CN114748075A

CN114748075A - Zebra fish in-vivo electrophysiological monitoring system for high-throughput drug screening

Info

Publication number: CN114748075A
Application number: CN202210329035.4A
Authority: CN
Inventors: 林旭东; 周雅静
Original assignee: Sun Yat Sen University
Current assignee: Guangdong Longsee Medical Technology Co ltd
Priority date: 2022-03-31
Filing date: 2022-03-31
Publication date: 2022-07-15

Abstract

The invention discloses a zebra fish in-vivo electrophysiological monitoring system for high-flux drug screening, which is mainly based on an integrated chip, wherein the integrated chip is composed of a micro-fluidic chip substrate and a micro-invasive electrode bottom plate, through the fixing action of the micro-fluidic chip and micro-needle electrodes in the micro-invasive electrodes, electroencephalogram signals of zebra fish juvenile fish can be accurately measured, and the problems of zebra fish damage or inaccurate signals and the like possibly brought in the traditional method are solved. Moreover, the invention creatively uses the selective manufacturing method of the patterned array type microneedle to realize the design of the microneedle with the micron level, and realizes the wide application of the microneedle array.

Description

Zebra fish in-vivo electrophysiological monitoring system for high-throughput drug screening

Technical Field

The invention belongs to the technical field of medical instruments, and particularly relates to a zebra fish in-vivo electrophysiological monitoring system for high-throughput drug screening.

Background

Zebra fish (Danio rerio) is a bony fish, native to south Asia. The zebra fish is a typical subtropical fresh water ornamental fish, and stripes with alternate blue and silver colors are distributed on the body surface of the fish, are similar to the stripes on the zebra body, and are called as the zebra fish. The zebra fish is slender in body shape, mild in temperament, easy to feed, low in water quality requirement and short in growth period, and can reach sexual maturity in about 3 months. The breeding cycle is usually about one week, and the egg laying amount at one time can reach hundreds of eggs. The ovum is fertilized in vitro, the in vitro development is carried out, the embryo body is transparent, the embryo development is synchronous and fast, and the growth and development conditions of each organ of the zebra fish embryo can be clearly observed on the premise of not damaging the embryo. The zebra fish juvenile fish has the characteristics of small volume, rapid development, transparency and the like, is an excellent model animal at present, and is commonly used for researches such as neuroscience, biochemistry and new drug research and development. Research in recent years shows that the acquisition of electrophysiological parameters of the zebra fish model is not only beneficial to the mechanism research of biology aspects such as animal neural circuits, tissue repair and behavior drive, but also has important application in the aspects of side effect detection, drug effect evaluation and the like in drug screening experiments from the viewpoint of interdiscipline such as engineering technology.

In the related art, most of the electrophysiological systems on the market are designed for rats, rabbits, monkeys, and even human larger animals. There is no report on the commercial electrophysiological system for zebrafish juvenile fish, which is a very small animal (several millimeters in length and less than 1 millimeter in width). Although there are many teams trying to overcome this problem, the reported techniques have several drawbacks. An integrated zebra fish analysis platform IZAP of a SoonGwenon team at the university of California in 2016 is used for realizing the recording of electric signals by closely contacting a plurality of surface electrodes with the heads of zebra fish. In 2018, a research team fixes zebra fish through a capillary glass tube and low-temperature agarose, and detects changes of electroencephalogram signals of juvenile fish in a chip along with light stimulation by applying an invasive single electrode. Although both systems realize electrophysiological recording of zebra fish, the IZAP system based on surface electrode recording is susceptible to environmental interference, unstable and less accurate than invasive electrodes. For a system using an invasive electrode, a single capillary glass tube electrode has high limitation and one-sidedness in signal capture, low flux and complex operation, and is not beneficial to large-scale detection.

Therefore, if a large amount of zebra fish are required to be subjected to electrophysiological tests, the development of a zebra fish electrophysiological system for performing low-cost, high-throughput, high-efficiency and automatic recording on zebra fish electroencephalogram is urgently needed.

Disclosure of Invention

The present invention has been made to solve at least one of the above-mentioned problems occurring in the prior art. Therefore, the invention provides a zebra fish in-vivo electrophysiological monitoring system for high-flux drug screening, which is mainly based on an integrated chip, wherein the integrated chip is composed of a micro-fluidic chip substrate and a micro-invasive electrode bottom plate, and through the fixing action of the micro-fluidic chip and micro-needle electrodes in the micro-invasive electrodes, the electroencephalogram signals of the zebra fish juvenile fish can be accurately measured, and the zebra fish health can not be damaged, so that the zebra fish in-vivo electrophysiological monitoring system has the technical advantages of low cost, high flux, high efficiency and automation.

In a first aspect of the invention, an integrated chip is provided.

According to a first aspect of the invention, in some embodiments of the invention, the integrated chip comprises a microfluidic chip substrate and a micro-invasive electrode back plane.

In some embodiments of the present invention, the microfluidic chip substrate has several fixing chambers thereon, and the fixing chambers are used for fixing zebra fish.

In some embodiments of the invention, the number of the fixed chambers can be changed according to actual use requirements, and the change of the number does not affect the realization of the functionality.

In some embodiments of the invention, the number of fixed chambers is 5.

In some embodiments of the present invention, the minimally invasive electrode base plate has a plurality of microneedles with electrode traces attached thereto.

In some embodiments of the invention, the height of the microneedle is 90-110 μm, the diameter of the microneedle is less than or equal to 100 μm, and the distance between the microneedles is 400-450 μm.

In some preferred embodiments of the present invention, the microneedles are 100 μm in height, 100 μm in diameter, and 450 μm apart.

According to the first aspect of the present invention, in some embodiments of the present invention, the microfluidic chip substrate further has a liquid inlet channel and a liquid outlet channel, the liquid inlet channel and the liquid outlet channel are parallel to each other and are connected by a fixing chamber perpendicular to the liquid inlet channel and the liquid outlet channel, respectively, and a distance between the fixing chamber and an adjacent fixing chamber is 4.5-5.5 mm.

In some preferred embodiments of the present invention, the spacing between the fixed chamber and the adjacent fixed chamber is 5 mm.

In some embodiments of the present invention, the liquid inlet channel and the liquid outlet channel both have a width of 900 to 1000 μm and a height of 500 to 600 μm.

In some preferred embodiments of the present invention, the width of the liquid inlet channel and the liquid outlet channel are both 900 μm, and the height of the liquid inlet channel and the liquid outlet channel are both 600 μm.

In some embodiments of the invention, the fixed chamber contains a tapered structure with a width decreasing from 900 μm to 150 μm in the direction of fluid flow.

In the invention, the micro-fluidic chip is made of Polydimethylsiloxane (PDMS).

Of course, those skilled in the art can appropriately select other materials to prepare the microfluidic chip according to the actual use requirement, including but not limited to Polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), glass, and gelatin. The material is selected to be transparent in color, easy to observe and free from influencing test operation and various detections.

The 2D engineering structure diagram of the microfluidic chip of the invention is shown in FIG. 1. In some embodiments, the liquid inlet channel and the liquid outlet channel are consistent in size, both have a width of 900 μm and a height of 600 μm, and the joint of the liquid inlet channel and the fixed chamber is of a round-angle structure (a circular arc with a radius of 150 μm) so as to avoid damage of zebra fish. The fixed cavity is sequentially divided into a fixed cavity front section, a middle section and a rear end along the fluid flowing direction. The front section of the fixed cavity is connected with the liquid inlet channel, the size of the liquid inlet channel is consistent with that of the liquid inlet channel, the width of the liquid inlet channel is 900 micrometers, the height of the liquid inlet channel is 600 micrometers, and the length of the front section of the fixed cavity is 4.2 mm. The middle section of the fixed cavity is of a conical structure, the width of the fixed cavity is gradually reduced along the flowing direction of the fluid (from 900 mu m to 150 mu m), and the height of the fixed cavity is 600 mu m. The length of the middle section is about 0.8-1.4 mm. The rear section of the fixed cavity is connected with the liquid outlet channel, the length is 2.59mm, the height is 600 μm, and the width is 150 μm.

In some embodiments of the present invention, the middle fixing cavity section is used for fixing the head of the zebra fish juvenile fish, and the rear fixing cavity section can only pass through the tail of the zebra fish juvenile fish for limiting the tail movement of the zebra fish juvenile fish.

In some embodiments, the micro-invasive electrode base plate further comprises a working electrode and a reference electrode.

In some embodiments of the present invention, the working electrode has an epitaxial line width of 90 to 100 μm, an epitaxial electrical contact length of 7.5 to 8mm, and a width of 5.5 to 6 mm;

in some embodiments of the present invention, the reference electrode has a length of 4.5 to 5mm and a width of 10 to 12 mm.

Of course, the parameters of the working electrode and the reference electrode can be reasonably adjusted by those skilled in the art according to actual use requirements so as to meet the use requirements of different scenes in actual detection.

In a second aspect of the present invention, there is provided a method for preparing a micro-invasive electrode pad in an integrated chip according to the first aspect of the present invention, comprising the following steps:

(1) taking a monocrystalline silicon microneedle array as a template, using a flexible material for turnover, adding an electrical insulating resin material for reverse molding, and pouring again by using the flexible material to obtain a microneedle array base plate;

(2) Preparing an electrode mask plate according to the size of the electrode in the first aspect of the invention, aligning a circuit in the electrode mask plate with the micro-needle in the micro-needle array base plate prepared in the step (1), and sputtering 18-20 nm of chromium and 190-200 nm of gold to obtain the electrode.

According to a second aspect of the present invention, in some embodiments of the present invention, the flexible material comprises at least one of polydimethylsiloxane, polymethylmethacrylate, gelatin.

According to a second aspect of the present invention, in some embodiments of the present invention, the electrically insulating resin material comprises at least one of an epoxy resin, a silicone resin.

In some embodiments of the present invention, the method for preparing the micro-invasive electrode backplane specifically comprises:

(1) taking a monocrystalline silicon microneedle array (the space between needles is 400 mu m, the height is 100 mu m, the needles are rectangular pyramids, the bottom side length of a cone is 30 mu m) as a mould, soaking the mould in absolute ethyl alcohol for ultrasonic cleaning, carrying out vacuum drying, pouring PDMS (polydimethylsiloxane), and removing bubbles at room temperature in vacuum. And placing in an oven at 70 ℃ for baking for more than 4h for full curing, and removing the cured PDMS to obtain the microneedle array female die. And (3) manufacturing a mask plate by using a stainless steel plate with the thickness of 1mm through laser engraving, wherein the mask plate is only provided with a rectangular hollow at a position corresponding to the head fixing cavity of the zebra fish. The rectangular hollow space is consistent with the hollow space of the zebra fish fixing cavity and is 5 mm. The rectangular hollow part of the mask plate has the length of 400 mu m and the width of 800 mu m. Covering the mask plate on the microneedle array female die, pouring epoxy resin on the mask plate, covering the glass slide, and fully curing to obtain the microneedle array base plate.

(2) And manufacturing an electrode mask plate on a stainless steel plate with the thickness of 0.5mm by utilizing a laser etching technology. The epitaxial line width of the working electrode (namely an invasive electrode) on the electrode mask plate is 100 mu m, and the length and the width of the epitaxial electric contact are 8mm and 6 mm. The reference electrode is 5mm long and 12mm wide. Aligning an electrode mask plate with the microneedle array base plate under a body type microscope through a micro-operation instrument, and sputtering chromium with the thickness of 20nm and gold with the thickness of 200nm on the microneedle array base plate on the basis of the electrode mask plate through a magnetron sputtering technology to obtain the micro-invasive electrode base plate.

In the invention, selective mold turning is realized through rectangular hollow in the step (1), and a substrate with a specific number and shape of array micro-needles at a specific position is obtained, so that two micro-needles longitudinally arranged are contained in each fixed chamber, and accurate determination of the zebra fish brain electrical signals can be realized.

In the invention, selective mold turning is carried out through rectangular hollow in the step (1), the height of the obtained micro-needles is about 100 micrometers, the diameter is less than or equal to 100 micrometers, the distance between the micro-needles is 450 micrometers, and the distribution of the micro-needles can just correspond to the positions of forebrains and hindbrains of the juvenile zebra fish to be fixed. The configuration of the microneedles is not limited, and may be entirely perpendicular to the bottom surface, or may be appropriately inclined.

In some preferred embodiments of the present invention, after the epoxy resin casting and complete curing in step (1), the plane is treated with plasma and PDMS is cast to avoid unevenness in the plane of the resulting microneedle array, thereby affecting the accuracy of the assay.

In a third aspect of the present invention, there is provided a method for preparing the integrated chip of the first aspect of the present invention, comprising the steps of:

(1) preparing a microfluidic chip substrate:

according to the dimensions of each channel in the microfluidic chip substrate in the first aspect of the invention, after a model is built by using three-dimensional modeling software and simulation is completed, a high-precision CNC (computerized numerical control) machine tool is used for performing die machining on the microfluidic channel on a metal plate (such as a common copper plate) or performing die 3D printing directly. And (3) thoroughly cleaning the manufactured template, and pouring the template by using Polydimethylsiloxane (PDMS) after ensuring that no stain exists in the gap. And after the pouring is finished, placing the cast product in a vacuum pump to completely remove air bubbles, and then placing the cast product in an oven to bake for 4-6 hours at the temperature of 80 ℃. And after the PDMS is completely solidified, taking out and cooling to room temperature, then taking off the PDMS from the mold, and punching all the inlets and outlets by using a 1.6mm puncher.

(2) Preparation of a micro-invasive electrode base plate:

reference is made to the above method for preparing a micro-invasive electrode base plate.

(3) Assembling the microfluidic chip and the micro-invasive electrode array:

and (3) carrying out plasma treatment on the PDMS microfluidic chip obtained in the step (1) and the micro-invasive electrode array obtained in the step (2), and then carrying out permanent bonding according to the corresponding position of the micro-needle.

In some embodiments of the present invention, the specific operation of step (3) is: under a microscope, the micro-needle array is aligned to the front end of a fixed cavity of the micro-fluidic chip by using a micro manipulator (the central point of the central line of the intersection of a tail fixed cavity (rear section of the fixed cavity) and a head fixed cavity (middle section of the fixed cavity) in the chip is taken as an origin, and the distance between the origin and the micro-needle array is 400-450 mu m). And after accurate positioning, obtaining the product through a permanent bonding mode.

In a fourth aspect of the present invention, there is provided a zebrafish in-vivo electrophysiological monitoring system, which comprises the integrated chip of the first aspect of the present invention, the zebrafish delivery device and the electrical signal collection device.

In some embodiments of the present invention, the zebrafish conveying device is a power device using fluid as a conveying medium, such as a power pump.

In some preferred embodiments of the present invention, the electric signal collecting means is a digital source meter. And connecting a digital source meter with a channel electrode and a reference electrode of the micro-invasive electrode array, and testing the volt-ampere characteristic curve of the micro-invasive electrode array. Wherein, the reference electrode is connected with the negative pole of the source meter, the channel electrode is connected with the positive pole of the source meter, and the voltage range is 0-1V.

In design, the inventor preliminarily positions the brain of the zebra fish by preparing the size of the microneedle and the specific space: in order to obtain a microneedle array with large area, high cost performance and moderate rigidity, the invention firstly creates a preparation method of silicon-based microneedle template-PDMS mold turning-epoxy resin mold turning-PDMS re-pouring to prepare a microelectrode main body, and by the preparation method, a substrate with only microneedle bulges and flat other areas can be obtained.

In a fifth aspect of the present invention, there is provided a use of the integrated chip according to the first aspect of the present invention in any one of the following items (1) to (2);

(1) screening drugs;

(2) zebra fish brain electrical signals are collected or monitored.

In the related art, the disclosed in-vivo electrophysiological recording technology is basically only suitable for animals with larger sizes, and no system suitable for zebra fish juvenile fish exists, which has two technical problems: firstly, zebrafish are difficult to be fixed on a large scale and accurately carry out micro-invasive electrophysiological recording in critical brain areas; secondly, there are extremely high design difficulties and preparation difficulties in designing and preparing three-dimensional barb microelectrodes (namely, customizing according to needs and generating microneedle arrays with different shapes at specific positions) at specific fine (micron level) positions in the microchip. Moreover, the current invasive electrode detection system is complex to operate and low in flux, and the animals are often fixed by means of anesthesia, gel and the like, so that zebra fish is influenced or easily damaged, and the feasibility, the continuity and the accuracy of the test are influenced. Based on the problems, the integrated chip provided by the invention is characterized in that a specially designed micro-fluidic chip is matched with a micro-needle electrode obtained by corresponding design, the zebra fish is fixed above the micro-needle array without anesthesia based on the fluid dynamics principle, and the micro-needle array is prepared by a selective die-turning method, so that the large-flux nondestructive monitoring of the zebra fish is realized.

In addition, a micro-needle design is achieved by using a selective manufacturing method of the patterned array micro-needle creatively, and a micro-needle array with a specific shape is prepared at a specific position of the substrate based on a preparation method of silicon-based micro-needle template-PDMS (polydimethylsiloxane) flip-mold-epoxy resin flip-mold-PDMS re-pouring. The method can be used for developing the metal electrode micro-needle substrate suitable for the micro-fluidic chips with different sizes and different shape requirements.

The beneficial effects of the invention are:

1. the integrated chip is formed by organically combining a micro-fluidic technology and a micro-needle array technology, so that the large-flux high-efficiency zebra fish electroencephalogram signal acquisition under the conditions of no anesthesia and no gel fixation is realized, and the problems of zebra fish damage or inaccurate signals and the like possibly caused by the traditional method are solved.

2. The invention creatively uses the selective manufacturing method of the patterned array micro-needle to realize the micro-needle design, and combines the preparation mode of silicon-based micro-needle template-PDMS mold turning-epoxy resin mold turning-PDMS recasting to realize the preparation of the micro-needle array with a specific shape at a specific position of the substrate, thereby providing a method basis for developing the metal electrode micro-needle substrate suitable for micro-fluidic chips with different sizes and different shape requirements.

3. The in-vivo electrophysiological system suitable for the zebra fish juvenile fish can be used for screening drugs by collecting zebra fish behavioral information and analyzing the zebra fish electroencephalogram signals to a certain extent, so that the three advantages of simplicity in operation, miniaturization of equipment and convenience in recording of the zebra fish electroencephalogram signals are realized.

Drawings

Fig. 1 is a 2D engineering structure diagram of a microfluidic chip according to an embodiment of the present invention, wherein the dimension unit is mm.

Fig. 2 is a schematic flow chart illustrating a process for manufacturing a micro-invasive electrode array according to an embodiment of the present invention.

Fig. 3 is an engineering structure diagram of a microneedle array mask in an embodiment of the present invention, wherein the dimension unit is mm.

Fig. 4 is a pictorial view of a microneedle array in an example of the invention.

Fig. 5 is an engineering structure diagram of an electrode mask in an embodiment of the present invention, wherein a dimension unit is mm.

Fig. 6 is a schematic flow chart illustrating a process for manufacturing a micro-invasive electrode array according to an embodiment of the present invention.

Fig. 7 is a pictorial view of a minimally invasive electrode array in an embodiment of the present invention.

FIG. 8 is a diagram illustrating an integrated chip according to an embodiment of the present invention.

FIG. 9 is a diagram of an integrated chip in an embodiment of the invention.

Fig. 10 is a schematic and physical diagram of a microneedle in an integrated chip.

Fig. 11 is a current-voltage characteristic curve of an integrated chip according to an embodiment of the invention.

Fig. 12 is a zebra fish electroencephalogram monitored by using the integrated chip in the embodiment of the present invention.

FIG. 13 is a graph comparing survival (A) and abnormal rate (B) of zebrafish larvae at four days after 2h monitoring using the integrated chip in the example of the present invention.

FIG. 14 is a graph comparing the amplitudes of electrophysiological signals of zebrafish over time in the blank group and different drug groups.

FIG. 15 is a comparison graph of the amplitude spectrum of the electrophysiological signals of zebra fish under the effect of the blank group and different drug groups.

Fig. 16 is a comparison of data acquisition of the integrated chip (a) using the surface electrode and the integrated chip (B) in the present embodiment.

Detailed Description

In order to make the objects, technical solutions and technical effects of the present invention more clear, the present invention will be described in further detail with reference to specific embodiments. It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.

The experimental materials and reagents used are, unless otherwise specified, all consumables and reagents which are conventionally available from commercial sources.

In-vivo electrophysiological system suitable for zebra fish juvenile fish

In this embodiment, the in-vivo electrophysiological system for zebra fish juvenile fish comprises an integrated chip formed by combining a microfluidic chip and a micro-invasive electrode array.

(1) Designing and preparing a microfluidic chip:

the micro-fluidic chip is made of Polydimethylsiloxane (PDMS). The micro-fluidic chip is provided with a plurality of channels, including a liquid inlet channel, a plurality of fixed chambers and a liquid outlet channel. The liquid inlet channel and the liquid outlet channel adopt a parallel framework mode and are vertically connected with a plurality of fixed chambers in series. The fixed chamber is 5mm apart from the fixed chamber.

The 2D engineering structure of the microfluidic chip is shown in fig. 1, and in this embodiment, 5 fixed chambers are vertically connected in series between the liquid inlet channel and the liquid outlet channel. The liquid inlet channel and the liquid outlet channel are consistent in size, the width of the liquid inlet channel is 900-1000 micrometers (900 micrometers in the embodiment), the height of the liquid inlet channel is 500-600 micrometers (600 micrometers in the embodiment), and the joint of the liquid inlet channel and the fixed cavity is of a fillet structure (a circular arc with the radius of 100-150 micrometers) so as to avoid damage of zebra fish. The fixed cavity is sequentially divided into a fixed cavity front section, a middle section and a rear end along the fluid flowing direction. The front section of the fixed cavity is connected with the liquid inlet channel, the size of the front section of the fixed cavity is consistent with that of the liquid inlet channel, the width of the front section of the fixed cavity is 900-1000 micrometers (900 micrometers in the embodiment), the height of the front section of the fixed cavity is 500-600 micrometers (600 micrometers in the embodiment), and the length of the front section of the fixed cavity is 4-4.5mm (4.2 mm in the embodiment). The middle section of the fixed cavity is a conical structure, the width of the middle section is gradually reduced along the flowing direction of the fluid (from 900 μm to 150 μm), and the height of the middle section is kept constant (600 μm in the embodiment). The length of the middle section is about 0.8-1.4 mm. The rear section of the fixed cavity is connected with the liquid outlet channel, the length is 2.5-3 mm, the height is kept unchanged (600 μm in the embodiment), and the width is 150 μm.

The preparation method of the microfluidic chip in the embodiment of the invention is to copy the microfluidic hollow channel in the zebra fish microfluidic chip from the mold to prepare the microfluidic hollow channel. And processing the copper plate according to the size by using a machine tool to obtain the micro-fluidic chip template, cleaning, pouring by using PDMS (polydimethylsiloxane), and punching after curing is completed (punching by using a 1.6mm puncher).

(2) Designing and preparing a micro-invasive electrode array:

the method of manufacturing the micro-invasive electrode array in this embodiment is shown in fig. 2.

Taking a monocrystalline silicon microneedle array (the distance between needles is 400 mu m, the height is 100 mu m, the needles are rectangular pyramids, the bottom side length of a cone is 30 mu m) as a mold, soaking the mold in absolute ethyl alcohol, ultrasonically cleaning for 5min, carrying out vacuum drying, pouring PDMS (PDMS prepolymer and PDMS cross-linking agent are mixed according to the proportion of 10: 1), and removing bubbles in vacuum at room temperature. And placing the microneedle array female die in a 70 ℃ oven to be baked for more than 4 hours for full curing, and removing the cured PDMS to obtain the microneedle array female die.

A microneedle array selective flip mask plate (a mask plate is manufactured by processing a stainless steel plate with the thickness of 1mm by laser engraving) is manufactured by utilizing a laser etching technology, and an engineering drawing of the mask plate is shown in figure 3. The mask plate is designed based on the zebra fish microfluidic chip, and only the rectangular hollow part is arranged at the position corresponding to the zebra fish head fixing cavity. The rectangular hollow space is 5mm consistent with the hollow space of the zebra fish fixing cavity. The rectangular hollow part of the mask plate has the length of 400 mu m and the width of 800 mu m. Covering the mask plate on the microneedle array female die, pouring epoxy resin on the mask plate and covering the glass slide, curing overnight at room temperature, and further enabling one side of the mask plate to be tightly bonded with the glass slide, and enabling the other side of the mask plate to be an epoxy resin plane layer with the microneedle array fixed on a specific position. The physical diagram is shown in FIG. 4. By the selective mold-turning method, a substrate with a specific number and shape of the array microneedles at specific positions is obtained, so that two microneedles arranged longitudinally are contained in each fixing chamber. The height of the micro-needles is about 100 mu m, the diameter is less than or equal to 100 mu m (100 mu m in the embodiment), the distance between the micro-needles is 450 mu m, and the distribution of the micro-needles can exactly correspond to the positions of the forebrain and the hindbrain of the zebra fish juvenile fish to be fixed. The configuration of the microneedles is not limited, and may be entirely perpendicular to the bottom surface, or may be appropriately inclined.

Due to the surface tension of the liquid, the thickness of the mask plate and the like, the micro-needle array plane poured by epoxy resin in the conventional method has the problem of being not smooth enough. Therefore, in the embodiment of the present invention, after the plane is treated by using plasma, PDMS casting is performed to obtain a substrate with only micro-needle protrusions and flat other regions.

An electrode mask (a stainless steel plate with the thickness of 0.5 mm) is manufactured by using a laser etching technology, and an engineering drawing of the electrode mask is shown in fig. 5. The epitaxial line width of a working electrode (namely an invasive electrode) on the electrode mask plate is 100 mu m, and the length and the width of an epitaxial electric contact are 8mm and 6 mm. The reference electrode was 5mm long and 12mm wide. An electrode mask plate is aligned with the microneedle array base plate under a body type microscope through a micro-operation instrument, and chromium with the thickness of 20nm and gold with the thickness of 200nm are sputtered on the microneedle array base plate on the basis of the electrode mask plate through a magnetron sputtering technology to realize the manufacturing of a micro-invasive electrode array. The specific preparation process is shown in figure 6. A physical diagram of the prepared micro-invasive electrode array is shown in fig. 7.

In the design, the micro-invasive electrode array is mainly obtained by the design of the size and the specific interval of a specific micro-needle, and the preparation of a micro-invasive electrode array main body is realized by utilizing a micro-needle array with large area, high cost performance and moderate rigidity based on the positioning of the brain of the zebra fish and based on a preparation method of silicon-based micro-needle template-PDMS (polydimethylsiloxane) rollover-epoxy resin reverse mold-PDMS re-pouring.

(3) Assembling the microfluidic chip and the micro-invasive electrode array:

and (3) carrying out plasma treatment on the PDMS microfluidic chip obtained in the step (1) and the micro-invasive electrode array obtained in the step (2), and then carrying out permanent bonding according to the corresponding position of the micro-needle. Wherein, specifically: under a microscope, the micro-needle array is aligned to the front end of a fixed cavity of the micro-fluidic chip by using a micro manipulator (the central point of the central line of the intersection of a tail fixed cavity (rear section of the fixed cavity) and a head fixed cavity (middle section of the fixed cavity) in the chip is taken as an origin, and the distance between the origin and the micro-needle array is 400-450 mu m). After accurate positioning, the integrated electrophysiological system suitable for the zebra fish juvenile fish is prepared in a permanent bonding mode. The assembly process is shown in fig. 8. The physical diagram of the resulting integrated chip is shown in fig. 9. The schematic diagram of the microneedle in the microfluidic chip channel is shown in fig. 10.

The integrated chip utilizes the microfluidic channel layer to realize anesthesia-free and gel-free fixation of the zebra fish, and the basal layer of the microneedle array realizes the preparation of an invasive electrode through the magnetron sputtering technology, so that the purpose of recording different brain areas of the zebra fish in real time is achieved. And the micro-fluidic technology is organically combined with the invasive electrode prepared by the micro-needle array, so that the zebra fish brain current collection with simple operation, small equipment and convenient recording is realized. The integrated chip organically combined by the micro-fluidic chip and the micro-invasive electrode array can realize the long-time real-time recording of the spontaneous electroencephalogram activity of different brain areas of the zebra fish under the condition of no anesthesia and no gel fixation.

Practical use effect of in-vivo electrophysiological system suitable for zebra fish juvenile fish

(1) And (3) testing the conductivity of an electrode array of the integrated chip and the reading capability of the integrated chip on the zebra fish brain electrical signals:

before testing the conductivity of the electrode array of the integrated chip, the integrated chip prepared in the above embodiment is taken, water is injected into the inlet of the microfluidic chip in the integrated chip, so that the whole channel is filled with liquid and all bubbles in the microfluidic chip are removed. And (2) connecting a channel electrode (working electrode) and a reference electrode of the micro-invasive electrode array by using a digital source meter of the Gishili Source Meter series to test the volt-ampere characteristic curve of the micro-invasive electrode array, wherein the reference electrode is connected with the negative electrode of the source meter, the channel electrode is connected with the positive electrode of the source meter, and the voltage range is 0-1V.

The results are shown in FIG. 11.

It can be found that after the power supply is switched on, the voltage and the current of the micro-invasive electrode array are in a linear relation, which indicates that the electrode conductivity is good.

After the electrode conductivity is proved to be good, the zebra fish is fixed by using the integrated chip and the function of monitoring the electroencephalogram signal of the zebra fish is tested. The method comprises the following specific steps: taking the integrated chip prepared in the embodiment, injecting water into the inlet of the microfluidic chip in the integrated chip to fill the whole channel with liquid and remove all bubbles in the microfluidic chip. The inlet of the microfluidic chip is connected with an external control system for controlling zebra fish transportation through a hose. During connection, the air in the hose is exhausted and the outlet of the microfluidic chip is ensured to be in a liquid-sealed state, so that gas is prevented from being introduced into the chip channel. And opening the conveying device, and conveying the zebra fish from the inlet of the microfluidic chip through the transportation control system based on the hydrodynamic design of the microfluidic chip. When the zebra fish is sent into the fixing chamber, the abdomen of the zebra fish is kept upward, so that the micro-needle connected with the micro-invasive electrode array is contacted with and inserted into the head of the zebra fish, and the zebra fish is fixed. And connecting a channel electrode and a reference electrode of the micro-invasive electrode array by using a digital source meter of the Giusell Sourcemeter series to test the volt-ampere characteristic curve of the micro-invasive electrode array, wherein the reference electrode is connected with the negative electrode of the source meter, the channel electrode is connected with the positive electrode of the source meter, and the voltage range is 0-1V. Setting a test mode in control software as a V-T curve (voltage-time curve), setting the sampling frequency as 10KHz, and recording and storing the zebra fish brain electrical signals. And importing the data into analysis software SPIKE 2, and carrying out operations such as filtering and cluster analysis on the acquired data.

The results are shown in FIG. 12.

Fig. 12 is a zebra fish electroencephalogram obtained by the integrated chip. As can be seen from the figure, the baseline obtained by detection is relatively stable, and the peak of the action potential can be obviously seen, so that the integrated chip can successfully monitor the zebra fish brain electrical signals.

(2) The health hazard evaluation of the integrated chip on the zebra fish during monitoring of the electroencephalogram signals:

in order to prove that the integrated chip formed by the microfluidic chip and the micro-invasive electrode array in the embodiment is used for monitoring the electroencephalogram, the damage to the zebra fish is actually extremely small, the inventor carries out 2h continuous electroencephalogram signal detection on the zebra fish according to the operation in the embodiment, and then carries out health assessment on the monitored zebra fish for four days (the survival rate and the abnormal rate of the zebra fish are respectively counted). Zebrafish not monitored by brain electricity was used as a control.

Wherein the evaluation criteria for abnormalities include functional and morphological abnormalities. The evaluation criteria for function include visual confirmation of normal heartbeat and reflex response to touch stimulus. Morphological evaluation criteria include spinal curvature (i.e., lordosis, kyphosis, and scoliosis) and craniofacial abnormalities, among others.

The results are shown in FIG. 13.

The test result shows that the survival rate and the abnormal rate of the zebra fish monitored by the integrated chip are not obviously different from the normal survival rate and the abnormal rate of the zebra fish, and the test result proves that the integrated chip in the embodiment can realize long-time data acquisition on the premise of ensuring the health condition of the zebra fish.

Application of in-vivo electrophysiological system suitable for zebra fish juvenile fish in drug screening

In order to explain the function of the integrated chip in the above embodiment in drug screening, the inventor performed screening tests on 5 to 7dpf of zebra fish using drugs with existing clinical information (specifically, the drug use cases are shown in table 1), and read the zebra fish brain electrical signals before and after drug treatment using the integrated chip.

TABLE 1

In this embodiment, the use concentrations of the drugs are: AH is 0.15-0.25 mmol/L; FM is 0.1 mmol/L; DH is 0.1 mmol/L; YD is 0.2 mmol/L; MK is 0.2 mmol/L; ph is 10 mmol/L; bi is 0.1 mmol/L; as is 20-50 mmol/L; 4-A is 10 mmol/L.

In the step of collecting the zebra fish brain electrical signals by using the system and carrying out drug analysis, as the zebra fish is used as a vertebrate, the individual difference is obvious, in order to enable the test result to be more accurate, 5 zebra fish juvenile fishes are detected in each group, the obtained data is subjected to transverse comparison among different fishes and longitudinal comparison analysis of the same fish in different time periods, and therefore the reliability and the accuracy of recording the data in each step are improved. A blank control was set for each drug group.

The results are shown in FIGS. 14 and 15.

By analyzing the amplitude and frequency of the blank group, the amplitude and the frequency of the signal acquired and recorded by using the integrated chip are very similar. In the analysis of the drug action signals, the great difference between the drug group and the blank group can be seen from the amplitude histogram, the great difference also exists between different drugs, and the difference of the action of each drug can be seen from the frequency analysis. For example, Amitriptyline hydrochloride in a test drug is used as a drug with depression resistance and a certain pain relieving effect, and by using the drug, the situation that the zebra fish after being added with the drug generates continuous and regular discharge, the discharge frequency and amplitude of the zebra fish are increased, and the effect of the drug is met. After the 4-aminobutric acid is used as an inhibitory drug, the amplitude and the frequency of the zebra fish brain electrical signals are lower after the drug acts, and the integrated chip can accurately record and analyze the effect difference among different drugs and can be used for screening and classifying the drugs.

Comparison of in-vivo electrophysiological system suitable for zebra fish juvenile fish with conventional detection method

In this embodiment, in order to highlight the technical advantages of the in vivo electrophysiological system suitable for zebra fish juvenile fish in the above embodiment, the inventors selected a common surface electrode detection method disclosed in the prior art for comparison.

In this embodiment, the surface electrode used is an integrated chip obtained by assembling the microneedle-free electrode array and the microfluidic chip, and the parameters of the detection method, the detection environment and each part of the integrated chip are also consistent.

To compare the electrophysiological signals collected from the two electrodes, the electrophysiological signals of zebrafish in PTZ (Pentylenetetrazol, 10mmol/L) solution were monitored and recorded as described in the above examples. A blank control was set. PTZ acts to induce an epileptic response and, therefore, produces large fluctuations in the electrical signal.

The results are shown in FIG. 16.

The blank group of contrast can find that the integrated chip that adopts the micropin electrode does not have obvious difference with the integrated chip that adopts the surface electrode, but in the experimental group, the electrophysiological signal that adopts the integrated chip of surface electrode to gather because environmental stability is relatively poor, and noise interference is more, is difficult for gathering action potential. The electrophysiological signals acquired by the integrated chip adopting the micro-needle electrode (namely, the in-vivo electrophysiological system suitable for the zebra fish juvenile fish in the above embodiment) are slightly interfered by the environment, the action potential can be clearly captured, the recording performance of the electric signals is stable, and the data result is clearer and more stable. The inventor believes that the difference of the accuracy is mainly benefited by the fact that the microneedle electrodes in the integrated chip in the embodiment of the invention can enter the zebra fish brain, the contact area is smaller and more accurate, so that the microneedle electrodes are not easily interfered by the environment, the collected noise is less, and the signal to noise ratio is higher.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

1. An integrated chip is characterized by comprising a microfluidic chip substrate and a micro-invasive electrode baseplate;

the microfluidic chip substrate is provided with a plurality of fixing chambers for fixing zebra fish;

the micro-invasive electrode base plate is provided with a plurality of micro-needles connected with electrode circuits;

the height of the micro-needles is 90-110 mu m, the diameter of the micro-needles is less than or equal to 100 mu m, and the distance between the micro-needles is 400-450 mu m.

2. The integrated chip of claim 1, wherein the microfluidic chip substrate further comprises a liquid inlet channel and a liquid outlet channel, the liquid inlet channel and the liquid outlet channel are parallel to each other and are connected by a fixing chamber perpendicular to the liquid inlet channel and the liquid outlet channel, and the distance between the fixing chamber and an adjacent fixing chamber is 4.5-5.5 mm.

3. The integrated chip of claim 2, wherein the liquid inlet channel and the liquid outlet channel have a width of 900-1000 μm and a height of 500-600 μm.

4. The integrated chip of claim 2, wherein the fixed chamber comprises a tapered structure with a width decreasing from 900-1000 μm to 120-150 μm in a direction of fluid flow.

5. The integrated chip according to claim 1, wherein the micro-invasive electrode bottom plate further comprises a working electrode and a reference electrode, the working electrode has an epitaxial line width of 90-100 μm, an epitaxial electrical contact length of 7.5-8 mm and a width of 5.5-6 mm; the reference electrode is 4.5-5 mm long and 10-12 mm wide.

6. The method for preparing a micro-invasive electrode backplane in an integrated chip of claim 5, comprising the steps of:

(2) preparing an electrode mask plate according to the electrode size of claim 5, aligning a line in the electrode mask plate with the micro-needles in the micro-needle array base plate prepared in the step (1), and sputtering 18-20 nm thick chromium and 190-200 nm thick gold to obtain the electrode mask plate.

7. The method of claim 6, wherein the flexible material comprises at least one of polydimethylsiloxane, polymethylmethacrylate, and gelatin.

8. The method according to claim 6, wherein the electrically insulating resin material includes at least one of an epoxy resin and a silicone resin.

9. A zebra fish in-vivo electrophysiological monitoring system, comprising the integrated chip of any one of claims 1 to 5, a zebra fish conveying device, and an electrical signal collecting device;

the zebra fish conveying device is a power device which takes fluid as a conveying medium.

10. Use of the integrated chip according to any one of claims 1 to 5 in any one of the following (1) to (2);

(1) screening drugs;

(2) zebra fish brain electrical signals are collected or monitored.