CN114289082B - Microfluidic chip device for stimulating nematodes by electric shock - Google Patents
Microfluidic chip device for stimulating nematodes by electric shock Download PDFInfo
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- CN114289082B CN114289082B CN202111601536.5A CN202111601536A CN114289082B CN 114289082 B CN114289082 B CN 114289082B CN 202111601536 A CN202111601536 A CN 202111601536A CN 114289082 B CN114289082 B CN 114289082B
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Abstract
The invention discloses a microfluidic chip device for stimulating nematodes by electric shock, which comprises a main channel and 8 side channels, wherein the side channels are coplanar with the bottom surface of the main channel and have different heights, the lower transverse channel is the main channel, and the nematodes are introduced into the main channel for electric stimulation; the opening of 8 side channels is had to one side of main passageway, and side channel and main passageway intercommunication, and 8 side channels are all perpendicular with main passageway only in the junction with main passageway, then are radial arranging, and side channel can link to each other with external power source, has the electric conduction effect, and side channel total length and general structure are unanimous, guarantee that the resistance is the same, and 8 side channels are 8 electrodes and divide into 7 sections with the nematode, can carry out the electric stimulation to arbitrary section or section group. The invention adopts the micro-fluidic chip technology, can realize the flexible control of nematodes by using the excellent control technology in micro-nano scale, and the micro-channel is also the basic structure of the micro-fluidic chip, and the micro-channel is used for conducting electric stimulation, so that the experimental principle is simple, the chip design is simple, and the feasibility is strong.
Description
Technical Field
The invention belongs to the technical field of microfluidic chip analysis, relates to the research fields of nematode cell voltage-gated ion channels, cell membrane potential, nerve electric impulse, motor neuron control muscle contraction and the like, and in particular relates to a microfluidic chip device for electrically stimulating nematodes.
Background
The model animal caenorhabditis elegans (Caenorhabditis elegans, C.elegans) has the advantages of rapid growth and propagation, easy culture, tiny individual, transparent body, higher homology with the genome of higher animals, capability of performing molecular biological operation and the like, and has been widely applied to research in the fields of neurobiology, developmental biology, molecular biology, pharmacological toxicology and the like. The individual nematodes are tiny, the length of the adult nematodes is 1 millimeter, the width of the adult nematodes is about 80 microns, the adult nematodes are difficult to detect by naked eyes, and the fixed observation, the micromanipulation and the capture of the nematode are difficult. The micro-fluidic chip technology which is vigorously developed has been widely applied to the operation control of micro-scale mode animal nematodes with excellent control capability in micro-nano scale, such as the fixation operation, capturing, sorting, chemical drug stimulation of the nematodes, and the micro-injection technology of the nematodes on the micro-fluidic chip published in 2013, and the development of the nematode research technology is greatly promoted by the advent of the micro-fluidic chip technology.
The method has the advantages that the electric stimulation is difficult to be carried out on organisms with the length of less than 1 mm, the report of nematode electric stimulation research is less, and the problem can be well solved by the appearance of a microfluidic chip technology. The nematode body has 302 neurons, accounting for about 30% of total cells, which is a better neurobiological research model, and the electrical stimulation is one of the technical means of neurobiological research. There are numerous ion channels on nematode cells, including voltage-gated ion channels, whose electrical stimulation can alter channel function, causing changes in ion levels in the cell fluid, which will produce what biological effects? What is the voltage threshold to initiate voltage-gated ion channel opening? The electrical stimulation may cause muscle contraction, the contraction of the nematode muscle cells is dominated by motor neurons, the electrical signal may cause muscle contraction, how does its molecular mechanism? These can all be studied on model animal nematodes, and microfluidic chip technology can provide electrical stimulation means for these studies.
The electrode implantation method can be used for the electric stimulation of animals in modes such as rabbits, mice, frogs, primates and the like, but the electrode implantation can bring about wounds, and has the defect that the micro-fluidic chip can be used for the electrode insertion invasive electric stimulation by the microinjection method proposed in 2013, and can also be used for guiding the electric stimulation by taking a micro-channel filled with electrolyte solution as an electrode to contact with nematode epidermis, so that the wounds can be avoided.
Disclosure of Invention
The invention aims to: in order to solve the defects in the prior art, the invention provides a microfluidic chip device for electrically and locally stimulating adults of nematodes, which realizes the operation of electrically stimulating the nematodes and records and analyzes corresponding physiological effects by using an optical imaging analysis technology.
The technical scheme is as follows: a microfluidic chip device for stimulating nematodes by electric shock comprises a main channel and 8 side channels, wherein the side channels are coplanar with the bottom surface of the main channel and have different heights, the lower transverse channel is the main channel, and the nematodes are introduced into the main channel for electric stimulation; the device is characterized in that 8 side channels are arranged on one side of the main channel, the side channels are communicated with the main channel, the 8 side channels are perpendicular to the main channel only at the joint of the side channels and the main channel and are radially arranged, the side channels can be connected with an external power supply, the electric conduction effect is achieved, the total length and the general structure of the side channels are consistent, the same resistance is ensured, the 8 side channels, namely 8 electrodes, divide nematodes into 7 sections, and electric stimulation can be carried out on any section or section group.
As an optimization: the front and back of the main channel are respectively provided with a bottleneck structure with the width of 70 microns, and the channel between the two bottleneck structures is a nematode residence area and is used for electric stimulation.
As an optimization: the width of the main channel is 80 micrometers, and the height is 80 micrometers.
As an optimization: the main channel is used for nematode sampling, fixing, electric stimulation, signal acquisition and nematode sampling.
As an optimization: the width of the opening of the side channel is 20 micrometers, and the height is 30-40 micrometers.
As an optimization: the rear end of the side channel has a height of 80 microns and a width of 100-600 microns, and the design can reduce the resistance and ensure enough conductive liquid in the electrode channel.
As an optimization: one surface channel wall of the side channel is not straight, but is in an arc-shaped structure which can be matched with the nematode posture so as to maintain the nematode normal posture.
As an optimization: negative pressure can be applied in the side channel to suck nematode epithelial tissue into the channel, so that the nematode epithelial tissue has a sealing effect and prevents electric leakage.
The beneficial effects are that: the individual nematodes of model animals are small and living and are difficult to electrically stimulate using conventional electrodes and microelectrodes. The invention adopts the micro-fluidic chip technology, can realize the flexible control of nematodes by using the excellent control technology in micro-nano scale, and the micro-channel is a basic structure of the micro-fluidic chip, and the micro-channel is used for conducting electric stimulation, so that the invention has the advantages of simple experimental principle, simple chip design, strong feasibility, and optical imaging analysis function, and the technical advantages of the micro-fluidic chip can be brought into play by introducing the electric stimulation technology into nematode research.
Drawings
FIG. 1 is a schematic diagram of a microfluidic chip design for nematode electrical stimulation in accordance with the present invention; the upper diagram is an integral structure diagram of the chip, and a transverse channel below the diagram is a main channel; the lower graph is an enlarged graph of the electric stimulation working area, and the main channel is used for nematode sample injection, fixation, electric stimulation, signal acquisition and nematode sample discharge. The main channel design was 80 microns in height and 80 microns in width. The side channels are also electrode channels and are arranged radially on one side of the main channel, and are communicated with the main channel and vertically open to the main channel.
FIG. 2 is a finished product diagram of the nematode electro-stimulation chip of the invention; one end of the metal tube is connected with the plastic tube, the other end is inserted into the PDMS internal connection electrode channel, and the metal tube can be clamped with an electrode and electrified. The plastic pipe can be filled with solution or connected with a negative pressure source to adsorb nematodes.
Fig. 3 is a pictorial view of the present invention showing nematodes in the main channel. The nematodes are adsorbed by the electrode channel, part of tissues block the opening of the electrode channel, the electrode channel is sealed, and the electric stimulation is waited for. The scale length is 80 microns.
FIG. 4 is a schematic diagram of the calcium ion change of the invention, wherein the electric stimulation can cause the appearance of calcium peaks of nematode intestinal cells, the intestinal cells express calcium ion fluorescent protein probe marks, and the fluorescence change can be detected by applying 12V voltage, namely the calcium ion change.
Detailed Description
The following technical solutions in the embodiments of the present invention will be clearly and completely described so that those skilled in the art can better understand the advantages and features of the present invention, thereby making a clearer definition of the protection scope of the present invention. The described embodiments of the present invention are intended to be only a few, but not all embodiments of the present invention, and all other embodiments that may be made by one of ordinary skill in the art without inventive faculty are intended to be within the scope of the present invention.
Examples
In the microfluidic chip capable of realizing accurate control of micro-nano channel fluid, the micro-scale micro-channel is of a basic structure, electrolyte buffer solution is filled in the micro-channel to have a conductive function, and external current can be led into a nematode body to realize electric stimulation after the micro-channel is contacted with the nematode.
The micro-fluidic chip micro-channel structure designed as shown in fig. 1 is as follows: the lower transverse channel is the main channel into which nematodes will be introduced for electrical stimulation, the main channel having a width of 80 microns and a height of 80 microns, with reference to the individual dimensions of the nematodes. The side channel is communicated with the main channel, the width of the side channel opening is 20 micrometers, the height is 30-40 micrometers, and the side channel is lower than the main channel, so that nematodes can be prevented from entering the side channel, and the width and the height of all the side channel openings are consistent. The side channels may be connected to an external power source and have an electrical conduction effect, and are called electrode channels. Negative pressure can be applied in the side channel to suck nematode epithelial tissue into the channel, so that the nematode epithelial tissue has a sealing effect and prevents electric leakage. The rear end of the side channel has a height of 80 micrometers and a width of 100-600 micrometers, and the design can reduce the resistance and ensure that enough conductive liquid exists in the electrode channel.
The 8 side channels are perpendicular to the main channel only at the connection part with the main channel, and then are radially arranged, and the total length and the general structure of the side channels are consistent so as to ensure that the resistances are the same. The side channels are coplanar with the bottom surface of the main channel and have different heights. 8 side channels, namely 8 electrodes divide the nematode into 7 sections, and can electrically stimulate any section or section group, thereby improving the accuracy of the stimulation site.
The main channel has a bottleneck structure, the width of which is 70 microns, and the channel between the two structures is the nematode residence area and the place where the electric stimulation is carried out. The design is to prevent other channels in the chip from causing nematode loss to the interference of the main channel. One channel wall of the side channel is not straight, but is in an arc-shaped structure which can be matched with the nematode posture so as to maintain the nematode normal posture.
As shown in figure 2, after the chip is perforated, a metal tube of a needle head of a No. 7 syringe is inserted into the hole to connect the side channel with the PE tube, wherein the metal tube has a connecting function and is also an application point of an electrode, and the electrode can be connected with an electrolyte solution in the chip channel by being applied to the metal tube, so that electric stimulation can be applied to the wire.
Before the voltage is applied for stimulation, enough electrolyte solution is ensured to be communicated in the electrode channel. When the negative pressure in the side channel is large, gas molecules can penetrate through the PDMS to enter the channel, and electric conduction can be possibly blocked, so that the negative pressure in the electrode channel cannot be too large, and gas inhalation is prevented. When gas is introduced, the negative pressure can be properly reduced, and the electric stimulation is implemented after the gas is exhausted.
The electrical stimulation can be synchronously carried out to record the nematode physiological behavior by optical imaging, and experimental analysis is carried out. For example, calcium ion imaging analysis, etc., as shown in fig. 3.
The whole nematode is electrically stimulated, two electrodes are respectively connected to two ends of the intestinal part of the nematode, 12V direct current stable voltage is applied, the stimulation is carried out once for 50 seconds, the instant stimulation is not delayed, meanwhile, calcium imaging of intestinal cells is carried out, and the occurrence of calcium concussion synchronous with the electrode stimulation can be seen, as shown in fig. 4. If the time for applying the electric stimulus is prolonged, a calcium signal is generated when the voltage is applied, the cells do not have corresponding calcium signals after the electric stimulus is removed, and the nematode intestinal cells are insensitive to the electric stimulus removal. The low voltage does not cause the appearance of a calcium peak at the time of electrical stimulation, and when the voltage value breaks through 5V, the calcium peak appears, which may be because the opening of ion channels on the endoplasmic reticulum membrane requires the external stimulus to reach a specific threshold.
The invention adopts the micro-fluidic chip technology, can realize the flexible control of nematodes by using the excellent control technology in micro-nano scale, and the micro-channel is a basic structure of the micro-fluidic chip, and the micro-channel is used for conducting electric stimulation, so that the invention has the advantages of simple experimental principle, simple chip design, strong feasibility, and optical imaging analysis function, and the technical advantages of the micro-fluidic chip can be brought into play by introducing the electric stimulation technology into nematode research.
Claims (1)
1. A microfluidic chip device for electrically shocking and stimulating nematodes, which is characterized in that: the nematode-killing device comprises a main channel and 8 side channels, wherein the side channels are coplanar with the bottom surface of the main channel and have different heights, the lower transverse channel is the main channel, and nematodes are introduced into the main channel for electric stimulation; the nematode electric stimulation device is characterized in that an opening of 8 side channels is formed in one side of the main channel, the side channels are communicated with the main channel, the 8 side channels are perpendicular to the main channel only at the joint of the side channels and the main channel and are radially arranged, the side channels are connected with an external power supply, the electric conduction effect is achieved, the total length and the general structure of the side channels are consistent, the same resistance is ensured, the 8 side channels, namely the 8 electrode channels, divide nematodes into 7 sections, and electric stimulation can be carried out on any section or section group;
the front and the back of the main channel are respectively provided with a bottleneck structure with the width of 70 microns, and the channel between the two bottleneck structures is a nematode residence area for electric stimulation;
the width of the main channel is 80 microns, and the height is 80 microns;
the main channel is used for nematode sampling, fixing, electrical stimulation, signal acquisition and nematode sampling;
the width of the opening of the side channel is 20 micrometers, and the height is 30-40 micrometers;
the rear end of the side channel has the height of 80 microns and the width of 100-600 microns, and the design can reduce the resistance and ensure that enough conductive liquid exists in the electrode channel;
one surface channel wall of the side channel is not straight, but is in an arc-shaped structure which can be matched with the nematode posture so as to maintain the nematode normal posture;
negative pressure is applied in the side channel to suck the nematode epithelial tissue into the channel, so that the nematode epithelial tissue has a sealing effect and prevents electric leakage.
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| CN202111601536.5A CN114289082B (en) | 2021-12-24 | 2021-12-24 | Microfluidic chip device for stimulating nematodes by electric shock |
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| CN202111601536.5A CN114289082B (en) | 2021-12-24 | 2021-12-24 | Microfluidic chip device for stimulating nematodes by electric shock |
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| CN114289082B true CN114289082B (en) | 2023-05-05 |
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| US20200016592A1 (en) * | 2018-06-25 | 2020-01-16 | Nemametrix Inc. | High-throughput microfluidic-based methods for recording defecation motor program (dmp) events in nematodes |
| CN111735852B (en) * | 2020-05-29 | 2022-03-29 | 东南大学 | Micro-fluidic chip for monitoring movement behaviors and physiological characteristics of nematodes |
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