WO2018045482A1

WO2018045482A1 - Ultrasonic nerve regulation system

Info

Publication number: WO2018045482A1
Application number: PCT/CN2016/098158
Authority: WO
Inventors: 郑海荣; 周伟; 孟龙; 王凯悦; 牛丽丽; 黄小伟; 蔡飞燕; 李飞; 王辰
Original assignee: 深圳先进技术研究院
Priority date: 2016-09-06
Filing date: 2016-09-06
Publication date: 2018-03-15

Abstract

An ultrasonic nerve regulation system comprises a signal generator (100), a transducer (300), a microscope (400), a recording device (500), and a storage device (200). The storage device (200) is used for storing an object, the object being a nematode or a cell or a brain slice. The signal generator (100) is used for outputting a sine wave electrical signal. The transducer (300) is used for generating a wave signal according to the sine wave electrical signal. The recording device (500) is used for recording a behavior change of the object by means of the microscope (400), the object being exposed to the wave signal. A nerve is regulated by means of an ultrasound; the system does not need to be in contact with the nerve, and an electrode and a drug injection pump also do not need to be implanted; the ultrasonic nerve regulation system is a noninvasive nerve regulation means.

Description

Ultrasonic neuromodulation system

Technical field

The invention relates to the field of neuroscience and technology, in particular to a research device for ultrasonic neuromodulation, in particular to an ultrasound neuromodulation system.

Background technique

For a long time, mental illnesses (motor disorders, pain, epilepsy, Parkinson's disease, mental illness, and angina) have seriously affected human health and quality of life. Although new antipsychotic drugs are being used in clinical practice, a considerable number of patients are still not sensitive to drug treatment or are not satisfied with the efficacy. The neuromodulation treatment method is a popular treatment method in recent years, and has a good therapeutic effect on various neurological diseases, and the development speed is fast. Neuromodulation exerts excitement, inhibition or regulation of neuronal or neuronal signal transduction in the central nervous system, peripheral nervous system, and autonomic nervous system adjacent or distant parts by implantable or non-implantable techniques, electrical or chemical means. The role of biomedical engineering technology to improve the quality of life of patients and improve neurological function.

At present, neuromodulation mainly uses electrical stimulation and drug means. The most common neuromodulation techniques used in electrical stimulation include deep brain stimulation (DBS), spinal cord stimulation (SCS), and vagus nerve stimulation (VNS). These techniques are achieved by implanting electrodes into the brain, spinal cord, and vagus nerves of the human body, using electrical signals to stimulate the nerves for therapeutic purposes. The following is introduced one by one.

Deep brain electrical stimulation, also known as brain pacemaker therapy, is a technique in which brain stereotactic surgery is used to implant electrodes in specific brain nuclei in the brain, and neurons can be inhibited by high-frequency electrical stimulation. Thereby playing a role in healing. The deep stimulation of deep brain stimulation for Parkinson's disease is the globus pallidus and the thalamus intermediate nucleus (Vim). With the deepening of the study, the subthalamic nucleus (STN) electrical stimulation can also significantly relieve the tremor of Parkinson's disease patients. Muscle rigidity and bradykinesia symptoms.

Vagus nerve stimulation is a neuromodulation technique that entangles a spiral stimulating electrode in the left cerebral vagus nerve trunk and stimulates the vagus nerve through long-term and intermittent stimulation. The principle may be related to the extensive projection of the vagus nerve, which can be projected through the solitary tract nucleus to the thalamus, amygdala and forebrain, and projected through the spinal cord network to the cerebral cortex. Therefore, vagus nerve stimulation can regulate the excitability of the cerebral cortex and thus control seizures. A number of studies have shown that the efficacy of the vagus nerve in controlling seizures is significant over time and has a good effect on seizures in children.

The spinal cord electrical stimulation system places the stimulating electrode (strip electrode or needle-shaped puncture electrode) in the posterior part of the spinal epidural space, adjacent to the posterior column of the spinal cord, and then connected to the pulse generator implanted under the skin of the ankle. Stimulate the posterior column of the spinal cord and the sensory neurons in the posterior horn of the spinal cord for therapeutic purposes.

The drug neuromodulation technology is mainly used to treat cancer pain, Parkinson's disease, Alzheimer's disease (AD) by implanting a drug-loaded drug micro pump into brain tissue or spinal canal and slowly releasing the drug through a drug micro pump. ), refractory sputum, etc.

It can be seen from the above description that electrical stimulation of nerve regulation on the one hand, electrical stimulation of neuromodulation technology requires implanting electrodes in the human body to cause certain damage to the human body. On the other hand, electrical stimulation technology stimulates a certain area of the brain without focusing. To a single neuron, the principles of neurobiology are elucidated from the perspective of neurons. Drug neuromodulation, the implantation of the drug micropump will cause certain damage to the human body, and the method only uses long-term slow injection of drugs into the nerve tissue, thereby treating and alleviating neurological diseases, and has not been able to Neurological diseases are treated from the perspective of neuromodulation mechanisms.

Ultrasound neuromodulation is a new non-invasive brain stimulation and regulation technique in recent years. Based on the mechanical effects of ultrasound, the technique stimulates or inhibits the central nervous system of the stimulation site through different intensities, frequencies, pulse repetition frequencies, pulse widths, and durations. Effect, a reversible change in bidirectional regulation of neural function. In 1955, Fry et al. developed focused ultrasound that not only treats pain and Parkinson's disease, but also studies the structure and function of the brain circuit. In 2010, the University of Arizona team demonstrated for the first time through live animal experiments to achieve neuromodulation using low-frequency, low-pressure ultrasound. Therefore, it is very important to study the mechanism of ultrasound neuromodulation. The in-depth understanding of the mechanism of ultrasound neuromodulation also helps to improve the safety, effectiveness and accuracy of ultrasound neuromodulation, which is of great significance for promoting the clinical application of ultrasound neuromodulation. However, the current lack of ultrasound neuromodulation can be applied to the scientific tools of neuroscience and brain research. In addition, the physical and neurobiological mechanisms of ultrasound neuromodulation are still unclear.

Summary of the invention

In order to overcome the above technical problems existing in the prior art, the present invention provides an ultrasonic neuromodulation system including a signal generator, a transducer, a microscope, a recording device, and a storage device, by storing a guest on a storage device, The signal generator emits a sinusoidal electric signal, which generates a wave signal after passing through the transducer, exposes the object to the wave signal, and then uses the wave signal to stimulate the object. Finally, the recording device records the behavior change of the object through the microscope, and the subsequent recordable The information is quantified to achieve regulation of the nerves through the wave signal and to study the mechanism of the ultrasound nerve regulation, without the need to contact with the nerve, nor to implant the electrode and the drug injection pump.

It is an object of the present invention to provide an ultrasound neuromodulation system, the system comprising a signal generator, a transducer, a microscope, a recording device and a reservoir, wherein the reservoir is for storing a guest, The object is a nematode or a cell or a brain slice; the signal generator is configured to output a sinusoidal electrical signal; the transducer is configured to generate a wave signal according to the sinusoidal electrical signal; and the recording device is configured to pass The microscope records changes in the behavior of the object, the object being exposed to the wave signal.

In one embodiment, the system further includes a power amplifier for power amplifying the sinusoidal electrical signal and transmitting the amplified sinusoidal electrical signal to the transducer.

In one embodiment, the sinusoidal electrical signal has an amplitude greater than 150 millivolts and the amplified sinusoidal electrical signal has a power greater than 38 decibels.

In one embodiment, the recording device is a high speed image sensor.

In one embodiment, the transducer is a bulk wave transducer or an interdigital transducer, and when the transducer is a bulk wave transducer, the wave signal is a body wave signal; When the energy device is an interdigital transducer, the wave signal is an ultrasonic surface wave signal.

In one embodiment, the interdigital transducer includes a piezoelectric substrate and a plurality of interdigital electrodes plated on the piezoelectric substrate.

In one embodiment, the number of the interdigital electrodes is 1 or 2 or 4 or 8.

In one embodiment, the piezoelectric substrate is 128°, YX double-sided polished lithium niobate or zinc oxide or aluminum nitride.

In one embodiment, when the guest is a nematode or a cell, the reservoir is a polydimethylsiloxane PMDS channel, and the PMDS channel is bonded to a piezoelectric substrate of the interdigital transducer on.

In one embodiment, the PMDS channel is tapered.

In one embodiment, when the guest is a nematode, an M9 solution is placed in the PMDS lumen, and the nematode is placed in the M9 solution.

In one embodiment, when the guest is a nematode, the reservoir is an agar plate, and the agar plate is placed on a piezoelectric substrate of the interdigital transducer.

In one embodiment, when the object is a nematode, the behavioral change includes a number of return times of the nematode and a change in the frequency of the oscillation.

In one embodiment, when the guest is a cell or a brain slice, the reservoir is a slide placed on a piezoelectric substrate of the interdigital transducer.

The present invention has an advantageous effect of providing an ultrasonic neuromodulation system including a signal generator, a transducer, a microscope, a recording device, and a storage device. The sine wave is emitted by the signal generator by storing a guest on the storage device. The electrical signal generates a wave signal after passing through the transducer, exposing the object to the wave signal and then stimulating the object by using the wave signal, and finally recording the behavior change of the object by the recording device through the microscope, and then the quantitative analysis can be performed by the recorded information, thereby The regulation of nerves by wave signals and the study of ultrasound neural regulation mechanisms do not require contact with the nerves, nor the need for implanted electrodes and drug injection pumps.

The above and other objects, features, and advantages of the present invention will become more apparent and understood by the appended claims appended claims

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 described below. Obviously, the drawings in the following description are only It is a certain embodiment of the present invention, and other drawings can be obtained from those skilled in the art without any creative work.

1 is a structural block diagram of Embodiment 1 of an ultrasound neural control system according to an embodiment of the present invention;

2 is a structural block diagram of Embodiment 2 of an ultrasound neural control system according to an embodiment of the present invention;

3 is a structural block diagram of an interdigital transducer in an ultrasonic neuromodulation system according to an embodiment of the present invention;

4 is a structural block diagram of Embodiment 1 of a storage device in an ultrasound neuromodulation system according to an embodiment of the present invention;

FIG. 5 is a structural block diagram of Embodiment 2 of a storage device in an ultrasound neuromodulation system according to an embodiment of the present invention;

6a to 6e are schematic views showing a manufacturing process of the interdigital transducer;

6f to 6j are schematic views showing a process of fabricating a PDMS channel;

Figure 6k is a schematic diagram showing the results of the combination of PDMS and interdigital transducer after plasma treatment;

Figure 7 is a schematic view showing the experiment of C. elegans when PMDS is bonded to the interdigital transducer;

Figure 8 shows the actual use of the ultrasound neuromodulation system;

Figure 9a shows a schematic view of the morphology of nematodes in a normal state;

Figure 9b to Figure 9d show three states of the nematode when the nematode is turned back;

Figure 10a shows a schematic of an ultrasound neuromodulation experiment on an agar plate;

Figures 10b to 10e show schematic diagrams of nematode behavior without ultrasound;

Figures 10f to 10i show a schematic diagram of the behavior of the nematode avoiding reaction after application of ultrasound;

FIG. 11 is a structural block diagram of Embodiment 3 of a storage device in an ultrasound neuromodulation system according to an embodiment of the present invention.

detailed description

The technical solutions in the embodiments of the present invention are clearly and completely described in the following with reference to the accompanying drawings in the embodiments of the present invention. It is obvious that the described embodiments are only a part of the embodiments of the present invention, but not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative efforts are within the scope of the present invention.

At present, the existing neural regulation methods will cause certain damage to the human body. Ultrasonic neuromodulation provides us with a non-invasive method of neuromodulation. It is necessary to accurately and effectively use ultrasound for neuromodulation, and to study the mechanism of ultrasound neuromodulation. important. Ultrasound neuromodulation lacks the scientific tools that can be applied to neuroscience and brain research. In addition, the physical and neurobiological mechanisms of ultrasound neuromodulation are still unclear. Conventional ultrasound stimulation uses an ultrasound probe as an excitation source with a range of millimeters. It is used to study microscopic single-cell stimulation and specific specific neurons that stimulate the nematode. The traditional ultrasound system is difficult to integrate with the microscope. Dynamic responses to nerve cells, such as calcium imaging, cannot be observed in real time. In addition, conventional ultrasound systems cannot perform patch clamp recording of single cells.

In view of the above disadvantages, the present invention provides an ultrasonic neuromodulation system which has good light transmittance and is compatible with a conventional microscope, and can quantitatively record calcium imaging of a nerve cell and a change of a single cell membrane potential in real time, and the chip generates The sound field can be micron-sized, similar to the size of nerve cells, and can precisely stimulate a single cell and a certain neuron of a nematode. We can use this system to study the ultrasound neuromodulation from the perspective of neurons. This system is mainly used in the neuromodulation study of the neurological model C. elegans (C. elegans), and can also be applied to the neuroregulation of cells, brain slices, etc. researching.

1 is a structural block diagram of Embodiment 1 of an ultrasound neuromodulation system according to an embodiment of the present invention. As shown in FIG. 1, the system includes a signal generator 100, a storage device 200, a transducer 300, a microscope 400, and Recording device 500.

The storage unit 200 is configured to store a guest, and the object is a nematode or a cell or a brain slice. In a specific embodiment, the nematode can be a model organism such as Caenorhabditis elegans.

The signal generator 100 is configured to output a sinusoidal electrical signal. In a specific embodiment, the amplitude value of the sinusoidal electrical signal is greater than 150 millivolts in order to ensure the intensity of the stimulation signal.

The transducer 300 is configured to generate a wave signal according to the sinusoidal electrical signal. In a specific embodiment, the transducer is a bulk wave transducer or an interdigital transducer 301. When the energy device is a bulk wave transducer, the wave signal is a body wave signal; when the transducer is an interdigital transducer, the wave signal is an ultrasonic surface wave signal.

Ultrasound probes used in hospital B-mode devices are one type of body wave transducers. Typically, a bulk wave transducer consists of a housing, a matching layer, a piezoceramic disk transducer, a backing, an extraction cable, and a Cymbal array receiver.

The recording device 500 is configured to record a change in behavior of the object by the microscope 400, the object being exposed to the wave signal. In a specific embodiment, the recording device may be a high-speed image sensor CCD, and the movement of the nematode or the cell or the brain slice is recorded by the microscope, and the number of returning times of the nematode and the variation of the swing frequency may be analyzed through the recorded video and the like. Changes in cells, brain slices, or brain slices.

As shown above, the present invention provides a neuromodulation system for a model organism (C. elegans), a nerve cell, a brain slice, including a signal generator, a transducer, a microscope, a recording device, and a storage device. A guest object is stored on the storage device, and a signal generator generates a sinusoidal electric signal, and after the transducer generates a wave signal, the object is exposed to the wave signal, and the wave signal is used to stimulate the object, and finally the recording device passes through the microscope. Recording changes in the behavior of the object, which can be quantified and analyzed by the recorded information, thereby realizing the regulation of the nerve through the wave signal and studying the mechanism of the ultrasonic nerve regulation, without contacting the nerve, and without implanting the electrode and the drug injection pump. .

2 is a structural block diagram of Embodiment 2 of an ultrasound neuromodulation system according to an embodiment of the present invention. As shown in FIG. 2, the system further includes:

The power amplifier 600 is configured to perform power amplification on the sinusoidal electrical signal and transmit the amplified sinusoidal electrical signal to the transducer. In a specific embodiment, to ensure the intensity of the stimulation signal, the power of the amplified sinusoidal electrical signal is greater than 38 decibels. This ensures a signal of sufficient strength to be transmitted to the surface acoustic wave microfluidic chip.

3 is a structural block diagram of an interdigital transducer 301 in an ultrasonic neuromodulation system according to an embodiment of the present invention. As can be seen from FIG. 3, the interdigital transducer 301 includes a piezoelectric substrate 3011 and the pressure. An interdigital electrode 3012 is plated on the electrical substrate.

In order to obtain a large electromechanical coupling coefficient, in a specific embodiment, 128° YX double-sided polished lithium niobate or zinc oxide or aluminum nitride is used as the piezoelectric substrate, and the number of the interdigital electrodes is 1 or 2 or 4 or 8. Making an interdigital finger The process of the transducer mainly includes the processes of coating, lithography, coating, peeling and the like. The fabrication process of the interdigital transducer in a specific embodiment is briefly described below, and the manufacturing process of the interdigital transducer is shown in FIGS. 6a to 6e.

(1) Gluing: On the surface of the completely cleaned piezoelectric substrate material, the positive photoresist AZ4620 was spin-coated at 5000 rpm for 30 s, and the chip was placed on a 120 ° C hot plate for baking for 3 min. We used a step meter to test the thickness of the photoresist. The thickness of the photoresist is about 5 μm, as shown in Figure 6a.

(2) Exposure and development: Then, the film film shown in Fig. 6b is covered on the above surface of Fig. 6a for exposure, and the pattern portion is opaque, the pattern portion is transparent, and the portion having light transmission is Curing, the solidified portion was not dissolved when developing with AZ400, the non-cured portion was dissolved, and after baking, it was baked on a hot plate at 150 ° C for 10 minutes to form a pattern as shown in Fig. 6c.

(3) Sputtering: The substrate on which the pattern transfer has been completed is subjected to magnetron sputtering to grow a metal layer having a thickness of about 200 nm, as shown in Fig. 6d.

(4) Degumming: The substrate on which the aluminum film is grown is placed in an acetone solution, and the photoresist is stripped by ultrasonic vibration of the ultrasonic cleaning machine to complete the fabrication of the surface acoustic wave device, as shown in Fig. 6e.

7 is an experimental physical diagram of C. elegans when PMDS is bonded to an interdigital transducer. Referring to FIG. 7, it can be seen that in the specific use scenario, there are two interdigital electrodes 3012 distributed in the PMDS cavity. side.

4 is a structural block diagram of Embodiment 1 of a storage device 200 in an ultrasound neuromodulation system according to an embodiment of the present invention. As shown in FIG. 4, in the first embodiment, the storage device is a PMDS channel, The PMDS channel is bonded to the piezoelectric substrate of the interdigital transducer.

When the guest is a nematode or a cell, the reservoir is a polydimethylsiloxane PMDS channel. In a specific embodiment, the PDMS channel can be fabricated in the following manner. Figures 6f to 6j show the fabrication process of the PDMS.

(1) Pretreatment: The residual impurities on the surface of the silicon substrate, such as dust and organic adsorbate, are removed by pickling, alcohol washing and water washing, and finally the silicon wafer is placed in a clean place to dry.

(2), gluing and pre-baking: SU-8 (50) negative photoresist was spin-coated with a glue applicator, 3000 rpm, 30 s, SU-8 (50) thickness was about 50 μm. After the coating is completed, the silicon wafer is horizontally placed on a heating plate at 90 ° C for 1 h, and the solvent in the photoresist is volatilized to enhance the adhesion between the photoresist and the silicon wafer, and the pattern of FIG. 6f is obtained.

(3) Exposure and development: The film sheet which has been patterned is placed on Fig. 6f as shown in Fig. 6f, and the photoresist is exposed by an exposure machine at an exposure dose of 600 cJ/cm2 for a duration of 30 s. The exposed silicon wafer was immersed in the developing solution, the photoresist in the unexposed area was dissolved, and the photoresist in the exposed region was left to remain. After development, it was baked on a hot plate at 150 ° C for 10 minutes to obtain a pattern in FIG. 6h.

(4) Casting PDMS: PDMS A glue and B glue are mixed according to mass ratio of 10:1, mixed evenly, poured into the petri dish where the silicon wafer is located, vacuum the culture dish to remove bubbles in PDMS, and finally The Petri dish was placed in an oven at 80 ° C for 30 min to cure the PDMS, as shown in Figure 6i.

(5) Peeling PDMS: The PDMS containing the pattern was cut with a scalpel and completely peeled off from the silicon wafer. Finally, the microcavity was perforated by a puncher to make an inlet and an outlet.

The prepared interdigital transducer and the PDMS channel are plasma-treated, the plasma processing power is 150W, the duration is 70s, and then the PDMS cavity end is glued down on the interdigital transducer for bonding, 80 Bake in a °C oven for 20 min. A surface acoustic wave microfluidic chip for experimentation prepared as shown in Fig. 6k was obtained.

In a specific embodiment, such as the imaging of neuronal calcium, the PMDS lumen is tapered. An M9 solution is placed in the PMDS channel, and the nematode is placed in the M9 solution.

FIG. 5 is a structural block diagram of Embodiment 2 of a storage device in an ultrasonic neuromodulation system according to an embodiment of the present invention. As shown in FIG. 5, when the object is a nematode, the storage device may also be an agar plate. The agar plate is placed on the piezoelectric substrate of the interdigital transducer.

11 is a structural block diagram of Embodiment 3 of a storage device in an ultrasound neuromodulation system according to an embodiment of the present invention. As shown in FIG. 11, when the object is a cell or a brain slice, the storage device is a slide. The slide is placed on a piezoelectric substrate of the interdigital transducer.

The technical solution of the present invention will be described in detail below with reference to specific embodiments. In the following embodiments, the interdigitated transducer and the nematode are mainly taken as an example, and the present invention provides a neuromodulation system that can be used for model organisms (C. elegans), nerve cells, and brain slices, which can be quantified. The angle of the neurological regulation of nematodes was analyzed. The utility model comprises a signal generator, a transducer, a microscope, a recording device, a storage device and a power amplifier. The sinusoidal electric signal is generated by the signal generator by storing the nematode on the storage device, and the wave signal is generated after the transducer is generated. The object is exposed to the wave signal and then the wave signal is used to stimulate the object. Finally, the recording device records the behavior change of the object through the microscope, and then the recorded information can be quantitatively analyzed, thereby realizing the regulation of the nerve through the wave signal and regulating the nerve. The mechanism is studied without the need for contact with the nerves or the implantation of electrodes and drug injection pumps. The application scenarios of the present invention under several specific embodiments are described below.

Embodiment 1

In this embodiment, the PMDS cavity is bonded to the interdigital transducer to form a surface acoustic wave microfluidic chip. Figure 7 is an experimental physical diagram of C. elegans when PMDS is bonded to an interdigital transducer. The PMDS is bonded to the interdigital transducer and connected to the wireworm container at one end of the PDMS cavity. The nematode suspended in the M9 solution, the other end of the channel is connected to the syringe through a thin tube, and the nematode is sucked into the PDMS channel by a pulling operation. Figure Figure 8 shows the actual use of the ultrasound neuromodulation system. The fabricated experimental chip was placed under a stereo microscope, connected to a power amplifier and a signal generator, and the results of the experiment were recorded by a high-speed CCD. specific:

(1) Preparation of an interdigital transducer. The processing method of the surface acoustic wave chip is studied from the thickness of the coating, the exposure time, and the thickness of the coating. The influence of these parameters on the insertion loss and the device bandwidth of the device is studied by adjusting the metal film material, the logarithm of the finger, and the acoustic aperture size.

(2) Preparation and bonding of PDMS channels. The structure of the PDMS channel is designed, and a copy of the channel is made using photolithography. The PDMS channel is then formed by steps of pouring, curing, and punching. A plasma treatment method is used to bond the channels to the already fabricated chips to fabricate the equipment used in the experiment.

(3) Configuration of M9 solution and injection of nematodes. In the course of the experiment, it is necessary to configure the M9 solution and put the nematode into the M9 solution for experiment, which can provide a good living environment for the nematode. The nematode can then be injected into the channel we have prepared by syringe to perform the experiment. To make a 1 L solution of M9, 6 g of Na ₂ HPO ₄ , 3 g of KH ₂ PO ₄ , 5 g of NACL and 0.25 g of MgSO ₄ ·7H ₂ 0 were added to 1 L of double distilled water, and they were mixed into a solution and sterilized by high temperature and high pressure. It is then stored at 4 ° C and taken out at the time of use. Figure 7 shows a schematic diagram of the experiment. The PMDS is bonded to the interdigital transducer. At one end of the PDMS channel, a container for carrying nematodes is attached. The inside of the container is filled with nematodes suspended in M9 solution, and the other end of the channel is fine. The tubing is attached to the syringe, and the nematode is drawn into the PDMS lumen by a pulling operation, and then the excimer transducer excitation signal can be generated to generate ultrasonic waves to stimulate the nematode.

(4) Quantitative analysis of changes in nematode behavior in M9 solution. The behavioral changes of nematodes were observed under a microscope. Under normal circumstances, the nematode is constantly oscillating in the M9 solution, and it is found that the nematode will turn back and accelerate the swing during the ultrasonic surface wave stimulation. By adjusting the parameters of the signal generator, the time of signal generator emission and stop is fixed, and the number of times the nematode returns and the variation of the nematode swing frequency are observed during the period to achieve the purpose of quantitative analysis. On the other hand, nematodes that have been knocked out by specific neurons can be stimulated by ultrasonic surface waves to quantify the behavior of nematodes, thereby studying whether individual neurons are sensitive to surface acoustic waves.

Specifically, quantitative analysis of changes in nematode behavior in M9 solution. Figure 8 shows the entire experimental system. By recording a high-speed CCD in a stereo microscope to record the behavior of the nematode, the signal generator's signal sine wave electrical signal amplitude is greater than 150 mV, amplified by the power amplifier, sinusoidal The power of the signal is greater than 38 dbm, and is transmitted to the surface acoustic wave chip as a signal of sufficient strength for the surface acoustic wave chip. During the experiment, the time to control the nematode exposure to ultrasound (such as 0.5-1 minute) is the same as the time not exposed to ultrasound, compared with the number of times the nematode recurs and the variation of the nematode swing frequency in the same time, as shown in the figure. As shown in 9a, it is a nematode under normal conditions. In the state, Figures 9b to 9d show the morphology of the nematode when it turns back. In addition, it is also possible to study whether these neurons are sensitive to ultrasound stimulation by selecting different nematode knockout nematodes.

Embodiment 2

In this embodiment, the PMDS cavity is bonded to the interdigital transducer to form a surface acoustic wave microfluidic chip. Figure 7 is an experimental physical diagram of C. elegans when PMDS is bonded to an interdigital transducer. The PMDS is bonded to the interdigital transducer and connected to the container of the wireworm at one end of the PDMS cavity. There is a nematode suspended in the M9 solution, and the other end of the channel is connected to the syringe through a thin tube, and the nematode is sucked into the PDMS channel by a pulling operation. Figure 8 shows a diagram of the actual use of an ultrasound neuromodulation system. The fabricated experimental chip was placed under a stereo microscope, connected to a power amplifier and a signal generator, and the results of the experiment were recorded by a high-speed CCD. specific:

(2) Preparation and bonding of PDMS channels. The structure of the PDMS channel is designed, and a copy of the channel is made using photolithography. The PDMS channel is then formed by steps of pouring, curing, and punching. A plasma treatment method is used to bond the channels to the already fabricated chips to fabricate the equipment used in the experiment. The PDMS channel used in this embodiment is a tapered channel with a narrower width of the channel. The widest point can be 60 to 100 microns, and the narrowest can be 40 microns. When the nematode is inhaled into the lumen, it is fixed at the tip of the channel.

(3) Configuration of M9 solution and injection of nematodes. In the course of the experiment, it is necessary to configure the M9 solution and put the nematode into the M9 solution for experiment, which can provide a good living environment for the nematode. The nematode can then be injected into the channel we have prepared by syringe to perform the experiment. To make a 1 L solution of M9, 6 g of Na ₂ HPO ₄ , 3 g of KH ₂ PO ₄ , 5 g of NACL and 0.25 g of MgSO ₄ ·7H ₂ 0 were added to 1 L of double distilled water, and they were mixed into a solution and sterilized by high temperature and high pressure. It is then stored at 4 ° C and taken out at the time of use. Figure 7 shows the experimental physical map. The PMDS is bonded to the interdigital transducer. At one end of the PDMS cavity, a container carrying the nematode is connected. The inside of the container is filled with nematodes suspended in the M9 solution, and the other end of the channel passes. The thin tube is connected to the syringe, and the nematode is sucked into the PDMS channel by a pulling operation, and then the excitation signal of the interdigital transducer can be generated to generate ultrasonic waves to stimulate the nematode.

(4) Neuronal calcium imaging. The behavioral changes of nematodes were observed under a microscope. The nematodes were stimulated with ultrasound, and the calcium ion imaging of nematode neurons was observed in real time under a microscope to determine which neurons were activated. It is possible to determine which neurons are sensitive to ultrasound stimuli by the state in which the neurons are activated. In addition, stimulation of a single neuron can be achieved by using focused ultrasound, and the working state of the neuron is judged by observing neuronal calcium imaging.

Embodiment 3

In this embodiment, the nematode depositor is an agar plate, and the agar plate is placed on the interdigital transducer to study changes in nematode behavior on the agar plate. Figure 10a shows a schematic of an ultrasound neuromodulation experiment on an agar plate. Figure 8 shows a diagram of the actual use of an ultrasound neuromodulation system. The fabricated experimental chip was placed under a stereo microscope, connected to a power amplifier and a signal generator, and the results of the experiment were recorded by a high-speed CCD. specific:

(2) Preparation of agar plates. Agar plates can be prepared by prior art methods. After preparation, agar plates are placed on the interdigital transducers and the nematodes are picked onto agar plates for ultrasonic stimulation.

(3) Changes in nematode behavior. The behavioral changes of nematodes were observed under a microscope. The system is mainly by placing an agar plate on a micro-ultrasound neuromodulation chip and picking the nematode onto an agar plate for ultrasonic stimulation. Figures 10b to 10e show schematic diagrams of nematode behavior without ultrasound; Figures 10f to 10i show a schematic diagram of the behavior of nematodes with avoidance response after application of ultrasound. When the nematode walks in a straight line on the agar plate, applying ultrasound to stimulate the nematode, the nematode will retreat or pause, and combined with neuronal calcium ion imaging can study those neurons that play a leading role in these behaviors.

The above is a neuroregulatory system which can be used for model organisms (C. elegans), nerve cells and brain slices, and can analyze the neurological regulation of nematodes from a quantitative perspective. The utility model comprises a signal generator, a transducer, a microscope, a recording device, a storage device and a power amplifier. The sinusoidal electric signal is generated by the signal generator by storing the nematode on the storage device, and the wave signal is generated after the transducer is generated. The object is exposed to the wave signal and then the wave signal is used to stimulate the object. Finally, the recording device records the behavior change of the object through the microscope, and then the recorded information can be quantitatively analyzed, thereby realizing the regulation of the nerve through the wave signal and regulating the nerve. The mechanism is studied without the need for contact with the nerves or the implantation of electrodes and drug injection pumps.

The invention uses the surface acoustic wave chip as a platform, combines the PDMS cavity and the neural model C. elegans, quantifies the results of ultrasonic neuromodulation, and completes the research on the mechanism of ultrasonic neuromodulation. The key innovations of the present invention are:

(1) Non-invasive, ultrasound neuromodulation is the regulation of nerves by ultrasound, no need to contact with nerves, nor need to implant electrodes and drug injection pumps, is a non-invasive neuromodulation.

(2) Accuracy, the present invention combines a surface acoustic wave chip with a PDMS cavity and a microscope that can be quantized from the angle To analyze the effects of ultrasound neuromodulation, and we use neuronal knockout nematodes to achieve ultrasound neural regulation mechanisms that are accurate to a single nerve. Combining surface acoustic wave chips with agar plates and microscopes allows for the study of nematode behavioral changes and is accurate to those neurons that play a leading role in nematode behavior.

(3) Repeatability, the preparation process of the surface acoustic wave microfluidic chip is a standard MEMS process, and the device performance has good consistency, which lays a foundation for the repeatability of the experiment. Moreover, the nematodes of the same medium in the process of nematode culture are uniformly cultured from the fertilized egg period. At the same time, the nematodes are in the same growth period, and multiple nematodes can be continuously tested.

(4) Compatibility of optics and patch clamps, in the process of ultrasound nerve stimulation, it is necessary to record the activity of neurons to observe the working state of nematode neurons. Since the PMDS channel has good light transmission, it can be traditional The combination of optical microscopy and patch clamps (which measure the current of individual ion channels on neuronal cells) records neuronal changes in real time.

The point of protection of the present invention is to study the mechanism of ultrasound neuromodulation using a surface acoustic wave chip.

(1) This concept and method of neuromodulation using ultrasound.

(2) As a tool for nerve stimulation, surface acoustic wave chip realizes the research on the mechanism of ultrasonic neuromodulation from the microscopic point of view by combining surface acoustic wave chip and PDMS cavity.

(3) A method of stimulating a model organism using a surface acoustic wave chip. The agar or PDMS channel was placed on a surface acoustic wave chip to study the nematode behavior.

(4) Prepare multi-level sound field, realize multi-point simultaneous stimulation, and study neural network.

The beneficial effects of the invention are:

At present, the existing methods all need to implant corresponding devices in the human body, which cause certain damage to the human body, and they all regulate a large number of nerve nuclei in the human body without focusing on a single nerve. Yuan, explores neuromodulation from the perspective of neurons. The ultrasonic neuromodulation used in the present invention does not require implantation of any device in the human body, and is a non-invasive regulation method. Moreover, with the continuous advancement of MEMS technology, microfluidic chips have been rapidly developed, and the combination of microfluidic chips and surface acoustic wave devices has received extensive attention. Combined with surface acoustic wave chips and PDMS channels, ultrasound can be used to study the mechanism of ultrasound neuromodulation from the perspective of microscopic neurons.

The present invention can be applied to studies of neuromodulation mechanisms such as cells and brain slices in addition to application in neuromodulation studies of Caenorhabditis elegans. The cavity of the PDMS can be flexibly designed. In the specific embodiment, a circular channel can be used to make the nematode move flexibly in the circular cavity. The tapered channel can be used to fix the nematode, and other shapes can be used. The lumen thus functions to accommodate nematodes or fixed nematodes. The surface acoustic wave chip-interdigital transducer can be flexibly designed in size and shape. Currently, lithium niobate single crystal is used as the piezoelectric substrate. Thin film piezoelectric materials such as zinc oxide and aluminum nitride. A bulk wave transducer prepared based on a piezoelectric thin film material such as zinc oxide or aluminum nitride can also be used.

One of ordinary skill in the art can understand that all or part of the process of implementing the above embodiments may be completed by a computer program to instruct related hardware, and the program may be stored in a general computer readable storage medium. When executed, the flow of an embodiment of the methods as described above may be included. The storage medium may be a magnetic disk, an optical disk, a read-only memory (ROM), or a random access memory (RAM).

Those skilled in the art will also appreciate that the various functions listed in the embodiments of the present invention are implemented by hardware or software depending on the design requirements of the particular application and the overall system. A person skilled in the art can implement the described functions using various methods for each specific application, but such implementation should not be construed as being beyond the scope of the embodiments of the present invention.

The principles and embodiments of the present invention have been described in connection with the specific embodiments of the present invention. The description of the above embodiments is only for the understanding of the method of the present invention and the core idea thereof. At the same time, for those skilled in the art, according to the present invention The present invention is not limited by the scope of the present invention.

Claims

An ultrasonic neuromodulation system, characterized in that the system comprises a signal generator, a transducer, a microscope, a recording device and a storage device,

Wherein the depositor is for storing a guest, the object being a nematode or a cell or a brain slice;

The signal generator is configured to output a sinusoidal electric signal;

The transducer is configured to generate a wave signal according to the sinusoidal electrical signal;

The recording device is configured to record a change in behavior of the object by the microscope, the object being exposed to the wave signal.
The system of claim 1 wherein said system further comprises:

And a power amplifier for performing power amplification on the sinusoidal electrical signal and transmitting the amplified sinusoidal electrical signal to the transducer.
The system of claim 2 wherein said sinusoidal electrical signal has an amplitude greater than 150 millivolts and said amplified sinusoidal electrical signal has a power greater than 38 decibels.
The system of claim 3 wherein said recording device is a high speed image sensor.
The system of claim 4 wherein said transducer is a bulk wave transducer or an interdigital transducer, and wherein said transducer is a bulk wave transducer, said wave signal is a body wave signal; when the transducer is an interdigital transducer, the wave signal is an ultrasonic surface wave signal.
The system of claim 5 wherein said interdigital transducer comprises a piezoelectric substrate and a plurality of interdigital electrodes plated on said piezoelectric substrate.
The system of claim 6 wherein the number of said interdigital electrodes is 1 or 2 or 4 or 8.
The system of claim 6 wherein said piezoelectric substrate is 128°, YX double-sided polished lithium niobate or zinc oxide or aluminum nitride.
The system according to claim 7 or 8, wherein when the object is a nematode or a cell, the reservoir is a polydimethylsiloxane PMDS channel, and the PMDS cavity is bonded in the chamber. On the piezoelectric substrate of the interdigital transducer.
The system of claim 9 wherein said PMDS channel is tapered.
The system of claim 10 wherein when said guest is a nematode, said M9 solution is placed in said PMDS lumen, said nematode being placed in said M9 solution.
A system according to claim 7 or 8, wherein said storage is when said object is a nematode The device is an agar plate placed on the piezoelectric substrate of the interdigital transducer.
The system according to claim 7 or 8, wherein when said object is a nematode, said behavioral change comprises a number of return times of the nematode and a change in the frequency of the oscillation.
The system according to claim 7 or 8, wherein when said object is a cell or a brain slice, said reservoir is a slide, said slide being placed on the piezoelectric of said interdigital transducer On the substrate.