged and respectively communicated with the liquid distributor (31);
the flushing liquid storage device (221), the magnetic particle storage device (222), the washing liquid storage device (223) and the eluent storage device (224) respectively convey the stored reaction liquid to the liquid distributor (31) under the control of the liquid feeding driver (21), and realize the one-way circulation of the corresponding reaction liquid to the solution input cavity (11) under the control of the distribution controller (32);
the outlet of the composite sample storage (225) is communicated with the other inlet of the solution input cavity (11), and the stored composite sample solution is directly conveyed into the solution input cavity (11) under the control of the liquid feeding driver (21).
6. The apparatus for purifying a low load of pathogen nucleic acid according to claim 4,
the micro-fluidic chip (1) is provided with a solution input cavity (11), a bubble capturing mechanism (12) and an enrichment and purification cavity (13) which are sequentially communicated; the inlet of the solution input cavity (11) is respectively communicated with the outlet of the composite sample storage device (225) and the outlet of the liquid distributor (31); the outlet of the enrichment and purification cavity (13) is communicated with the acquisition mechanism (5).
7. The device for purifying low-load pathogen nucleic acid according to claim 6, wherein the collection mechanism (5) comprises an enrichment sample collector (51), a waste liquid collector (52) and a collection controller (53), the nucleic acid solution enriched and purified in the enrichment and purification chamber (13) is introduced into the enrichment sample collector (51) under the control of the collection controller (53), and the magnetic bead waste liquid, the sample waste liquid, the washing waste liquid and the flushing waste liquid of the system pipeline are sequentially introduced into the waste liquid collector (52).
8. The device for purifying low-load pathogen nucleic acid according to claim 1, wherein the magnetic field generating mechanism (4) is arranged at a position on the microfluidic chip (1) where the magnetic substances are required to be mixed, fixed or separated, preferably, the magnetic field generating mechanism (4) is arranged above and/or below the enrichment and purification cavity (13).
9. The apparatus for purifying a low load of pathogen nucleic acids according to claim 8, wherein the magnetic field generating means (4) comprises a magnet driver (41) and two sets of magnets (42, 43), the two sets of magnets (42, 43) being symmetrically distributed above and below the enrichment and purification chamber (13).
10. The apparatus for purifying a low load of pathogen nucleic acid according to claim 2, wherein the flow channel is a straight line structure flow channel or a curved line structure flow channel.
Preferably, the flow channel is a micro flow channel or a capillary flow channel.
Preferably, the width of the same flow channel is the same or different at different positions.
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