CN113418857B - Marine microorganism identification device and method combining alternating current-dielectrophoresis and coulter counting - Google Patents
Marine microorganism identification device and method combining alternating current-dielectrophoresis and coulter counting Download PDFInfo
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- 238000003940 alternating current dielectrophoresis Methods 0.000 title claims abstract description 17
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- 238000004720 dielectrophoresis Methods 0.000 claims abstract description 14
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Abstract
The invention relates to a marine microorganism identification device and method combining alternating current-dielectrophoresis and Coulter counting, wherein the invention applies uneven electric field to enable microorganisms to be acted by dielectrophoresis force, utilizes a Coulter counting module to count the number of microorganisms under positive dielectrophoresis force and the number of microorganisms under negative dielectrophoresis force, and adjusts the frequency of an alternating current signal, when the number of the microorganisms under positive dielectrophoresis force accounts for 50% of the total number of the microorganisms, the frequency of the externally-added alternating current signal is the specific frequency of the microorganisms, thereby establishing a specific frequency database of various microorganisms in the sea. When identifying the microorganisms in the ocean, distinguishing and identifying different types of microorganisms in the ocean is realized by measuring the specific frequency of the microorganisms and comparing the specific frequency with the parameters in the specific frequency database. The technical scheme of the invention solves the problems of large equipment volume, complex operation, time consumption, incapability of on-site detection and the like of the conventional marine microorganism detection equipment and method.
Description
Technical Field
The invention relates to the technical field of marine microorganism identification, in particular to a marine microorganism identification device and method combining alternating current-dielectrophoresis and coulter counting.
Background
There are a large number of microorganisms in the ocean, which exist as single cells or in groups, and which are capable of living independently, including viruses, bacteria, fungi, unicellular algae, etc. The marine microorganism can eliminate pathogenic bacteria of terrestrial origin, and its powerful decomposing ability can purify various types of pollution, and can provide antibiotics and other biological resources. However, some pathogenic microorganisms can cause diseases to marine organisms and human beings, and pose a great threat to public health and mariculture in coastal areas. Therefore, the method has important significance for detecting and identifying the marine microorganisms.
The microfluidic chip is a technology characterized by fluid manipulation in a micro-scale space, and among various methods of using the microfluidic chip, dielectrophoresis is one of the most effective methods. Dielectrophoresis refers to the movement of polarizable particles suspended in a solution generated in an uneven electric field, has the advantages of no marking, flexible chip design, portability in use, low power consumption, easiness in parallel operation and integration and the like, and is widely applied to the manipulation and identification of particles and cells.
The coulter counting principle means that when insulating particles in a suspension solution flow through a small hole, the particles change the resistance of a small hole circuit, a voltage pulse signal appears, and the particles can be counted by counting the number of pulse voltages. The coulter counting principle has high measurement accuracy and high sensitivity, can judge the size of cells by the amplitude of pulse voltage, and is widely applied to counting of cells.
The traditional marine microorganism detection equipment and method comprises a flow cytometer, a fluorescence microscope, a biochemical method and the like, but the methods have the disadvantages of large equipment volume, complex operation, time consumption, incapability of on-site detection and the like.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a marine microorganism identification device and method combining alternating current-dielectrophoresis and coulter counting. The invention applies uneven electric field to make the microbe be acted by dielectrophoresis force, and uses the Coulter counting module to count the microbe quantity acted by positive dielectrophoresis force and the microbe quantity acted by negative dielectrophoresis force, and adjusts the frequency of the alternating current signal, when the two quantities are approximately equal, namely the microbe quantity acted by positive dielectrophoresis force is 50% of the total microbe quantity, the frequency of the additional alternating current signal is the specific frequency of the microbe, and then establishes the specific frequency database of various microbes in the sea. When marine microorganisms are identified, the specific frequency of the microorganisms is measured and compared with the parameters in the specific frequency database, so that the microorganisms of different types in the sea are distinguished and identified.
The technical means adopted by the invention are as follows:
a marine microorganism identification device combining ac-dielectrophoresis and coulter counting, comprising: the method comprises the following steps: the ITO glass substrate layer, the 3D electrode layer and the PDMS cover plate layer; the 3D electrode layer is deposited on the ITO glass substrate layer; bonding the PDMS cover plate layer with the glass substrate layer;
the ITO glass substrate layer comprises a plurality of ITO electrodes, and two ITO electrodes are used as wires between the power supply line and the 3D electrode layer; all the rest ITO electrodes are used as electrodes of a Coulter counting module;
the 3D electrode layer comprises a first 3D electrode and a second 3D electrode, and the first 3D electrode and the second 3D electrode are respectively connected with two ITO electrodes serving as wires between a power line and the 3D electrode layer in the ITO glass substrate layer;
the PDMS cover layer comprises a plurality of inlets, a plurality of sample feeding channels respectively communicated with the inlets, a main channel communicated with the sample feeding channels, a plurality of sub-channels communicated with the main channel, a plurality of coulter counting modules correspondingly connected with the sub-channels, a plurality of sample discharging channels correspondingly connected with the coulter counting modules and a plurality of outlets communicated with the sample discharging channels.
Further, the ITO glass substrate layer comprises a first ITO electrode, a second ITO electrode, a third ITO electrode, a fourth ITO electrode, a fifth ITO electrode, a sixth ITO electrode, a seventh ITO electrode and an eighth ITO electrode; the first ITO electrode and the second ITO electrode are used as wires between the power line and the 3D electrode layer; and the third ITO electrode, the fourth ITO electrode, the fifth ITO electrode, the sixth ITO electrode, the seventh ITO electrode and the eighth ITO electrode are used as Coulter counting module electrodes.
Furthermore, the first 3D electrode is connected with the first ITO electrode, and the second 3D electrode is connected with the second ITO electrode.
Further, the first 3D electrode is connected to the main channel through two small holes, and the second 3D electrode is directly connected to the main channel.
Furthermore, the PDMS cover sheet layer includes a first inlet, a second inlet, a third inlet, a first sample channel communicated with the first inlet, a second sample channel communicated with the second inlet, a third sample channel communicated with the third inlet, a main channel communicated with the first sample channel, the second sample channel, and the third sample channel, a first sub-channel communicated with the main channel, a second sub-channel communicated with the main channel, a first coulter counting module connected with the first sub-channel, a second coulter counting module connected with the second sub-channel, a first sample outlet channel correspondingly connected with the first coulter counting module, a second sample outlet channel correspondingly connected with the second coulter counting module, a first outlet communicated with the first sample outlet channel, and a second outlet communicated with the second sample outlet channel.
Further, the first coulter counting module comprises the third ITO electrode, a fourth ITO electrode, a fifth ITO electrode, and a first coulter counting channel; the second Coulter counting module comprises a sixth ITO electrode, a seventh ITO electrode, an eighth ITO electrode and a second Coulter counting channel; the fourth ITO electrode and the seventh ITO electrode are connected with a signal source, and the third ITO electrode, the fifth ITO electrode, the sixth ITO electrode and the eighth ITO electrode output voltage signals.
Further, the first inlet is communicated with the main channel through the first sample feeding channel, the second inlet is communicated with the main channel through the second sample feeding channel, and the third inlet is communicated with the main channel through the third sample feeding channel.
Further, the main channel is connected with the first coulter counting module through the first sub-channel, and the main channel is connected with the second coulter counting module through the second sub-channel.
Further, the first coulter counting channel is communicated with the first outlet through the first sampling channel, and the second coulter counting channel is communicated with the second outlet through the second sampling channel.
The invention also provides a marine microorganism identification method based on the marine microorganism identification device, which comprises the following steps:
s1, injecting a suspension containing microorganisms into a second inlet, injecting a buffer solution into a first inlet and a third inlet, wherein the injection speeds of the three inlets are the same, simultaneously starting a signal source, and applying alternating current signals to a first ITO electrode and a second ITO electrode;
s2, detecting the number of microorganisms flowing through the first Coulter counting module according to a voltage signal output by the first Coulter counting module, and detecting the number of microorganisms flowing through the second Coulter counting module according to a voltage signal output by the second Coulter counting module;
s3, adjusting the frequency of the alternating current signal, wherein the number of microorganisms flowing through the first Coulter counting module and the second Coulter counting module is equal in the same time, namely the number of microorganisms subjected to positive dielectrophoresis force accounts for 50% of the total number of microorganisms, and the frequency of the externally-added alternating current signal is the specific frequency of the microorganisms;
s4, repeatedly executing the steps S1-S3, and establishing a specific frequency database of various microorganisms in the ocean;
and S5, when identifying the microorganisms in the sea, executing the steps S1-S3, comparing the measured specific frequency with the parameters of the database, and identifying the microorganisms.
Compared with the prior art, the invention has the following advantages:
1. the marine microorganism identification device provided by the invention is small in size, portable and capable of being used for field real-time detection.
2. The marine microorganism identification device provided by the invention identifies and distinguishes microorganisms by detecting the specific frequency of the microorganisms, and has high accuracy.
3. The marine microorganism identification device provided by the invention only needs to adjust the frequency of the alternating current signal, is simple to operate and has high detection speed.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the description of the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 is a schematic view of the overall structure of the apparatus of the present invention.
FIG. 2 is a schematic structural diagram of an ITO glass substrate layer of the present invention.
Fig. 3 is a schematic structural diagram of a second coulter counting module.
In the figure: 1. an ITO glass substrate layer; 2. a first ITO electrode; 3. a second ITO electrode; 4. a third ITO electrode; 5. a fourth ITO electrode; 6. a fifth ITO electrode; 7. a sixth ITO electrode; 8. a seventh ITO electrode; 9. an eighth ITO electrode; 10. a 3D electrode layer; 11. a first 3D electrode; 12. a second 3D electrode; 13. a PDMS cover sheet layer; 14. a first inlet; 15. a second inlet; 16. a third inlet; 17. a first sample introduction channel; 18. a second sample introduction channel; 19. a third sample inlet channel; 20. a main channel; 21. a first sub-channel; 22. a second sub-channel; 23. a first coulter counter module; 24. a second coulter counter module; 25. a first coulter count channel; 26. a second coulter count channel; 27. a first sample outlet channel; 28. a second sampling channel; 29. a first outlet; 30. a second outlet;
Detailed Description
It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict. The present invention will be described in detail below with reference to the accompanying drawings in conjunction with embodiments.
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. The following description of at least one exemplary embodiment is merely illustrative in nature and is in no way intended to limit the invention, its application, or uses. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
The relative arrangement of the components and steps, the numerical expressions and numerical values set forth in these embodiments do not limit the scope of the present invention unless it is specifically stated otherwise. Meanwhile, it should be understood that the sizes of the respective portions shown in the drawings are not drawn in an actual proportional relationship for the convenience of description. Techniques, methods, and apparatus known to those of ordinary skill in the relevant art may not be discussed in detail but are intended to be part of the specification where appropriate. Any specific values in all examples shown and discussed herein are to be construed as exemplary only and not as limiting. Thus, other examples of the exemplary embodiments may have different values. It should be noted that: like reference numbers and letters refer to like items in the following figures, and thus, once an item is defined in one figure, it need not be discussed further in subsequent figures.
In the description of the present invention, it is to be understood that the directions or positional relationships indicated by the directional terms such as "front, rear, upper, lower, left, right", "lateral, vertical, horizontal" and "top, bottom", etc., are generally based on the directions or positional relationships shown in the drawings for the convenience of description and simplicity of description, and that these directional terms, unless otherwise specified, do not indicate and imply that the device or element so referred to must have a particular orientation or be constructed and operated in a particular orientation, and therefore should not be considered as limiting the scope of the invention: the terms "inner and outer" refer to the inner and outer relative to the profile of the respective component itself.
For ease of description, spatially relative terms such as "over 8230 \ 8230;,"' over 8230;, \8230; upper surface "," above ", etc. may be used herein to describe the spatial relationship of one device or feature to another device or feature as shown in the figures. It will be understood that the spatially relative terms are intended to encompass different orientations of the device in use or operation in addition to the orientation depicted in the figures. For example, if a device in the figures is turned over, devices described as "above" or "on" other devices or configurations would then be oriented "below" or "under" the other devices or configurations. Thus, the exemplary terms "at 8230; \8230; 'above" may include both orientations "at 8230; \8230;' above 8230; 'at 8230;' below 8230;" above ". The device may be otherwise variously oriented (rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein interpreted accordingly.
It should be noted that the terms "first", "second", and the like are used to define the components, and are only used for convenience of distinguishing the corresponding components, and the terms have no special meanings unless otherwise stated, and therefore, the scope of the present invention should not be construed as being limited.
As shown in fig. 1, the present invention provides a marine microorganism identification apparatus combining ac-dielectrophoresis and coulter count, comprising: the ITO glass substrate layer 1, the 3D electrode layer 10 and the PDMS cover plate layer 13; the 3D electrode layer 10 is deposited on the ITO glass substrate layer 1; the PDMS cap layer 13 is bonded to the glass substrate layer 1. Wherein: the ITO glass substrate layer 1 comprises a plurality of ITO electrodes, two of the ITO electrodes are used as conducting wires between a power supply line and the 3D electrode layer 10; all the remaining ITO electrodes were used as coulter counter module electrodes. The 3D electrode layer 10 includes a first 3D electrode 11 and a second 3D electrode 12, and the first 3D electrode 11 and the second 3D electrode 12 are respectively connected to two ITO electrodes of the ITO glass substrate layer 1 as a conducting wire between the power line and the 3D electrode layer 10. The PDMS cap layer 13 includes a plurality of inlets, a plurality of sample channels respectively connected to the plurality of inlets, a main channel connected to the plurality of sample channels, a plurality of sub-channels connected to the main channel, a plurality of coulter counting modules correspondingly connected to the plurality of sub-channels, a plurality of sample outlets correspondingly connected to the plurality of coulter counting modules, and a plurality of outlets connected to the plurality of sample outlets.
In specific implementation, as a preferred embodiment of the present invention, as shown in fig. 2, the ITO glass substrate layer 1 includes a first ITO electrode 2, a second ITO electrode 3, a third ITO electrode 4, a fourth ITO electrode 5, a fifth ITO electrode 6, a sixth ITO electrode 7, a seventh ITO electrode 8, and an eighth ITO electrode 9; the first ITO electrode 2 and the second ITO electrode 3 are used as conducting wires between a power line and the 3D electrode layer 10; the third ITO electrode 4, the fourth ITO electrode 5, the fifth ITO electrode 6, the sixth ITO electrode 7, the seventh ITO electrode 8, and the eighth ITO electrode 9 serve as coulter counter module electrodes.
In specific implementation, as a preferred embodiment of the present invention, with reference to fig. 1, the first 3D electrode 11 is connected to the first ITO electrode 2, and the second 3D electrode 12 is connected to the second ITO electrode 3. The first 3D electrode 11 is connected to the main channel 20 through two small holes, and the second 3D electrode 12 is directly connected to the main channel 20.
In specific implementation, as a preferred embodiment of the present invention, with reference to fig. 1, the PDMS cover sheet layer 13 includes a first inlet 14, a second inlet 15, and a third inlet 16, a first sample channel 17 communicated with the first inlet 14, a second sample channel 18 communicated with the second inlet 15, a third sample channel 19 communicated with the third inlet 16, a main channel 20 communicated with the first sample channel 17, the second sample channel 18, and the third sample channel 19, a first sub-channel 21 communicated with the main channel 20, a second sub-channel 22 communicated with the main channel 20, a first coulter counter module 23 connected with the first sub-channel 21, a second coulter counter module 24 connected with the second sub-channel 22, a first sample outlet channel 27 correspondingly connected with the first coulter counter module 23, a second sample outlet channel 28 correspondingly connected with the second coulter counter module 24, a first outlet 29 communicated with the first sample outlet channel 27, and a second outlet 30 communicated with the second sample outlet channel 28.
In specific implementation, as a preferred embodiment of the present invention, with reference to fig. 1, the first inlet 14 is communicated with the main channel 20 through the first sample channel 17, the second inlet 15 is communicated with the main channel 20 through the second sample channel 18, and the third inlet 16 is communicated with the main channel 20 through the third sample channel 19.
In specific implementation, as a preferred embodiment of the present invention, as shown in fig. 3, the first coulter counter module 23 includes the third ITO electrode 4, the fourth ITO electrode 5, the fifth ITO electrode 6, and a first coulter counter channel (25); the second Coulter counting module (24) comprises a sixth ITO electrode (7), a seventh ITO electrode (8), an eighth ITO electrode (9) and a second Coulter counting channel (26); the fourth ITO electrode (5) and the seventh ITO electrode (8) are connected with a signal source, and the third ITO electrode (4), the fifth ITO electrode (6), the sixth ITO electrode (7) and the eighth ITO electrode (9) output voltage signals.
In specific implementation, as a preferred embodiment of the present invention, referring to fig. 1, the main channel 20 is connected to the first coulter counting module 23 through the first sub-channel 21, and the main channel 20 is connected to the second coulter counting module 24 through the second sub-channel 22. The first coulter counter channel 25 is in communication with the first outlet 29 through the first outlet channel 27, and the second coulter counter channel 26 is in communication with the second outlet 30 through the second outlet channel 28.
The invention also provides a marine microorganism identification method based on the marine microorganism identification device, which comprises the following steps:
s1, injecting a suspension containing microorganisms into a second inlet 15, injecting a buffer solution into a first inlet 14 and a third inlet 16, wherein the injection speeds of the three inlets are the same, simultaneously starting a signal source, and applying alternating current signals to a first ITO electrode 2 and a second ITO electrode 3;
s2, detecting the number of microorganisms flowing through the first coulter counting module 23 according to a voltage signal output by the first coulter counting module 23, and detecting the number of microorganisms flowing through the second coulter counting module 24 according to a voltage signal output by the second coulter counting module 24;
s3, adjusting the frequency of the alternating current signal, wherein the number of microorganisms flowing through the first Coulter counting module 23 and the second Coulter counting module 24 is almost equal in the same time, namely the number of microorganisms subjected to positive dielectrophoresis force accounts for 50% of the total number of microorganisms, and the frequency of the externally-applied alternating current signal is the specific frequency of the microorganisms;
s4, repeatedly executing the steps S1-S3, and establishing a specific frequency database of various microorganisms in the ocean;
and S5, when identifying the microorganisms in the ocean, executing the steps S1-S3, comparing the measured specific frequency with the parameters of the database, and identifying the microorganisms.
Example (b):
the suspension containing chlorella is injected into the second inlet 15 and the buffer is injected into the first inlet 14 and the third inlet 16 at the same rate. Simultaneously starting a signal source, and applying alternating current signals to the first ITO electrode 2 and the second ITO electrode 3;
the number of chlorella that flow through the first coulter counter module 23 is detected based on the voltage signal output from the first coulter counter module 23, and the number of chlorella that flow through the second coulter counter module 24 is detected based on the voltage signal output from the second coulter counter module 24.
The frequency of the alternating current signal is adjusted, when the number of the chlorella flowing through the first coulter counter module 23 and the second coulter counter module 24 are approximately equal in the same time, the number of the chlorella subjected to positive dielectrophoresis force accounts for 50% of the total number of the chlorella, and the frequency of the alternating current signal is the specific frequency of the chlorella.
Repeating the above operations to build a specific frequency database of various microorganisms in the ocean.
When identifying a microorganism in the sea, if the specific frequency of a certain microorganism is measured to be the same as the specific frequency of chlorella, the microorganism is chlorella.
Claims (9)
1. A marine microorganism identification device combining ac-dielectrophoresis and coulter count, comprising: the ITO glass substrate layer (1), the 3D electrode layer (10) and the PDMS cover sheet layer (13); a 3D electrode layer (10) is deposited on the ITO glass substrate layer (1); the PDMS cover sheet layer (13) is bonded with the glass substrate layer (1);
the ITO glass substrate layer (1) comprises a plurality of ITO electrodes, and two ITO electrodes are used as wires between a power line and the 3D electrode layer (10); all the rest ITO electrodes are used as Coulter counting module electrodes; the ITO glass substrate layer (1) comprises a first ITO electrode (2), a second ITO electrode (3), a third ITO electrode (4), a fourth ITO electrode (5), a fifth ITO electrode (6), a sixth ITO electrode (7), a seventh ITO electrode (8) and an eighth ITO electrode (9); the first ITO electrode (2) and the second ITO electrode (3) are used as leads between a power supply line and the 3D electrode layer (10); a third ITO electrode (4), a fourth ITO electrode (5), a fifth ITO electrode (6), a sixth ITO electrode (7), a seventh ITO electrode (8) and an eighth ITO electrode (9) are used as Coulter counting module electrodes;
the 3D electrode layer (10) comprises a first 3D electrode (11) and a second 3D electrode (12), and the first 3D electrode (11) and the second 3D electrode (12) are respectively connected with two ITO electrodes serving as wires between a power supply line and the 3D electrode layer (10) in the ITO glass substrate layer (1);
the PDMS cover layer (13) comprises a plurality of inlets, a plurality of sample feeding channels respectively communicated with the inlets, a main channel communicated with the sample feeding channels, a plurality of sub-channels communicated with the main channel, a plurality of Coulter counting modules correspondingly connected with the sub-channels, a plurality of sample discharging channels correspondingly connected with the Coulter counting modules and a plurality of outlets communicated with the sample discharging channels;
the marine microorganism identification method based on the marine microorganism identification device combining alternating current-dielectrophoresis and coulter counting comprises the following steps:
s1, injecting a suspension containing microorganisms into a second inlet (15), injecting a buffer solution into a first inlet (14) and a third inlet (16), wherein the injection speeds of the three inlets are the same, simultaneously starting a signal source, and applying an alternating current signal to a first ITO electrode (2) and a second ITO electrode (3);
s2, detecting the number of microorganisms flowing through the first Coulter counting module (23) according to the voltage signal output by the first Coulter counting module (23), and detecting the number of microorganisms flowing through the second Coulter counting module (24) according to the voltage signal output by the second Coulter counting module (24);
s3, adjusting the frequency of the alternating current signal, wherein the number of microorganisms flowing through the first Coulter counting module (23) and the second Coulter counting module (24) is equal in the same time, namely the number of microorganisms subjected to positive dielectrophoresis force accounts for 50% of the total number of microorganisms, and the frequency of the externally-applied alternating current signal is the specific frequency of the microorganisms;
s4, repeatedly executing the steps S1-S3, and establishing a specific frequency database of various microorganisms in the ocean;
and S5, when identifying the microorganisms in the sea, executing the steps S1-S3, comparing the measured specific frequency with the parameters of the database, and identifying the microorganisms.
2. The marine microorganism recognition device combining alternating current-dielectrophoresis and coulter count according to claim 1, the ITO glass substrate layer (1) comprising a first ITO electrode (2), a second ITO electrode (3), a third ITO electrode (4), a fourth ITO electrode (5), a fifth ITO electrode (6), a sixth ITO electrode (7), a seventh ITO electrode (8) and an eighth ITO electrode (9); the first ITO electrode (2) and the second ITO electrode (3) are used as leads between a power supply line and the 3D electrode layer (10); the third ITO electrode (4), the fourth ITO electrode (5), the fifth ITO electrode (6), the sixth ITO electrode (7), the seventh ITO electrode (8) and the eighth ITO electrode (9) are used as Coulter counting module electrodes.
3. The marine microorganism identification device combining alternating current-dielectrophoresis and coulter counting according to claim 2, wherein the first 3D electrode (11) is connected to the first ITO electrode (2), and the second 3D electrode (12) is connected to the second ITO electrode (3).
4. The marine microorganism recognition apparatus combining AC-dielectrophoresis and Coulter counting according to claim 3, wherein the first 3D electrode (11) is connected to the main channel (20) through two small holes, and the second 3D electrode (12) is directly connected to the main channel (20).
5. The marine microorganism identification device combining ac-dielectrophoresis and coulter counting according to claim 1, wherein the PDMS cover layer (13) comprises a first inlet (14), a second inlet (15) and a third inlet (16), a first sample channel (17) communicating with the first inlet (14), a second sample channel (18) communicating with the second inlet (15) and a third sample channel (19) communicating with the third inlet (16), a main channel (20) communicating with the first sample channel (17), the second sample channel (18) and the third sample channel (19), a first sub-channel (21) communicating with the main channel (20), a second sub-channel (22) communicating with the main channel (20), a first coulter counting module (23) connecting with the first sub-channel (21), a second coulter counting module (24) connecting with the second sub-channel (22), a first outlet channel (27) connecting with the first coulter counting module (23), a second coulter counting module (28) connecting with the second sub-channel (24), and a second outlet (28) communicating with the first outlet (28).
6. A marine microorganism identification means combining AC-dielectrophoresis and Coulter counting according to claim 5, the first Coulter counting module (23) comprising the third ITO electrode (4), the fourth ITO electrode (5), the fifth ITO electrode (6), the first Coulter counting channel (25); the second Coulter counting module (24) comprises a sixth ITO electrode (7), a seventh ITO electrode (8), an eighth ITO electrode (9) and a second Coulter counting channel (26); the fourth ITO electrode (5) and the seventh ITO electrode (8) are connected with a signal source, and the third ITO electrode (4), the fifth ITO electrode (6), the sixth ITO electrode (7) and the eighth ITO electrode (9) output voltage signals.
7. The marine microorganism identification device combining AC-dielectrophoresis and Coulter counting according to claim 5, wherein the first inlet (14) communicates with the main channel (20) through the first sample channel (17), the second inlet (15) communicates with the main channel (20) through the second sample channel (18), and the third inlet (16) communicates with the main channel (20) through the third sample channel (19).
8. The device for marine microorganism identification combining ac-dielectrophoresis and coulter counting according to claim 7, wherein the main channel (20) is connected to the first coulter counting module (23) through the first sub-channel (21), and the main channel (20) is connected to the second coulter counting module (24) through the second sub-channel (22).
9. The marine microorganism recognition device combining ac-dielectrophoresis and coulter counting according to claim 8, wherein the first coulter counting channel (25) communicates with the first outlet (29) through the first sample outlet channel (27), and the second coulter counting channel (26) communicates with the second outlet (30) through the second sample outlet channel (28).
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