CN110835603A - Device and method for rapidly realizing reversible damage of cell membrane - Google Patents
Device and method for rapidly realizing reversible damage of cell membrane Download PDFInfo
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Abstract
The invention relates to a device and a method for quickly realizing reversible damage of cell membranes, which are used for realizing the following steps: collecting photoacoustic signals of a silanization culture tank, filtering the photoacoustic signals, extracting peak values, and sending the filtered photoacoustic signals to a computer device for analysis; the computer device extracts values according to the characteristics, compares the values with a preset cell tolerance stress database and analyzes the values to judge whether the laser energy exceeds the tolerance threshold of the cells in the culture pond; if the laser energy does not exceed the preset value, reducing the laser energy or turning off the treatment, and if the laser energy does not exceed the preset value, increasing the laser energy of the laser emitting device. The invention has the beneficial effects that: the operation period of the system device is greatly shortened, and the universality of the system device is expanded; the non-contact and pollution-free laser is used as a pumping source, and the controllable, rapid, high-flux and automatic recoverable cell membrane operation on host cells can be realized.
Description
Technical Field
The invention relates to a device and a method for quickly realizing reversible damage of cell membranes, belonging to the field of computers and biomedicine.
Background
With the progress of scientific technology, human beings continuously and deeply explore life, understanding of life has been stepped into cell and subcellular levels, and more scientific researches hope that the control of tiny cells and organelles is realized under the condition of not damaging cell activity (reversible damage), so that the development of multiple subject fields such as cell communication, stem cell differentiation, gene therapy, embryonic development and the like is promoted.
The existing methods for realizing reversible damage of cell membranes by using physical mechanisms include an acoustic method, a thermal method, a magnetic method, an electrical method and the like, but the methods are limited in the application of reversible damage of cell membranes due to the problems of complex operation, poor controllability and the like.
At present, two devices for realizing reversible damage of cell membranes based on a laser technology are mainly used: one is to realize the target location of the pumping beam on the specific cell membrane by the tight focusing technology, and realize the reversible damage of the cell membrane by utilizing the nonlinear energy deposition of the tight focusing beam; the second is the chelation of the nanoparticles with the host cell, which results in reversible damage to the cell membrane by targeted energy deposition. Both of the above devices or systems have drawbacks that are difficult to overcome: the first device has the advantages that the pump light source module needs to carry out tight focusing beam shaping, so the device can only process one cell at a time, in addition, the focusing point is not easy to regulate and control, the radiation density of pump laser is high, irreversible damage is easy to cause, and the pulse width of an optional light source is very limited. In the second method, although high-throughput cell processing speed can be realized, it takes a long time to prepare host cell membrane surface antibody-modified nanoparticles, and the optimization exploration of parameters such as host cell and gold nanoparticle matching parameters, incubation time and optimal nanoparticle morphology is time-consuming and labor-intensive.
The two existing methods for realizing reversible damage of cell membranes have great limitations, so that the reversible damage of the cell membranes is difficult to realize effectively and quickly.
Disclosure of Invention
The invention aims to at least solve one of the technical problems in the prior art, and provides a device and a method for quickly realizing reversible damage of cell membranes, wherein the device is coupled with a special cell culture pond module after silanization treatment, so that the problems of selectivity of receptors on the surfaces of host cell membranes to nanoparticles, limitation of receptor sites and the like are solved, and the selection limitation of various parameters such as the morphology structure and concentration of the nanoparticles, host cell strains, incubation time of the nanoparticles and the host cells, a coupling mode of the nanoparticles and the host cells is also realized, so that the operation cycle of a system device is greatly shortened, and the universality of the system device is extended; the device is coupled with the photoacoustic early warning feedback module, and red warning reminding is set for the behavior that the cell membrane is possibly damaged irreversibly due to the operation of super (laser) dose; meanwhile, the device of the invention adopts non-contact and pollution-free laser as a pumping source, can realize controllable, rapid, high-flux and automatic recoverable cell membrane operation on host cells, and solves the problems described in the background technology.
The technical scheme of the invention comprises a device for quickly realizing reversible damage of cell membranes, which comprises a laser emitting device, a photoacoustic early warning feedback device and a culture pond device, and is characterized in that: the laser emitting device is used for sending instructions to the culture pond device and carrying out subsequent regulation and control on the energy of the laser beam according to the photoacoustic early warning feedback device; the culture pond device comprises a movable platform and a culture pond arranged on the platform, wherein the movable platform is used for controlling the movable platform to move in a three-dimensional space position, so that a target area of the culture pond is positioned in a focal spot area of a pump beam emitted by the laser emitting device; the photoacoustic early warning feedback device is used for collecting and analyzing the characteristic photoacoustic signals of the culture pond and dynamically regulating and controlling the energy of the laser emitted by the laser emitting device according to the analysis result.
According to the device of quick realization cell membrane reversibility damage, wherein laser emission device is including the pumping laser light source, the group of lenses of expanding beam, attenuation piece group, polaroid, spectroscope and the high power objective lens that connect gradually, its characterized in that: the pump laser light source is used for emitting pump laser and regulating and controlling the energy of the pump laser according to an instruction issued by the photoacoustic early warning feedback device; the beam expanding lens group, the attenuation sheet group, the polaroid, the spectroscope and the high power objective lens are respectively used for sequentially carrying out beam expanding, attenuation, polarization, beam splitting and focusing operations on the pump laser.
According to the device for rapidly realizing reversible damage of the cell membrane, the polaroid is used for splitting the laser beam attenuated by the attenuation sheet group into two beams of laser, one beam of laser is sent to the photoacoustic early warning feedback device, and the other beam of laser is sent to the spectroscope.
The device for rapidly realizing reversible damage of cell membranes is characterized in that the culture pond device is configured to be an XYZ precision electric displacement table and a silanized cell culture pond arranged on the XYZ precision electric displacement table, and the XYZ precision electric displacement table is used for enabling a focal plane of a laser beam emitted by the laser emitting device to be located on an aperture plane of the silanized cell culture pond through three-dimensional position adjustment.
The device for rapidly realizing reversible damage of cell membranes, wherein the silanized cell culture pond is configured to: the culture dish is characterized in that a sulfur-containing organosilane single-layer film is arranged on the inner wall of the culture dish, hydrogen-containing sulfur groups on the surface of the sulfur-containing organosilane single-layer film are sites for chemically adsorbing gold nanoparticles, and mediator nanoparticles are solidified on the sulfur-containing organosilane single-layer film.
In a preferred embodiment, the mediator nanoparticles are specifically gold nanoparticles.
According to the device for rapidly realizing reversible damage of cell membranes, wherein the photoacoustic early warning feedback device comprises a broadband photoacoustic probe, an ultrasonic pulse receiver, a power energy meter and a computer device, and is characterized in that: the broadband photoacoustic probe is arranged right above the culture pond and used for collecting photoacoustic signals generated by the culture pond, filtering the photoacoustic signals by the ultrasonic pulse receiver to obtain filtering signals and sending the filtering signals to the computer device for signal characteristic peak value extraction; the power energy meter is used for collecting the energy of the laser emitted by the laser emitting device and sending the energy to the computer device; and the computer device compares the filtering signal with a stored preset value, sends an instruction for regulating and controlling laser energy to the laser emitting device according to a comparison result, and simultaneously displays the instruction on an interactive interface.
In a preferred embodiment, the alarm device is further included, and the alarm device gives an alarm prompt according to the instruction of the calculator device, and the alarm device comprises but is not limited to an audio alarm device and/or a light alarm device.
According to the device for rapidly realizing reversible damage of the cell membrane, the computer device specifically comprises: and converting the filtering signal into a mechanical signal, comparing the mechanical signal with a stored mechanical force threshold database, if the mechanical signal exceeds the threshold, carrying out corresponding prompt and alarm, and if the mechanical signal is lower than the threshold, increasing the laser energy of the laser emitting device.
The technical scheme of the invention also comprises a method for quickly realizing reversible damage of cell membranes, which is used for executing any one of the devices, and is characterized in that: s10, collecting photoacoustic signals of the silanization culture tank, filtering the photoacoustic signals, extracting peak values, and sending the filtered photoacoustic signals to a computer device for analysis; s20, comparing the extracted value with a preset cell tolerance stress database by the computer device according to the characteristics, and judging whether the laser energy exceeds the tolerance threshold of the cells in the culture pond; and S30, if the laser energy is not higher than the preset value, reducing the laser energy or carrying out closing treatment, and if the laser energy is not higher than the preset value, increasing the laser energy of the laser emitting device.
The invention has the beneficial effects that: the problems of selectivity of a host cell membrane surface receptor to nanoparticles, limitation of receptor sites and the like are solved, the limitation of the selection of various parameters such as the morphology structure, concentration, host cell strain, incubation time of the nanoparticles and the host cells, a coupling mode of the nanoparticles and the host cells is limited, the operation cycle of a system device is greatly shortened, and the universality of the system device is extended; the device is coupled with the photoacoustic early warning feedback module, red warning reminding is set for the behavior that the cell membrane is possibly damaged irreversibly due to the operation of super (laser) dose, and controllable, rapid, high-flux and automatic recoverable cell membrane micro-operation on host cells can be realized by adopting non-contact and pollution-free laser as a pumping source.
Drawings
The invention is further described below with reference to the accompanying drawings and examples;
FIG. 1 is a block diagram illustrating the connection of a device according to an embodiment of the present invention;
FIG. 2 illustrates an overall flow diagram according to an embodiment of the invention;
FIG. 3 is a schematic view of a device connection according to an embodiment of the present invention;
FIG. 4 is a schematic view of a silanized cell culture cell according to an embodiment of the present invention;
FIGS. 5a, 5b, 5c and 5d are schematic diagrams illustrating the principle of gold nanoparticle-mediated reversible damage to cell membranes according to the embodiment of the present invention;
fig. 6 is a flowchart illustrating the operation of the photoacoustic warning feedback apparatus according to the embodiment of the present invention.
Detailed Description
Reference will now be made in detail to the present preferred embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like reference numerals refer to like elements throughout.
In the description of the present invention, it should be understood that the orientation or positional relationship referred to in the description of the orientation, such as the upper, lower, front, rear, left, right, etc., is based on the orientation or positional relationship shown in the drawings, and is only for convenience of description and simplification of description, and does not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and thus, should not be construed as limiting the present invention.
In the description of the present invention, the meaning of a plurality of means is one or more, the meaning of a plurality of means is two or more, and larger, smaller, larger, etc. are understood as excluding the number, and larger, smaller, inner, etc. are understood as including the number. If the first and second are described for the purpose of distinguishing technical features, they are not to be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated or implicitly indicating the precedence of the technical features indicated.
In the description of the present invention, unless otherwise explicitly limited, terms such as arrangement, installation, connection and the like should be understood in a broad sense, and those skilled in the art can reasonably determine the specific meanings of the above terms in the present invention in combination with the specific contents of the technical solutions.
Fig. 1 is a block diagram illustrating connection of devices according to an embodiment of the present invention. The device includes laser emission device, optoacoustic early warning feedback device and culture dish device, specifically lies in: the laser emitting device is used for emitting laser beams to the culture pond device and controlling the energy of the laser beams according to the instruction issued by the photoacoustic early warning feedback device; the culture pond device comprises a three-dimensional movable platform and a culture pond arranged on the platform, and the movable platform is used for controlling the movable platform to adjust the three-dimensional space position so as to align the relative positions of the culture pond and the laser emission device; the photoacoustic early warning feedback device is used for acquiring and analyzing photoacoustic signals of the culture pond device and dynamically regulating and controlling the energy of laser emitted by the laser emitting device according to an analysis result.
Fig. 2 shows a general flow diagram according to an embodiment of the invention. The process comprises the following steps: s10, collecting photoacoustic signals of the silanization culture tank, filtering the photoacoustic signals, extracting peak values, and sending the filtered photoacoustic signals to a computer device for analysis; s20, comparing the extracted value with a preset cell tolerance stress database by the computer device according to the characteristics, and judging whether the laser energy exceeds the tolerance threshold of the cells in the culture pond; and S30, if the laser energy is not exceeded, reducing the laser energy or closing the processing, and if the laser energy is not exceeded, increasing the laser energy of the laser emitting device.
Fig. 3 is a schematic view showing the connection of the apparatus according to the embodiment of the present invention. The device comprises a pump laser light source 001, a beam expanding lens group 002, an attenuation sheet group 003, a polaroid 004, a spectroscope 005, a high-power objective 006, an XYZ precision electric translation table 007, a silanized cell culture tank module 008, a photoacoustic probe 009, an ultrasonic pulse emitting/receiving device 010, a power energy meter 011 and an external computer 012. The beam expanding lens group 002 mainly performs beam expanding and shaping on the pump laser, and the laser after beam expanding and shaping is regulated and controlled through the attenuation sheet group 003. The polaroid 004 divides the regulated laser into two beams, and one beam reaches the power energy meter 011 and is used for monitoring and recording the pump laser light source in real time; the other beam is focused by the high-power objective lens 006 after passing through the spectroscope 005, the relative position of the focal plane and the silanized cell culture cell 008 is determined by the sample cell space site, the relative position can be regulated and controlled by the XYZ precision electric displacement table 007, and a corresponding photoacoustic alarm device can be properly added according to the requirement.
FIG. 4 is a schematic view of a silanized cell culture cell according to an embodiment of the present invention. Based on fig. 3, when designing the silanized cell culture cell module 008, in the specific implementation, a suitable nano-dielectric needs to be selected first, and the gold nanoparticles have tunable optical properties, good biocompatibility and low cell toxicity and are widely applied to biological tissue imaging, cancer diagnosis and treatment, so the gold nanoparticles are selected as the dielectric in the module. In specific implementation, mature chemical methods such as a sodium citrate method, a seed crystal synthesis method and the like can be adopted to prepare the gold nanoparticles or the commercially available gold nanoparticles after sterilization treatment.
The method comprises the steps of taking sulfur-containing organosilane which is easy to adsorb on the surface of a silicon-containing glass culture dish as a raw material, firstly preparing a sulfur-containing organosilane self-assembled monolayer film on the surface of the inner wall of the glass culture dish, then taking a hydrogen-containing sulfur group on the surface of the film as a site for chemically adsorbing gold nanoparticles, and finally solidifying mediator gold nanoparticles on the self-assembled film, so as to construct the silanized cell culture pool module in the device.
Fig. 5a, 5b, 5c and 5d are schematic diagrams illustrating the principle of gold nanoparticle-mediated rapid reversible damage of cell membranes according to the embodiment of the invention. Laser heats nanometer particles through surface plasma resonance effect, nanometer particle electrons absorb incident photons through resonance to cause peripheral energy deposition, the heated nanometer particles and peripheral media generate thermoelastic expansion, thermoelastic stress waves are emitted to the periphery, wherein the photothermal effect can cause local phase change of cell membrane phospholipid bilayers or cause denaturation of glycoprotein on the surface of the membrane to cause change of host cell membrane permeability, and the thermoelastic stress waves caused by the photothermal effect of the nanometer particles are one of the principles of the device for realizing reversible damage of the cell membrane; when the laser dose is further increased, the thermal effect caused by local energy deposition can cause the evaporation and phase change of surrounding substances to form micro-nano vacuoles, the vacuoles are broken to form micro jet flow, the jet flow is like a scalpel to cut micro-nano pores on the surface of a cell, the mechanical jet flow caused by the asymmetric movement of the vacuoles is another principle for quickly realizing the reversible damage of the cell membrane, the vacuoles generate acoustic signals in the volume change process, and the acoustic signals can represent the vacuole energy and reflect the mechanical stress generated when the vacuoles are asymmetrically broken.
Fig. 6 is a flowchart illustrating the operation of the photoacoustic warning feedback apparatus according to the embodiment of the present invention. Based on fig. 3, the technical solution principle of the process is as follows:
the photoacoustic early warning feedback module comprises a photoacoustic probe 009, an ultrasonic pulse transmitter/receiver 010 and an external computer 012. When the photothermal effect is generated, no matter thermoelastic stress wave emission or vacuole oscillation mechanical stress wave emission is induced, the generated photoacoustic conversion signal can be received by the broadband photoacoustic probe 009, and the intensity of the collected signal can be used for representing the degree of cell damage. The originally collected photoacoustic signal is subjected to low-pass filtering and denoising by the ultrasonic pulse transmitter/receiver 010, then automatically stored in the external computer 012, and the characteristic peak value of the photoacoustic signal is extracted, and then the characteristic peak value of the photoacoustic signal is compared with the constructed early warning parameter database to judge whether the characteristic peak value is higher than the early warning threshold value. If the characteristic peak value is lower than the early warning threshold value, the incident laser dose can be increased, and the operation efficiency of the system device is improved; if the temperature is higher than the early warning threshold, the laser irradiation can be stopped as required to avoid irreversible damage to the host cells.
The technical scheme of the invention also provides a disclosure of a scheme for constructing and operating the device, and with reference to fig. 3, the method comprises the following steps:
step one, device construction: placing the culture pond module 008 after silanization treatment on an XYZ three-dimensional precision adjustable electric translation table 007, and adjusting the XYZ three-dimensional space position to enable the focal radius of a pump light source 001 focused by a high power objective 006 and a YZ platform of the culture pond module 008 to coincide with the aperture of the sample pond 008 after beam expansion by a beam expansion lens group 002, attenuation by an attenuation sheet group 003, beam splitting by a spectroscope 005 and focusing by a high power objective lens 006; the spatial position of the broadband photoacoustic probe 009 is adjusted so that the probe target surface is parallel to the YZ plane, so that the focus of the probe coincides with the focus of the pump laser source 001.
Step two, setting an early warning parameter database: the photoacoustic early warning feedback module comprises a broadband photoacoustic probe 009, an ultrasonic pulse transmitter/receiver 010 and an external computer 012. Because the host cells of different cell lines have difference in tolerance to mechanical external force, in order to realize automatic controllability of controllable damage of cell membranes, the device needs to establish a mechanical force threshold database that common cells reach irreversible damage, and the threshold is set as an early warning parameter and used as a critical value for triggering early warning of the device. The silanized nanoparticles interact with the pump laser 001, and due to a thermoelasticity mechanism, an evaporation phase change mechanism, a photoinduced breakdown mechanism and the like, light-sound (mechanical) signal conversion can be realized, the converted mechanical signal can be acquired by the photoacoustic probe 009, and after being filtered by the ultrasonic pulse transmitter/receiver 010, the external computer 012 extracts a signal peak value and stores the signal peak value and makes a judgment on whether to give an early warning or not.
The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the gist of the present invention.
Claims (11)
1. The utility model provides a realize device that cell membrane reversibility harms fast, the device includes laser emission device, optoacoustic early warning feedback device and culture pond device, its characterized in that:
the laser emitting device is used for emitting laser beams to the culture pond device and regulating and controlling the energy of the laser beams according to instructions issued by the photoacoustic early warning feedback device;
the culture pond device comprises a movable platform and a culture pond placed on the platform, wherein the movable platform is used for controlling the movable platform to perform three-dimensional displacement, so that a target region of the culture pond is positioned in a pump laser focal spot region emitted by the laser emitting device; the photoacoustic early warning feedback device is used for acquiring and analyzing the laser characteristic signal of the laser emitting device and the acousto-optic signal of the culture pond, and dynamically regulating and controlling the energy of the laser emitted by the laser emitting device according to the analysis result.
2. The device for rapidly realizing reversible damage of cell membranes according to claim 1, wherein the laser emitting device comprises a pump laser source, a beam expanding lens group, an attenuating plate group, a polarizing plate, a spectroscope and a high power objective lens which are connected in sequence, and is characterized in that:
the pump laser light source is used for emitting pump laser and regulating and controlling the energy of the pump laser according to an instruction issued by the photoacoustic early warning feedback device;
the beam expanding lens group, the attenuation sheet group, the polaroid, the spectroscope and the high power objective lens are respectively used for sequentially carrying out beam expanding, attenuation, polarization, beam splitting and focusing operations on the pump laser.
3. The device for rapidly realizing reversible damage to cell membranes as claimed in claim 2, wherein the polarizer is configured to split the laser beam attenuated by the attenuator group into two laser beams, one of the two laser beams is sent to the photoacoustic pre-warning feedback device, and the other laser beam is sent to the spectroscope.
4. The device for rapidly achieving reversible damage of cell membrane according to claim 1, wherein the culture cell device is configured as an XYZ precision electric displacement stage and a silanized cell culture cell arranged on the XYZ precision electric displacement stage, and the XYZ precision electric displacement stage is used for enabling a focal plane of a laser beam emitted by the laser emitting device to be located on an aperture plane of the silanized cell culture cell through three-dimensional translation.
5. The device for rapidly achieving reversible damage of cell membranes according to claim 4, wherein the silanized cell culture tank is configured to: the culture dish is characterized in that a sulfur-containing organosilane single-layer film is arranged on the inner wall of the culture dish, hydrogen-containing sulfur groups on the surface of the sulfur-containing organosilane single-layer film are sites for chemically adsorbing gold nanoparticles, and mediator nanoparticles are solidified on the sulfur-containing organosilane single-layer film.
6. The device for rapidly realizing reversible damage of cell membranes, according to claim 5, wherein the mediator nanoparticles are mediator gold nanoparticles.
7. The device for rapidly realizing reversible damage of cell membranes according to claim 1, wherein the photoacoustic early warning feedback device comprises a broadband photoacoustic probe, an ultrasonic pulse receiver, a power energy meter and a computer device, and is characterized in that:
the broadband photoacoustic probe is arranged right above the culture pond and used for collecting photoacoustic signals generated by the culture pond, receiving and filtering the photoacoustic signals by the ultrasonic pulse receiver and sending the photoacoustic signals to the computer device for characteristic peak value extraction;
the power energy meter is used for collecting the energy of the laser emitted by the laser emitting device and sending the energy to the computer device;
and the computer device compares the filtering signal with a stored preset value, sends an instruction for regulating and controlling laser energy to the laser emitting device according to a comparison result, and simultaneously displays the instruction on an interactive interface.
8. The device for rapidly realizing reversible damage to cell membranes according to claim 7, further comprising an alarm device for giving an alarm prompt according to instructions of a calculator device, wherein the alarm device comprises but is not limited to an audio alarm device and/or a light alarm device.
9. The device for rapidly achieving reversible damage to cell membranes according to claim 7, further comprising a cell mechanical force tolerance threshold database.
10. The device for rapidly achieving reversible damage of cell membranes according to claim 7, wherein the computer device comprises:
and converting the filtering signal into a mechanical signal, comparing the mechanical signal with a stored mechanical force threshold database, if the mechanical signal exceeds the threshold, carrying out corresponding prompt and alarm, and if the mechanical signal is lower than the threshold, increasing the laser energy of the laser emitting device.
11. A method for rapidly achieving reversible damage to cell membranes, said method being based on a device according to any one of claims 1 to 10, wherein:
s10, collecting photoacoustic signals of the silanization culture tank, filtering the photoacoustic signals, extracting peak values, and sending the filtered photoacoustic signals to a computer device for analysis;
s20, comparing the extracted value with a preset cell tolerance stress database by the computer device according to the characteristics, and judging whether the laser energy exceeds the tolerance threshold of the cells in the culture pond;
and S30, if the laser energy is exceeded, reducing the laser energy or closing the treatment, and if the laser energy is not exceeded, increasing the laser energy of the laser emitting device.
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