CN114874907B - System for detecting biological effect of electromagnetic exposed cells in vitro - Google Patents
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Abstract
The invention discloses a system for detecting electromagnetic exposure cell biological effect in vitro, which adopts a mode of combining a microfluidic component and a cell incubator to generate a novel irradiation system of a monopole radio frequency antenna coupling microfluidic chip.
Description
Technical Field
The invention relates to the technical field of electromagnetic radiation biological effects, in particular to a system for detecting electromagnetic exposure cell biological effects in vitro.
Background
With the popularity of wireless communication devices, the widespread use of mobile phones inevitably raises public concerns about the potentially adverse biological effects of rf electromagnetic field exposure. With the development of technology, researchers have gradually gained in scientific knowledge about the biological effects induced by radio frequency electromagnetic fields. At present, a great deal of electromagnetic radiation research on the cell biological effect is carried out by bioelectromagnetism specialists at home and abroad, and the accumulation of scientific knowledge on the cell biological response to an external electric field is accelerated. The problem is that most of the effort to solve the relationship between radio frequency electromagnetic fields and biological effects relies on in vivo experiments or through cell-based models in culture dishes, which are either time consuming, expensive or reagent-intensive and only amenable to low-throughput analysis. For this reason, it is absolutely necessary to find a new technique to solve the prior art obstacle. With the development of microfluidic technology, microfluidics is defined as a microfabricated structure with sub-micron-sized microchannels, which can process and analyze very precisely on a fly-scale microfluidics, with the problems of breaking through bottlenecks such as high loss, high time consumption and low throughput encountered in the usual experimental process. Therefore, the microfluidic technology is adopted, so that the microfluidic chip is beneficial to researching the cell biological effect exposed by the in-vitro radio frequency electromagnetic field or breaking through the effective way of the bottleneck.
Disclosure of Invention
The invention aims at providing a system for detecting electromagnetic exposure cell biological effect in vitro aiming at the defects of the prior art, the invention adopts a mode of combining a microfluidic component and a cell incubator to generate a novel irradiation system of a monopole radio frequency antenna coupling microfluidic chip, the invention generates a controllable electromagnetic field radiation environment by an electromagnetic field radiation generating device, and the electromagnetic field is radiated to the microfluidic chip through the coupling of a monopole transmitting antenna and the microfluidic chip of the microfluidic component, and the system has the advantages of DC-40GHz in a multi-frequency range and multiple waveform modes, examples: under the working condition of continuous waves or pulse waves, the uniform distribution of gradient electric fields in the micro-cavity in the micro-fluidic chip is realized, the requirements of accurate detection and high-flux analysis of electromagnetic biological effects are met, and the research method for detecting the electromagnetic exposure cell biological effects in vitro is provided, and has the advantages of short time consumption, low price, less consumption of reagents and capability of meeting the high-flux analysis.
The specific technical scheme for realizing the aim of the invention is as follows:
the system for detecting the biological effect of the electromagnetic exposed cells in vitro is characterized by comprising an electromagnetic field radiation generating device, a microfluidic component, a cell incubator and a testing device;
the electromagnetic field radiation generating device consists of a computer, a radio frequency generator, a coaxial cable and a power amplifier;
the microfluidic component consists of a microfluidic chip and a grounding copper sheet;
the microfluidic chip is composed of polydimethylsiloxane, hydrogel and a high-transmittance optical glass slide; the grounding copper sheet is sheet-shaped, and a glass slide seat is arranged on the grounding copper sheet; the high-transmittance optical glass slide is arranged on a glass slide seat of the grounding copper sheet, and the polydimethylsiloxane is arranged on the high-transmittance optical glass slide and is chemically bonded through hydrogel to form a micro-channel and micro-chambers which are mutually separated;
the cell incubator consists of an incubator body, an SMA converter, a monopole transmitting antenna, an electric field measuring instrument and an infrared thermal imager;
a platform seat, a converter seat, an antenna socket, an electric field measuring instrument seat and a thermal imaging instrument seat are arranged in the incubation box body; the SMA converter, the monopole transmitting antenna, the electric field measuring instrument and the infrared thermal imaging instrument are sequentially arranged on the converter seat, the antenna socket, the electric field measuring instrument seat and the thermal imaging instrument seat;
the microfluidic component is arranged on a platform seat of the cell incubator;
the testing device consists of a fluorescence microscope, a confocal microscope and a fluorescence enzyme-labeled instrument;
the electromagnetic field radiation generating device and the testing device are arranged on the outer side of the cell incubator, and a fluorescence microscope, a confocal microscope and a fluorescence microplate reader of the testing device are sequentially arranged on the experiment table;
the microfluidic chip is respectively arranged on the fluorescence microscope, the confocal microscope and the fluorescence enzyme-labeling instrument;
the computer is in wireless communication connection with the radio frequency generator, and the radio frequency generator, the power amplifier, the SMA converter and the monopole transmitting antenna are connected through coaxial cables; the electric field measuring instrument and the infrared thermal imager are connected with external terminal receiving equipment through wireless communication.
The invention adopts a mode of combining a microfluidic component and a cell incubator to generate a novel irradiation system of a monopole radio frequency antenna coupling microfluidic chip, generates a controllable electromagnetic field radiation environment by an electromagnetic field radiation generating device, couples the monopole transmitting antenna with the microfluidic chip of the microfluidic component, radiates an electromagnetic field to the microfluidic chip, realizes uniform distribution of a gradient electric field in a micro-cavity in the microfluidic chip in a multi-frequency range DC-40GHz and multi-waveform mode, meets the requirements of accurate detection and high-flux analysis of electromagnetic biological effects, and provides a research method for detecting electromagnetic exposure cell biological effects in vitro.
The invention has the beneficial effects that:
the novel microfluidic technology and the electromagnetic exposure method are coupled for the first time for electromagnetic field measurement outside the body of the biological cell.
The electromagnetic field intensity can be accurately measured, the electromagnetic field direction is more definite, the uniformity of the electromagnetic field is better, the interference on micro-environment cell culture is small, and the measurement result is more accurate.
The electromagnetic cell biological effect detection device can work for a long time, can detect electromagnetic cell biological effects of different electromagnetic field strengths simultaneously, and can meet the high flux requirement of a cell biological effect detection experiment relative to a low flux macroscopic irradiation system.
The micro-fluidic chip is internally provided with micro-channels and micro-chambers which are mutually separated, so that intercellular interference existing in macroscopic cell culture can be avoided to a great extent, and the current effect detection research of cell electromagnetic exposure is mostly based on cell clusters, so that a plurality of uncertainties can be brought to the biological effect quantitative research of an electromagnetic field, and the invention can provide powerful technical support for biological effect detection of the electromagnetic field.
Drawings
FIG. 1 is a schematic block diagram of the structure of the present invention;
FIG. 2 is a schematic structural diagram of a microfluidic component of the present invention;
fig. 3 is a schematic structural diagram of a microfluidic assembly and a cell incubator according to the present invention.
Detailed Description
Referring to fig. 1, the invention comprises an electromagnetic field radiation generating device 1, a microfluidic component 2, a cell incubator 3 and a testing device 4;
the electromagnetic field radiation generating device 1 is composed of a computer 11, a radio frequency generator 12, a coaxial cable 13 and a power amplifier 14.
Referring to fig. 1 and 2, the microfluidic component 2 is composed of a microfluidic chip 21 and a grounding copper sheet 25;
the microfluidic chip 21 is composed of polydimethylsiloxane 22, hydrogel 23 and a high-transmittance optical glass slide 24; the grounding copper sheet 25 is sheet-shaped, and is provided with a glass slide seat; the high-transmittance optical glass slide 24 is arranged on a glass slide seat of the grounding copper sheet 25, and the polydimethylsiloxane 22 is arranged on the high-transmittance optical glass slide 24 and is chemically bonded through the hydrogel 23 to form a micro-channel and micro-chambers which are mutually separated.
Referring to fig. 1, 2 and 3, the cell incubator 3 is composed of an incubator body 31, an SMA converter 32, a monopole transmitting antenna 33, an electric field measuring instrument 34 and a thermal infrared imager 35;
the incubator body 31 is internally provided with a platform seat 311, a converter seat 312, an antenna socket 313, an electric field measuring instrument seat 314 and a thermal imaging instrument seat 315; the SMA transducer 32, monopole transmitting antenna 33, electric field meter 34 and infrared thermal imager 35 are sequentially disposed on the transducer mount 312, antenna socket 313, electric field meter mount 314 and thermal imager mount 315;
the microfluidic component 2 is arranged on a platform base 311 of the cell incubator 3.
Referring to fig. 1, 2 and 3, the testing device 4 is composed of a fluorescence microscope 41, a confocal microscope 42 and a fluorescence microplate reader 43;
the electromagnetic field radiation generating device 1 and the testing device 4 are arranged on the outer side of the cell incubator 3, and a fluorescence microscope 41, a confocal microscope 42 and a fluorescence microplate reader 43 of the testing device 4 are sequentially arranged on the experiment table; the microfluidic chip 21 is respectively arranged on the fluorescence microscope 41, the confocal microscope 42 and the fluorescence microplate reader 43;
the computer 11 is in wireless communication connection with the radio frequency generator 12, and the radio frequency generator 12, the power amplifier 14, the SMA converter 32 and the monopole transmitting antenna 33 are connected through the coaxial cable 13; the electric field measuring instrument 34 and the infrared thermal imager 35 are connected with an external terminal receiving device through wireless communication.
Further description of the invention:
the invention adopts wireless communication, the computer 11 controls the frequency and the wave form of the output signal of the radio frequency generator 12, emits electromagnetic field for the system, and realizes the control of the electromagnetic field radiation environment.
The radio frequency generator 12 transmits the output signal to the SMA converter 32 and the monopole transmitting antenna 33 in the cell incubator 3 through the power amplifier 14; the electromagnetic field is radiated to the microfluidic chip 21 by coupling the monopole transmitting antenna 33 with the microfluidic chip 21 of the microfluidic assembly 2.
In order to ensure the sterile incubation environment of in-vitro cells, ensure enough cell fusion degree and cell activity and minimize external interference, the cell incubator 3 is also provided with an electromagnetic shielding material on the inner surface, and a temperature and carbon dioxide control device and an ultraviolet lamp disinfection device are arranged on the top.
In the experiment, the injection pump is adopted to inject the inoculated cells and nutrient substances into the micro-chambers which are separated from each other through the micro-flow channels on the micro-fluidic chip 21;
the invention sets up the electric field measuring apparatus 34 in the incubator 31, is used for detecting the electric field in the micro-chamber in the micro-fluidic chip 21 in real time, the detection information is sent to the terminal receiving equipment through the wireless communication;
the infrared thermal imager 35 is arranged in the incubation box 31 and used for detecting temperature fluctuation in a micro-cavity of the micro-fluidic chip 21 in real time, and detection information is sent to the terminal receiving equipment through wireless communication;
the invention sets the fluorescence microscope 41, the confocal microscope 42 and the fluorescence microplate reader 43 of the testing device 4 on the experiment table, wherein the fluorescence microscope 41 is used for detecting the cell morphology and position change caused by electromagnetic exposure; confocal microscope 42 is used to detect intracellular microstructural responses caused by electromagnetic exposure; the fluorescent microplate reader 43 is used to detect changes in intracellular protein expression caused by electromagnetic exposure.
The invention also provides a biochemical kit for marking cells and exposing corresponding biological effects electromagnetically in the experimental process.
Example 1
Probing umbilical vein endothelial cells (HUVECs) with a system for in vitro probing biological effects of electromagnetic exposed cells at electromagnetic radiation parameters set to: frequency band: 1.8GHz; type (2): a continuous wave; electromagnetic irradiation time: 30 minutes; the resulting in vitro cell migration effect.
Referring to fig. 1, 2 and 3, the flow is as follows: first is the rf generator 12 parameter set-up and related platform set-up. The method comprises the following steps: the radio frequency generator 12 is controlled by the computer 11 control software LabVIEW in a wireless programming way, and parameters of electromagnetic radiation are set as follows: frequency band: 1.8GHz; type (2): a continuous wave; electromagnetic irradiation time: 30 minutes.
Referring to fig. 1, 2 and 3, a microfluidic assembly 2, an incubator 31 and a testing device 4 are constructed. The electromagnetic field radiation generating device 1 and the testing device 4 are arranged in the outer space of the incubation box 31; the microfluidic component 2, the electric field measuring instrument 34 and the thermal infrared imager 35 are disposed in the inner space of the incubation box 31.
Referring to fig. 1, 2 and 3, the incubation and electromagnetic radiation process of cell inoculation in the microfluidic chip 21 is next described. The method comprises the following steps: cell suspension (6.multidot.10) of cells with a certain degree of fusion was pumped by syringe 4 Individual/ml) is slowly inoculated into the micro-chamber through a plurality of micro-channel ports of the micro-fluidic chip 21, and relevant parameters of the injection pump for inoculating cells are as follows: flow rate: 1 microliter/minute; duration of time: 5 minutes; injection ofThe pump conveys the cell suspension to the micro-cavities which are separated from each other in the micro-channel in a negative pressure mode, and the cell inoculation density in the micro-cavities is uniform.
Referring to fig. 1, 2 and 3, after a suitable cell concentration is reached in the microcavity, the syringe pump is turned off and the cell suspension is allowed to rest in the incubator 31 for 15 minutes to achieve cell attachment. After the wall is attached, setting parameters of the injection pump again as follows: flow rate: 10 microliters/min; duration of time: 24 hours; and opened to continuously deliver nutrient solution to the adherent cells in the microcavities. The normal operation of the constant temperature and humidity ventilation system in the incubation box 31 is ensured in the incubation process, and the interference of environmental factors is eliminated, so that the temperature fluctuation in the micro-chamber is caused. After 24 hours of incubation, the computer 11 starts to control the radio frequency generator 12 and generate electromagnetic radio frequency signals, the emitted electromagnetic radio frequency signals are transmitted to the power amplifier 14 by the low-loss coaxial cable 13 to perform corresponding signal multiple amplification treatment, the amplified electromagnetic radio frequency signals are transmitted to the SMA converter 32 in the incubation box 31 by the low-loss coaxial cable 13, the electromagnetic radio frequency signals are finally transmitted to the monopole transmitting antenna 33 coupled with the microfluidic chip 21 of the microfluidic assembly 2 after conversion, the electromagnetic radio frequency signals on the monopole transmitting antenna 33 are excited at the top end of the monopole transmitting antenna, and the excited electromagnetic waves act on cells in a micro cavity in the microfluidic chip 21 in the form of electromagnetic fields. In this process: the electric field and temperature fluctuation in the micro-cavity of the micro-fluidic chip 21 are recorded in real time by the electric field measuring instrument 34 and the infrared thermal imager 35 platform respectively, and are displayed on the terminal receiving device in real time by wireless communication.
Referring to fig. 1, 2 and 3, after electromagnetic exposure is completed, the in vitro cell biological effect in the micro chamber in the microfluidic chip 21 is completed by the testing device 4. The method comprises the following steps: after electromagnetic exposure, the microfluidic chip 21 in the incubator 31 was taken out, and the cytoskeleton of umbilical vein endothelial cells was labeled with a biochemical kit on an ultra clean bench. After the labeling is completed, the microfluidic chip 21 is placed on a laboratory table of the test device 4, and cell morphology and position changes and relevant intracellular microstructure response observations are performed by using a fluorescence microscope 41 and a confocal microscope 42, respectively. The final electromagnetic exposure cell bioeffect data will be transmitted to the computer terminal. The data recorded by the computer terminal is shared through the Internet, so that the cell biological effect caused by electromagnetic exposure can be checked in real time. Finally, by analyzing and processing experimental data, obvious in-vitro biological effects generated by electromagnetic exposure of umbilical vein endothelial cells for 30 minutes can be detected.
Example 2
The system for detecting the biological effect of the electromagnetic exposed cells in vitro is used for detecting the human amniotic epithelial cells under the electromagnetic radiation parameters: frequency band: 4.8GHz; type (2): a pulse wave; electromagnetic irradiation time: 1 hour; and expressing the generated in vitro cell electromagnetic radiation related protein.
Referring to fig. 1, 2 and 3, the flow is as follows: the rf generator 12 parameter settings and associated platform set-up. The method comprises the following steps: firstly, the radio frequency generator 12 is controlled by the computer 11 control software LabVIEW in a wireless programming way, and parameters of electromagnetic radiation are as follows: frequency band: 4.8GHz; type (2): a pulse wave; electromagnetic irradiation time: and 1 hour. The microfluidic assembly 2, the incubator 31 and the test apparatus 4 were set up. The electromagnetic field radiation generating device 1 and the testing device 4 are arranged in the outer space of the incubation box 31; the microfluidic component 2, the electric field measuring instrument 34 and the thermal infrared imager 35 are all arranged in the inner space of the incubation box 31.
Referring to fig. 1, 2 and 3, the incubation and electromagnetic radiation process of cell inoculation in the microfluidic chip 21 is next described. The method comprises the following steps: cell suspension (2×10) of cells with a certain degree of fusion was pumped by syringe 5 At a rate of one milliliter) is slowly inoculated to the cell inlet of a micro-channel on polydimethylsiloxane 22 in the micro-fluidic chip 21, and then relevant parameters of an injection pump connected with the cell channel outlet on the micro-fluidic chip 21 are set as follows: flow rate: 0.5 microliters/minute; duration of time: 8 minutes. The injection pump conveys the cell suspension into the micro-cavities which are separated from each other in the micro-channel in a negative pressure mode, and the cell inoculation density in the micro-cavities is uniform.
Referring to fig. 1, 2 and 3, after a suitable cell concentration is reached in the microcavity, the syringe pump is turned off and the cell suspension is allowed to rest in the incubator 31 for 15 minutes to achieve cell attachment. After the wall is attached, setting parameters of the injection pump again: flow rate: 2 microliters/minute; duration of time: 1 hour; and the operation of the injection pump is realized every half hour, so that the nutrition supply of the adherent epithelial cells in the micro-chamber is ensured. The normal operation of the constant temperature and humidity ventilation system in the incubation box 31 is ensured in the incubation process, and the interference of environmental factors is eliminated, so that the temperature fluctuation in the micro-chamber is caused. After 24 hours, the computer control software 11 starts to control the radio frequency generator 12 and generate electromagnetic radio frequency signals, the emitted electromagnetic radio frequency signals are transmitted to the power amplifier 14 by the low-loss coaxial cable 13 to perform corresponding signal multiple amplification treatment, the amplified electromagnetic radio frequency signals are transmitted to the SMA converter 32 in the incubator body 31 by the low-loss coaxial cable 13, the electromagnetic radio frequency signals are finally transmitted to the monopole transmitting antenna 33 coupled with the microfluidic chip 21 of the microfluidic component 2, the electromagnetic radio frequency signals on the antenna are excited at the top end of the monopole transmitting antenna, and the excited electromagnetic waves act on cells in a micro cavity in the microfluidic chip 21 in the form of electromagnetic fields. In this process: the electric field and temperature fluctuation in the micro-cavity of the micro-fluidic chip 21 are recorded in real time by the electric field measuring instrument 34 and the infrared thermal imager 35 platform respectively, and are displayed on a terminal computer in real time.
Referring to fig. 1, 2 and 3, after electromagnetic exposure is completed, the in vitro cell biological effect in the micro chamber in the microfluidic chip 21 is completed by the testing device 4. The method comprises the following steps: after electromagnetic exposure, the microfluidic chip 21 in the incubator 31 is taken out, and the specific protein labeling of the epithelial cells related to electromagnetic radiation is realized on an ultra-clean bench by using a biochemical kit. After the labeling is finished, firstly, the cells in the micro-chamber are gently washed by using a buffer solution, then, the digestion of the adherent epithelial cells is realized by using a digestive solution, the digested cell suspension is collected by using a syringe pump, the collected cell suspension is subjected to the processes of centrifugation, resuspension and the like, then, the resuspended cells are dripped into a 96-well plate, and the cells in the well plate are subjected to detection of the human amniotic epithelial cells in electromagnetic radiation and electromagnetic wave parameters by using a fluorescent enzyme-labeled instrument 43 on a laboratory table of a testing device 4: frequency band: 4.8GHz; type (2): a pulse wave; electromagnetic irradiation time: and 1 hour. The generated in vitro cell electromagnetic radiation related protein quantity is expressed, and finally the electromagnetic exposure cell biological effect data is transmitted to a computer terminal. The data recorded by the computer is shared through the Internet, so that the cell biological effect caused by electromagnetic exposure can be checked in real time. Finally, through analysis and processing of experimental data, the obvious change of the expression of the in-vitro cell electromagnetic radiation related protein amount generated by the human amniotic epithelial cells after electromagnetic radiation exposure for 1 hour can be detected.
Claims (1)
1. The system for detecting the biological effect of the electromagnetic exposed cells in vitro is characterized by comprising an electromagnetic field radiation generating device (1), a microfluidic component (2), a cell incubator (3) and a testing device (4);
the electromagnetic field radiation generating device (1) is composed of a computer (11), a radio frequency generator (12), a coaxial cable (13) and a power amplifier (14);
the micro-fluidic component (2) is composed of a micro-fluidic chip (21) and a grounding copper sheet (25); the microfluidic chip (21) is composed of polydimethylsiloxane (22), hydrogel (23) and a high-transmittance optical glass slide (24); the grounding copper sheet (25) is sheet-shaped, and a glass slide seat is arranged on the grounding copper sheet; the high-permeability optical glass slide (24) is arranged on a glass slide seat of the grounding copper sheet (25), and the polydimethylsiloxane (22) is arranged on the high-permeability optical glass slide (24) and is chemically bonded through the hydrogel (23) to form a micro-channel and micro-chambers which are mutually separated;
the cell incubator (3) consists of an incubator body (31), an SMA converter (32), a monopole transmitting antenna (33), an electric field measuring instrument (34) and an infrared thermal imager (35); a platform seat (311), a converter seat (312), an antenna socket (313), an electric field measuring instrument seat (314) and a thermal imaging instrument seat (315) are arranged in the incubation box body (31); the SMA converter (32), the monopole transmitting antenna (33), the electric field measuring instrument (34) and the infrared thermal imager (35) are sequentially arranged on the converter seat (312), the antenna socket (313), the electric field measuring instrument seat (314) and the thermal imager seat (315);
the microfluidic component (2) is arranged on a platform seat (311) of the cell incubator (3);
the testing device (4) is composed of a fluorescence microscope (41), a confocal microscope (42) and a fluorescence enzyme-labeling instrument (43);
the electromagnetic field radiation generating device (1) and the testing device (4) are arranged on the outer side of the cell incubator (3), and a fluorescence microscope (41), a confocal microscope (42) and a fluorescence enzyme-labeled instrument (43) of the testing device (4) are sequentially arranged on the experiment table;
during testing, taking out the microfluidic chip (21) in the incubator body (31), and placing the microfluidic chip (21) on the fluorescence microscope (41), the confocal microscope (42) and the fluorescence enzyme labeling instrument (43);
the computer (11) is in wireless communication connection with the radio frequency generator (12), and the radio frequency generator (12), the power amplifier (14), the SMA converter (32) and the monopole transmitting antenna (33) are connected through the coaxial cable (13); the electric field measuring instrument (34) and the infrared thermal imager (35) are connected with external terminal receiving equipment through wireless communication.
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