CN114292742A - Integrated exosome source nucleic acid extraction system and method based on digital microfluidic - Google Patents
Integrated exosome source nucleic acid extraction system and method based on digital microfluidic Download PDFInfo
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Abstract
The invention provides an integrated exosome-derived nucleic acid extraction system and method based on digital microfluidics, comprising the following steps of: the micro-fluidic chip comprises an upper polar plate and a lower polar plate which are arranged at intervals, the upper polar plate is provided with a sample inlet, and an internal reaction cavity is formed between the upper polar plate and the partition plate, and between the lower polar plate and the partition plate; the magnet control unit comprises an electromagnet and a permanent magnet clamped at the top end of the electromagnet; the driving power supply comprises a chip driving power supply, a magnet driving power supply and a switch circuit, wherein the voltage input end of the switch circuit is respectively connected with the chip driving power supply and the magnet driving power supply, and the voltage output end of the switch circuit is respectively connected with the microfluidic chip and the magnet control unit; the control unit comprises a control interface and an MCU, the control interface is connected with the MCU, and the MCU is connected with the signal input end of the switch circuit. The invention realizes the high-efficiency integrated exosome enrichment and nucleic acid release and realizes the integration and automation of exosome source nucleic acid sample pretreatment.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to an integrated exosome-derived nucleic acid extraction system and method based on digital microfluidics.
Background
The traditional exosome separation and enrichment technology comprises several methods such as ultra-high speed centrifugation, ultrafiltration, Size Exclusion Chromatography (SEC), high-molecular polymer coprecipitation, immunization and the like, wherein an exosome separation means based on ultra-high speed centrifugation is a gold standard accepted in the field at present. However, the conventional separation method needs a larger amount of samples, takes longer time, or requires large-scale equipment, and thus cannot be well adapted to the requirements of micro-processing and rapid detection. Under the background, exosome separation and enrichment based on a microfluidic means is widely concerned, and related methods are widely researched, wherein the methods comprise immunization based on the driving of a traditional injection pump, a microfiltration column, magnetic beads, surface acoustic waves and the like, the methods usually need multiple pumps to inject samples or reaction liquid simultaneously or alternately, and in some cases, a micro valve on a chip and a physical field outside a flow channel need to be regulated and controlled simultaneously, so that the operability of the system is limited to a certain extent. However, the digital microfluidic platform is characterized by its high flexibility and automation, and can overcome the common problems in the conventional microfluidic field to some extent, so it is called an efficient platform for rapid pretreatment of trace exosomes.
The current digital microfluidic technology is mainly realized by a dielectric wetting (EWOD) technology, and the basic principle is that under the action of a strong electric field, a contact angle of a liquid drop on a solid surface is reduced, so that when a local high voltage is applied to a hydrophobic surface, the liquid drop tends to move to a high-voltage position under the action of surface tension, and thus, the driving and the control of the liquid drop are realized. At present, the dielectric wetting-based digital microfluidic technology has mature technology accumulation, a large-scale digital microfluidic chip can be prepared by utilizing a standard micro-electro-mechanical system (MEMS) process, and related application researches cover researches in the fields of biology and chemistry, including on-chip immunohistochemistry, Polymerase Chain Reaction (PCR), single cell culture and separation, pyrosequencing and various on-chip sample purification and preparation processes. However, there are still few reports on the research on the preparation and pretreatment of the digital microfluidic technology in the aspect of exosome sample.
Disclosure of Invention
In view of the above-mentioned shortcomings of the prior art, the present invention provides an integrated exosome-derived nucleic acid extraction system and method based on digital microfluidic, wherein exosome enrichment and nucleic acid release processes in an exosome sample are integrated on a digital microfluidic chip, so that the processed sample can be directly used for subsequent nucleic acid amplification and detection, thereby realizing pretreatment integration and automation of an exosome-derived nucleic acid sample.
To achieve the above and other related objects, the present invention provides an integrated exosome-derived nucleic acid extraction system based on digital microfluidics, comprising:
the microfluidic chip comprises an upper polar plate and a lower polar plate which are arranged at intervals, wherein the upper polar plate is provided with a sample inlet, two sides of the upper polar plate and the lower polar plate are respectively provided with a partition plate, an internal reaction cavity is formed between the partition plates, and an exosome sample is added into the internal reaction cavity from the sample inlet to extract nucleic acid;
the magnet control unit is arranged above the microfluidic chip and comprises an electromagnet and a permanent magnet clamped at the top end of the electromagnet, and the electromagnet is pushed and contracted in a direction vertical to the microfluidic chip to drive the permanent magnet to be close to or far away from the microfluidic chip;
the driving power supply comprises a chip driving power supply, a magnet driving power supply and a switch circuit, the voltage input end of the switch circuit is respectively connected with the chip driving power supply and the magnet driving power supply, the voltage output end of the switch circuit is respectively connected with the microfluidic chip and the magnet control unit, the chip driving power supply provides driving voltage for the microfluidic chip through the switch circuit, and the magnet driving power supply controls the push and the contraction of the electromagnet through the switch circuit;
the control unit comprises a control interface and an MCU, the control interface is connected with the MCU, the MCU is connected with the signal input end of the switch circuit, and the control interface controls the switch circuit through the MCU.
Preferably, the internal reaction cavity comprises a sample cavity, an incubation cavity, a releasing agent cavity, a waste liquid cavity and a standby cavity which are sequentially communicated through all the channels; the input end of the waste liquid cavity is connected with a first channel, the sample cavity is communicated with the first pipeline through a second channel, the incubation cavity is communicated with the first channel through a third channel, the releasing agent cavity is communicated with the first channel through a fourth channel, and the standby cavity is communicated with the first channel through a standby channel.
Preferably, the below of going up the polar plate is provided with conductive coating, conductive coating's below with the both sides of introduction port all coat and have the upper portion hydrophobic layer, the top of bottom plate is provided with the dielectric layer, the top coating of dielectric layer has the lower part hydrophobic layer, just the dielectric layer is inside to be inlayed and is equipped with a plurality of driving electrode, and is a plurality of driving electrode correspond respectively set up in the below of inside reaction chamber for the removal of drive liquid drop.
Preferably, the microfluidic chip is arranged on the clamp probe station, and a plurality of probes are arranged on the clamp probe station and are respectively and correspondingly connected with the plurality of driving electrodes.
Preferably, the switch circuit comprises a relay array, the relay array comprises a plurality of relays, each relay is connected with one path of the probe, and the relays correspondingly control the on-off of the driving electrodes through the probes.
Preferably, the conductive coating is an indium tin oxide conductive coating;
the lower electrode plate is a patterned electrode, and the patterned electrode is manufactured by adopting a standard MEMS (micro-electromechanical system) process;
the thickness of the spacing plate is 80-120 mu m.
Preferably, the chip driving power supply comprises a signal generator and a voltage amplifier, and a signal generated by the signal generator is amplified by the voltage amplifier and then is connected to the switching circuit.
Preferably, the control interface includes a control program and a graphical interface, the control program controls the switching circuit through the MCU, the graphical interface includes a manual mode and an automatic mode, the manual mode is used for a user to manually control the driving electrode, and the automatic mode is used for editing an action flow to be executed and then automatically executing the action flow.
An integrated exosome-derived nucleic acid extraction method based on digital microfluidics, comprising the following steps:
s1, providing the integrated exosome-derived nucleic acid extraction system based on digital microfluidics according to any one of claims 1 to 8;
s2, preparing an exosome sample solution, a magnetic bead suspension and a releasing agent solution;
s3, starting the MCU, the chip driving power supply and the magnet driving power supply, and opening a control interface;
s4, selecting a manual mode on a control interface, starting a driving electrode of the internal reaction cavity, and injecting the exosome sample solution, the magnetic bead suspension and the releasing agent solution prepared in the step S2 into the internal reaction cavity from the sample inlet respectively;
s5, adjusting the control interface to be in an automatic mode, setting an execution flow under the automatic mode, and automatically completing pretreatment and nucleic acid extraction of the exosome sample to obtain a nucleic acid release solution;
and S6, disconnecting the switch circuit, taking down the microfluidic chip, and collecting the obtained nucleic acid release solution.
Preferably, the step S5 of automatically completing the pretreatment and nucleic acid extraction of the exosome sample comprises the following steps:
s51, pushing the electromagnet to enable the permanent magnet to be close to the incubation cavity, separating magnetic beads from liquid, gathering the magnetic beads around the electromagnet and staying in the incubation cavity, and meanwhile, operating the driving electrode to transfer the liquid in the incubation cavity to the waste liquid cavity;
s52, contracting the electromagnet to enable the permanent magnet to be far away from the incubation cavity, transferring the exosome sample solution in the sample cavity into the incubation cavity, uniformly mixing the exosome sample solution with the magnetic beads, and incubating for 30-60 min to obtain a magnetic bead-exosome mixed solution;
s53, the electromagnet is pushed again to enable the permanent magnet to be close to the incubation cavity, the magnetic bead-exosome mixed product in the incubation cavity is separated from the rest sample solution, the magnetic bead-exosome mixed product is gathered around the electromagnet and stays in the incubation cavity, and meanwhile the driving electrode is controlled to transfer the rest sample solution to the waste liquid cavity;
s54, contracting the electromagnet again to enable the permanent magnet to be far away from the incubation cavity, transferring the releasing agent solution into the incubation cavity in the step S53, uniformly mixing the releasing agent solution with the mixed product of the magnetic beads and the exosomes in the incubation cavity, and incubating for 10min to obtain the nucleic acid releasing liquid.
As mentioned above, the integrated exosome-derived nucleic acid extraction system based on digital microfluidics and the method thereof have the following beneficial effects:
according to the invention, the process of exosome enrichment and nucleic acid release in an exosome sample is integrated on the microfluidic chip, so that efficient and integrated exosome enrichment and nucleic acid release are realized, the integration and automation of exosome source nucleic acid sample pretreatment are realized, and a rapid pretreatment means is provided for subsequent nucleic acid amplification, detection and analysis.
Compared with the traditional exosome enrichment and treatment mode, the microfluidic chip has the advantages of high automation degree and sufficient reaction when used for pretreating exosome samples, can automatically complete pretreatment of trace exosome samples within 90 minutes by utilizing the extraction system, enables the exosome enrichment efficiency to be remarkably higher than the enrichment efficiency of manual operation in a tube, has high flexibility, and can be used for nucleic acid extraction of exosome sources and other purposes.
Drawings
FIG. 1 is a schematic diagram of a nucleic acid extraction system according to an embodiment of the present invention.
FIG. 2 is a block diagram of a control procedure according to an embodiment of the present invention.
FIG. 3 is a block diagram of a graphical interface in an embodiment of the present invention.
Fig. 4 is a schematic structural diagram of a microfluidic chip according to an embodiment of the present invention.
Fig. 5 is a schematic structural layout of an internal reaction chamber of a microfluidic chip according to an embodiment of the present invention.
FIG. 6 is a graph showing the characterization of the remaining sample solution a, the exosome sample solution b and the remaining sample solution c in the tube after incubation in example 2 according to the particle size tracing analysis method in example 2 of the present invention.
FIG. 7 is a graph showing the results of the concentration of the remaining sample solution a, the exosome sample solution b and the remaining sample solution c in the tube after incubation in example 2, which were measured by particle size trace analysis in example 2 of the present invention.
FIG. 8 shows the results of the fluorescent quantitative PCR (qPCR) analysis of the in-tube release solution d collected from the in-tube mixed incubation group, the on-chip release solution e collected from example 3, and the blank sample f, respectively, in example 3 of the present invention.
FIG. 9 shows the results of the test of the in-tube released liquid d collected from the in-tube mixed incubation group using digital PCR analysis in example 3 of the present invention.
FIG. 10 shows the results of the test of the release liquid e on the sheet collected in this example 3 using the digital PCR analysis in example 3 of the present invention.
Description of the element reference numerals
100 microfluidic chip
101 upper polar plate
102 sample inlet
103 conductive plating
104 upper hydrophobic layer
105 partition plate
106 internal reaction chamber
1061 sample Chamber
1062 incubation cavity
1063 Release agent Chamber
1064 waste liquid cavity
1065 backup Chamber
107 lower pole plate
108 lower hydrophobic layer
109 dielectric layer
1091 drive electrode
200 magnet control unit
300 switching circuit
400 magnet driving power supply
501 signal generator
502 voltage amplifier
600 MCU
700 control interface
701 control program
702 graphic interface
Detailed Description
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
Please refer to fig. 10. It should be noted that the drawings provided in the present embodiment are only for illustrating the basic idea of the present invention, and the components related to the present invention are only shown in the drawings rather than drawn according to the number, shape and size of the components in actual implementation, and the type, quantity and proportion of the components in actual implementation may be changed freely, and the layout of the components may be more complicated.
According to the invention, the process of exosome enrichment and nucleic acid release in an exosome sample is integrated on a microfluidic chip, so that efficient and integrated exosome enrichment and nucleic acid release are realized, the integration and automation of exosome source nucleic acid sample pretreatment are realized, and a rapid pretreatment means is provided for subsequent nucleic acid amplification, detection and analysis; compared with the traditional exosome enrichment and treatment mode, the microfluidic chip has the advantages of high automation degree and sufficient reaction when used for pretreating exosome samples, can automatically complete pretreatment of trace exosome samples within 90 minutes by utilizing the extraction system, enables the exosome enrichment efficiency to be remarkably higher than the enrichment efficiency of manual operation in a tube, has high flexibility, and can be used for nucleic acid extraction of exosome sources and other purposes.
Example 1
The embodiment of the invention provides an integrated exosome source nucleic acid extraction system based on digital microfluidics, which comprises: the micro-fluidic chip 100, the magnet control unit 200, the driving power supply and the control unit; the microfluidic chip 100 comprises an upper polar plate 101 and a lower polar plate 107 which are arranged at intervals, wherein the upper polar plate 101 is provided with a sample inlet 102, two sides of the upper polar plate 101 and two sides of the lower polar plate 107 are respectively provided with a spacing plate 105, an internal reaction cavity 106 is formed between the spacing plates 105, and an exosome sample is added into the internal reaction cavity 106 from the sample inlet 102 for nucleic acid extraction; the magnet control unit 200 is arranged above the microfluidic chip 100, the magnet control unit 200 comprises an electromagnet and a permanent magnet clamped at the top end of the electromagnet, and the electromagnet pushes and contracts in the direction perpendicular to the microfluidic chip 100 to drive the permanent magnet to be close to or far away from the microfluidic chip 100; the driving power supply comprises a chip driving power supply, a magnet driving power supply 400 and a switch circuit 300, wherein the voltage input end of the switch circuit 300 is respectively connected with the chip driving power supply and the magnet driving power supply 400, the voltage output end of the switch circuit 300 is respectively connected with the microfluidic chip 100 and the magnet control unit 200, the chip driving power supply provides driving voltage for the microfluidic chip 100 through the switch circuit 300, and the magnet driving power supply 400 controls the push and the contraction of the electromagnet through the switch circuit 300; the control unit comprises a control interface 700 and an MCU600, the control interface 700 is connected with the MCU600, the MCU600 is connected with the signal input end of the switch circuit 300, and the control interface 700 realizes the control of the switch circuit 300 through the MCU 600.
Specifically, the electromagnet in the magnet control unit 200 is a push-pull electromagnet locked in two directions, and the relay of the switch circuit 300 controls the electromagnet to push and contract in the Z-axis direction (perpendicular to the microfluidic chip 100), so that the permanent magnet at the top end of the electromagnet is close to or away from the microfluidic chip 100, and the magnetic beads in the microfluidic chip 100 are concentrated and released. The one-way locking electromagnet is characterized in that a permanent magnet block is additionally arranged at one end of a product of a common push-pull electromagnet, the working principle is that the one-way locking electromagnet can be powered off immediately after being pushed (or pulled) by electrification and also can be kept at a terminal, the one-way locking electromagnet can be powered on to realize a pushing state just like a common push-pull electromagnet which is powered on for a long time, the one-way locking electromagnet can be powered off immediately after being powered on, mainly the one-way locking electromagnet is kept at the terminal by the retention force generated by the permanent magnet block inside, at the moment, the stroke and the force can be kept, and the one-way locking electromagnet needs to be powered on reversely after being reset; the two-way locking electromagnet has the same principle as the one-way locking electromagnet, but the inner part is designed with double coils, so that the two ends can generate holding force, and the strokes of the two ends and the holding force are the same.
Specifically, the MCU600 (microcomputer) employs Raspberry Pi 4B, and its corresponding control program 701 and its graphical interface 702 are developed based on Qt 5.12 and run under the Linux-based Raspberry Pi OS system, as shown in fig. 2 and 3. As an example, the internal reaction chamber 106 includes a sample chamber 1061, an incubation chamber 1062, a releasing agent chamber 1063, a waste liquid chamber 1064, and a spare chamber 1065, which are sequentially communicated through respective channels; the input end of the waste liquid cavity 1064 is connected with a first channel, the sample cavity 1061 is communicated with a first pipeline through a second channel, the incubation cavity 1062 is communicated with the first channel through a third channel, the releasing agent cavity 1063 is communicated with the first channel through a fourth channel, and the standby cavity 1065 is communicated with the first channel through a standby channel.
As an example, a conductive plating layer 103 is disposed below the upper plate 101, an upper hydrophobic layer 104 is coated below the conductive plating layer 103 and on both sides of the sample inlet 102, a dielectric layer 109 is disposed above the lower plate 107, a lower hydrophobic layer 108 is coated above the dielectric layer 109, and a plurality of driving electrodes 1091 are embedded inside the dielectric layer 109, and the driving electrodes 1091 are respectively disposed below the internal reaction chamber 106 and used for driving the movement of the droplets.
Specifically, the upper hydrophobic layer 104 and the lower hydrophobic layer 108 are both made of teflon af 2400 material, but other materials may be used in other embodiments, and are not limited herein. Referring to fig. 5, a plurality of driving motors are embedded in the dielectric layer 109, wherein three driving electrodes 1091 are correspondingly disposed below the incubation cavity 1062, and the three driving electrodes 1091 are alternately pressurized to uniformly mix the magnetic beads in the incubation cavity 1062 with the solution transferred into the incubation cavity 1062 and perform incubation; in addition, a plurality of driving electrodes 1091 are sequentially arranged below the first channel, the second channel, the third channel, the fourth channel and the spare channel, and the liquid drops can be transferred between different chambers in the internal reaction chamber 106 under the driving of the driving electrodes 1091. The number of the driving electrodes 1091 is not particularly limited.
As an example, the microfluidic chip 100 is disposed on a fixture probe stage, and a plurality of probes are disposed on the fixture probe stage, and are respectively connected to the plurality of driving electrodes 1091.
Specifically, electrode pins are respectively led out of the plurality of driving electrodes 1091, and are in contact with the probe, that is, the probe controls the on-off of the driving electrodes 1091 through the electrode pins, and the liquid drops in the internal reaction chamber 106 can be moved by switching on different driving electrodes 1091.
As an example, the switch circuit 300 includes a relay array, where the relay array includes a plurality of relays, each relay is connected to one of the probes, and the relay controls the on/off of the driving electrode 1091 through the corresponding probe.
Specifically, each relay in the switch circuit 300 is correspondingly connected with one path of probe, one path of probe is connected with one driving electrode 1091 through an electrode pin, and the on-off of the driving electrode 1091 on the microfluidic chip 100 is correspondingly controlled, so that the movement of liquid drops in the microfluidic chip 100 is controlled; however, the number of relays and the number of probes are not limited herein, and the one-to-one correspondence relationship between the relays and the probes is satisfied.
As an example, the conductive plating layer 103 is an Indium Tin Oxide (ITO) conductive plating layer 103; the lower electrode plate 107 is a patterned electrode, and the patterned electrode is manufactured by adopting a standard MEMS (micro-electromechanical systems) process; the thickness of the spacer 105 is 80 to 120 μm.
Specifically, the thickness of the spacer 105 may be 80 μm, 90 μm, 100 μm, 110 μm, 120 μm, or the like.
As an example, the chip driving power supply includes a signal generator 501 and a voltage amplifier 502, and a signal generated by the signal generator 501 is amplified by the voltage amplifier 502 and then is input to the switch circuit 300.
Specifically, the signal generator 501 is a device capable of providing electrical signals with various frequencies, waveforms and output levels, in this embodiment, preferably, the signal generator 501 generates a sine wave with a frequency of 1KHZ, the generated signal is amplified to 200 to 250Vrms by the voltage amplifier 502, and the amplified voltage is used as a driving voltage of the microfluidic chip 100.
By way of example, the control interface 700 includes a control program 701 and a graphical interface 702, the control program 701 controls the switch circuit 300 through the MCU600, and the graphical interface 702 includes a manual mode and an automatic mode, the manual mode is used for a user to manually control the driving electrode 1091, and the automatic mode is used for editing an action flow to be executed, and then automatically executing the action flow.
Specifically, the control program 701 controls the relay by directly calling a general purpose input/output interface (GPIO) of the MCU600, and allows a user to manually control a single electrode through the graphical interface 702, or to edit an action flow to be executed on site so as to be automatically executed; in the automatic execution mode, the program allows a user to save or read an action flow, and displays the current conditions of each driving electrode 1091 and the execution progress of the flow in real time through multithreading; in addition, in the control program 701, a user can freely select and define a GPIO pin to be used in an experiment on the raspberypi hardware, so that the process design is more flexible.
In order to better understand the digital microfluidic-based integrated exosome-derived nucleic acid extraction system in the embodiment of the present invention, an embodiment of the present invention further provides an integrated digital microfluidic-based exosome-derived nucleic acid extraction method, which includes the following steps:
s1, providing the integrated exosome-derived nucleic acid extraction system based on digital microfluidics according to any one of claims 1 to 8;
s2, preparing an exosome sample solution, a magnetic bead suspension and a releasing agent solution;
specifically, diluting an exosome sample solution, a magnetic bead suspension and a releasing agent solution, and then respectively adding 0.01% of tween-20;
s3, starting the MCU600, the chip driving power supply and the magnet driving power supply 400, and opening the control interface 700;
s4, selecting a manual mode on the control interface 700, starting the driving electrode 1091 of the internal reaction chamber 106, and injecting the exosome sample solution, the magnetic bead suspension and the releasing agent solution prepared in the step S2 into the internal reaction chamber 106 from the sample inlet 102;
specifically, in order to adapt to a standard 25 μ L fluorescent quantitative PCR reaction system, the total amount of a sample processed by the system at a time is 10 μ L, and in this embodiment, the addition amounts of the magnetic bead suspension and the nucleic acid releasing agent are both 10 to 20 μ L; the magnetic beads in the magnetic bead suspension are micron-sized magnetic beads, and the magnetic beads are one or a combination of magnetic beads captured by an immunization method, magnetic beads captured by an electrostatic force adsorption method and magnetic beads captured by a lipid molecule affinity method.
S5, adjusting the control interface 700 to an automatic mode, setting an execution flow under the automatic mode, and automatically completing pretreatment and nucleic acid extraction of the exosome sample to obtain a nucleic acid release solution;
s6, disconnecting the switch circuit 300, taking down the microfluidic chip 100, and collecting the obtained nucleic acid release solution.
Wherein, the step S5 of automatically completing the pretreatment and nucleic acid extraction of the exosome sample specifically comprises the following steps:
s51, the electromagnet is pushed to enable the permanent magnet to be close to the incubation cavity 1062, magnetic beads are separated from liquid, the magnetic beads are gathered around the electromagnet and retained in the incubation cavity 1062, and meanwhile the driving electrode 1091 is controlled to transfer the liquid in the incubation cavity 1062 to the waste liquid cavity 1064;
s52, contracting the electromagnet to enable the permanent magnet to be far away from the incubation cavity 1062, transferring the exosome sample solution in the sample cavity 1061 into the incubation cavity 1062, uniformly mixing the exosome sample solution with magnetic beads, and incubating for 30-60 min to obtain a magnetic bead-exosome mixed solution; specifically, three driving electrodes 1091 are correspondingly arranged below the incubation cavity 1062, and voltages are applied to the three driving electrodes 1091 in turn, so that the exosome sample solution is incubated with the magnetic beads in the process of keeping movement;
s53, pushing the electromagnet again to make the permanent magnet close to the incubation cavity 1062, separating the magnetic bead-exosome mixed product in the incubation cavity 1062 from the remaining sample solution, collecting the magnetic bead-exosome mixed product around the electromagnet and staying inside the incubation cavity 1062, and simultaneously controlling the driving electrode 1091 to transfer the remaining sample solution into the waste liquid cavity 1064;
s54, contracting the electromagnet again to enable the permanent magnet to be far away from the incubation cavity 1062, transferring the releasing agent solution into the incubation cavity 1062 in the step S53, uniformly mixing the releasing agent solution with the mixed product of the magnetic beads and the exosomes in the incubation cavity 1062, and incubating for 10min to obtain the nucleic acid releasing solution.
Example 2
The embodiment provides enrichment of an integrated exosome based on digital microfluidics, and the enrichment method comprises the following steps:
s1, providing the integrated exosome-derived nucleic acid extraction system based on digital microfluidics in example 1;
s2, preparing an exosome sample solution and a magnetic bead suspension; in this embodiment, the exosome sample is processedCarrying out gradient centrifugation, filtration and ultra-high speed centrifugation on the supernatant of the H1975 cell line cultured without serum, and separating and concentrating exosome in the supernatant to form the serum; the concentration of the exosome sample was 1.5 × 10 after particle size tracking analysis (NTA) testing the concentration8Perml (number of particles per mL 1.5X 10)8Tween-20 was added and formulated into an exosome sample solution with a concentration of 0.01% (v/v) (i.e., exosome sample was added to tween-20, the volume of tween-20 accounted for 0.01% of the total volume); the magnetic bead suspension was diluted once with commercial human CD9 antibody-modified magnetic beads (produced by Life Technologies Corporation), and tween-20 was added to obtain a magnetic bead suspension having a concentration of 0.01% (v/v) (when tween-20 was added to the human CD9 antibody-modified magnetic beads diluted once, the volume of tween-20 was 0.01% of the total volume);
s3, starting the MCU600, the chip driving power supply and the magnet driving power supply 400, and opening the control interface 700;
s4, selecting a manual mode on the control interface 700, turning on the driving electrode 1091 of the internal reaction chamber 106, and injecting 10. mu.L of the exosome sample solution and 20. mu.L of the magnetic bead suspension prepared in the step S2 into the sample chamber 1061 and the incubation chamber 1062 from the sample inlet 102, respectively;
s5, the control interface 700 is adjusted to an automatic mode, an execution flow is set in the automatic mode, and the pretreatment of the exosome sample is automatically completed;
the step S5 of automatically completing the pretreatment of the exosome sample specifically includes the following steps:
s51, the electromagnet is pushed to enable the permanent magnet to be close to the incubation cavity 1062, magnetic beads are separated from liquid, the magnetic beads are gathered around the electromagnet and retained in the incubation cavity 1062, and meanwhile the driving electrode 1091 is controlled to transfer the liquid in the incubation cavity 1062 to the waste liquid cavity 1064;
s52, contracting the electromagnet to enable the permanent magnet to be far away from the incubation cavity 1062, transferring the exosome sample solution in the sample cavity 1061 into the incubation cavity 1062, uniformly mixing the exosome sample solution with magnetic beads, and incubating for 30min to obtain a magnetic bead-exosome mixed solution;
s53, pushing the electromagnet again to make the permanent magnet close to the incubation cavity 1062, separating the magnetic bead-exosome mixed product in the incubation cavity 1062 from the remaining sample solution, collecting the magnetic bead-exosome mixed product around the electromagnet and staying inside the incubation cavity 1062, and simultaneously operating the driving electrode 1091 to transfer the remaining sample solution into the spare cavity 1065;
s6, opening the switch circuit 300, and removing the microfluidic chip 100, and collecting the sample solution left after incubation by using a pipette.
Performance testing
The residual sample solution a after incubation, the exosome sample solution b prepared in step S2, and the residual sample solution c in tube obtained from the mixed incubation group in tube collected in this example were tested separately using NTA.
The mixed incubation group in the tube specifically comprises the steps of putting a sample solution and magnetic beads in a centrifugal tube together according to a magnetic bead kit, standing at normal temperature or keeping shaking, and finally collecting the rest sample solution.
Referring to fig. 6 and 7, the test results show that the concentration of the residual sample solution a is 29.4% of the concentration of the prepared exosome sample solution b, and the efficiency of exosome sample enrichment in this example is estimated to be 70.6%, while the incubation efficiency of mixed incubation in the tube is 35.4%.
Example 3
The embodiment provides an integrated exosome-derived nucleic acid extraction method based on digital microfluidics, which is different from the extraction method in embodiment 2 in that:
the exosome sample solution and the magnetic bead suspension prepared in the step S2 are the same as those in the embodiment 1, and a diluent solution is also required to be prepared, wherein the diluent solution adopted in the embodiment is the saint xiang biological sample releasing agent S1014 diluted to 5% of the original concentration, and then tween-20 is added to prepare the diluent solution with the concentration of 0.01% (v/v);
10. mu.L of the releasing agent solution is also injected into the internal reaction chamber 106 in step S4;
step S5, automatically completing the pretreatment and nucleic acid extraction of the exosome sample in an automatic mode to obtain a nucleic acid release solution; wherein the step S5 of automatically completing the pretreatment and nucleic acid extraction of the exosome sample specifically comprises the following steps: the remaining sample solution is transferred to the waste liquid chamber 1064 in step S53; step S54, contracting the electromagnet again to enable the permanent magnet to be far away from the incubation cavity 1062, transferring the releasing agent solution into the incubation cavity 1062 in the step S53, uniformly mixing the releasing agent solution with the mixed product of the magnetic beads and the exosomes in the incubation cavity 1062, and incubating for 10min to obtain a nucleic acid releasing solution; the other steps are the same as the step of automatically finishing the pretreatment of the exosome sample in the embodiment 2, and are not described again;
in step S6, the microfluidic chip 100 is removed, and the nucleic acid releasing solution is collected by a pipette.
Other steps and methods are the same as those in embodiment 2, and are not described herein again.
Performance testing
Dividing the nucleic acid-releasing solution collected in step S6 into two parts; the in-tube release solution d collected by the in-tube mixed incubation group, the on-chip release solution e collected in the embodiment 3 and the blank sample f are respectively tested by adopting fluorescent quantitative PCR (qPCR) analysis; the release liquid d in the tube collected by the mixed incubation group in the tube and the release liquid e on the sheet collected in this example 3 were tested by digital PCR analysis. The gene to be detected in this example is glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, and the enzyme gene is housekeeping gene, and is widely used as an internal reference in nucleic acid detection; the digital PCR reaction adopts a one-step reverse transcription-amplification kit, so that RNA in an exosome sample and a small amount of DNA possibly existing in the exosome sample are not subdivided; the fluorescent quantitative PCR reaction adopts a fluorescent probe method, and front and rear primers and a probe are designed by self; each reaction system was 25. mu.L, which contained a premix, reverse transcription and DNA polymerase, primers and probe, RNase inhibitor and 5. mu.L of sample, and the remaining volume was made up with DEPC water, which was ultrapure water (first grade water) treated with DEPC (diethyl pyrocarbonate) and autoclaved at high temperature, as a colorless liquid.
Referring to the detection results in fig. 8-10, qPCR analysis shows that the Ct value of the nucleic acid release solution after pretreatment by the integrated exosome-derived nucleic acid extraction system based on digital microfluidics is 35.7, and the Ct value of the control group in the tube is 34.3;
the results of the digital PCR analysis and the results of the qPCR analysis are basically consistent, and the concentrations of the detected fragments are respectively 19.5 and 25.7 copies/mu L, which indicates that the RNA has certain loss but no magnitude difference in the total amount.
In summary, the exosome enrichment and nucleic acid release process in the exosome sample is integrated on the microfluidic chip, so that the efficient integration of exosome enrichment and nucleic acid release is realized, the integration and automation of exosome-derived nucleic acid sample pretreatment are realized, and a rapid pretreatment means is provided for subsequent nucleic acid amplification, detection and analysis; compared with the traditional exosome enrichment and treatment mode, the microfluidic chip has the advantages of high automation degree and sufficient reaction when used for pretreating exosome samples, can automatically complete pretreatment of trace exosome samples within 90 minutes by utilizing the extraction system, and ensures that the exosome enrichment efficiency is obviously higher than the enrichment efficiency of manual operation in a tube; the extraction method of the invention has high flexibility, and differential pretreatment results can be obtained according to different added reagents. Therefore, the invention effectively overcomes various defects in the prior art and has high industrial utilization value.
The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.
Claims (10)
1. An integrated exosome-derived nucleic acid extraction system based on digital microfluidics, comprising:
the microfluidic chip comprises an upper polar plate and a lower polar plate which are arranged at intervals, wherein the upper polar plate is provided with a sample inlet, two sides of the upper polar plate and the lower polar plate are respectively provided with a partition plate, an internal reaction cavity is formed between the partition plates, and an exosome sample is added into the internal reaction cavity from the sample inlet to extract nucleic acid;
the magnet control unit is arranged above the microfluidic chip and comprises an electromagnet and a permanent magnet clamped at the top end of the electromagnet, and the electromagnet is pushed and contracted in a direction vertical to the microfluidic chip to drive the permanent magnet to be close to or far away from the microfluidic chip;
the driving power supply comprises a chip driving power supply, a magnet driving power supply and a switch circuit, the voltage input end of the switch circuit is respectively connected with the chip driving power supply and the magnet driving power supply, the voltage output end of the switch circuit is respectively connected with the microfluidic chip and the magnet control unit, the chip driving power supply provides driving voltage for the microfluidic chip through the switch circuit, and the magnet driving power supply controls the push and the contraction of the electromagnet through the switch circuit;
the control unit comprises a control interface and an MCU, the control interface is connected with the MCU, the MCU is connected with the signal input end of the switch circuit, and the control interface controls the switch circuit through the MCU.
2. The digital microfluidic based integrated exosome-derived nucleic acid extraction system of claim 1, characterized in that: the internal reaction cavity comprises a sample cavity, an incubation cavity, a releasing agent cavity, a waste liquid cavity and a standby cavity which are sequentially communicated through all channels; the input end of the waste liquid cavity is connected with a first channel, the sample cavity is communicated with the first pipeline through a second channel, the incubation cavity is communicated with the first channel through a third channel, the releasing agent cavity is communicated with the first channel through a fourth channel, and the standby cavity is communicated with the first channel through a standby channel.
3. The digital microfluidic based integrated exosome-derived nucleic acid extraction system of claim 2, characterized in that: the utility model discloses a drive liquid drop device, including upper polar plate, lower polar plate, drive electrode, upper electrode, lower polar plate, upper electrode, lower electrode, upper electrode, lower polar plate, upper electrode, lower electrode, drive liquid drop's below for the removal of drive liquid drop is corresponding respectively, drive liquid drop is corresponding to set up in the below of the lower reaction chamber.
4. The digital microfluidic based integrated exosome-derived nucleic acid extraction system according to claim 3, characterized in that: the micro-fluidic chip is arranged on the clamp probe platform, a multi-path probe is arranged on the clamp probe platform, and the multi-path probe is respectively and correspondingly connected with the plurality of driving electrodes.
5. The digital microfluidic based integrated exosome-derived nucleic acid extraction system according to claim 4, characterized in that:
the switch circuit comprises a relay array, the relay array comprises a plurality of relays, each relay is connected with one path of the probe, and the relays correspondingly control the on-off of the driving electrodes through the probes.
6. The digital microfluidic based integrated exosome-derived nucleic acid extraction system of claim 1, characterized in that: the conductive coating is an indium tin oxide conductive coating;
the lower electrode plate is a patterned electrode, and the patterned electrode is manufactured by adopting a standard MEMS (micro-electromechanical system) process;
the thickness of the spacing plate is 80-120 mu m.
7. The digital microfluidic based integrated exosome-derived nucleic acid extraction system of claim 1, characterized in that: the chip driving power supply comprises a signal generator and a voltage amplifier, and signals generated by the signal generator are amplified by the voltage amplifier and then are connected to the switch circuit.
8. The digital microfluidic based integrated exosome-derived nucleic acid extraction system of claim 1, characterized in that:
the control interface comprises a control program and a graphical interface, the control program controls the switch circuit through the MCU, the graphical interface comprises a manual mode and an automatic mode, the manual mode is used for manually controlling the driving electrode by a user, and the automatic mode is used for editing an action flow to be executed so as to be automatically executed.
9. An integrated exosome-derived nucleic acid extraction method based on digital microfluidics is characterized in that: the nucleic acid extraction method comprises the following steps:
s1, providing the integrated exosome-derived nucleic acid extraction system based on digital microfluidics according to any one of claims 1 to 8;
s2, preparing an exosome sample solution, a magnetic bead suspension and a releasing agent solution;
s3, starting the MCU, the chip driving power supply and the magnet driving power supply, and opening a control interface;
s4, selecting a manual mode on a control interface, starting a driving electrode of the internal reaction cavity, and injecting the exosome sample solution, the magnetic bead suspension and the releasing agent solution prepared in the step S2 into the internal reaction cavity from the sample inlet respectively;
s5, adjusting the control interface to be in an automatic mode, setting an execution flow under the automatic mode, and automatically completing pretreatment and nucleic acid extraction of the exosome sample to obtain a nucleic acid release solution;
and S6, disconnecting the switch circuit, taking down the microfluidic chip, and collecting the obtained nucleic acid release solution.
10. The integrated exosome-derived nucleic acid extraction method based on digital microfluidics according to claim 9, characterized in that: in step S5, the pretreatment and nucleic acid extraction of the exosome sample are automatically completed, specifically including the following steps:
s51, pushing the electromagnet to enable the permanent magnet to be close to the incubation cavity, separating magnetic beads from liquid, gathering the magnetic beads around the electromagnet and staying in the incubation cavity, and meanwhile, operating the driving electrode to transfer the liquid in the incubation cavity to the waste liquid cavity;
s52, contracting the electromagnet to enable the permanent magnet to be far away from the incubation cavity, transferring the exosome sample solution in the sample cavity into the incubation cavity, uniformly mixing the exosome sample solution with the magnetic beads, and incubating for 30-60 min to obtain a magnetic bead-exosome mixed solution;
s53, the electromagnet is pushed again to enable the permanent magnet to be close to the incubation cavity, the magnetic bead-exosome mixed product in the incubation cavity is separated from the rest sample solution, the magnetic bead-exosome mixed product is gathered around the electromagnet and stays in the incubation cavity, and meanwhile the driving electrode is controlled to transfer the rest sample solution to the waste liquid cavity;
s54, contracting the electromagnet again to enable the permanent magnet to be far away from the incubation cavity, transferring the releasing agent solution into the incubation cavity in the step S53, uniformly mixing the releasing agent solution with the mixed product of the magnetic beads and the exosomes in the incubation cavity, and incubating for 10min to obtain the nucleic acid releasing liquid.
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