CN115856271A - Method and system for detecting virus or pseudovirus infected cells - Google Patents
Method and system for detecting virus or pseudovirus infected cells Download PDFInfo
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Abstract
The invention discloses a method for detecting virus or pseudovirus infected cells, which comprises the steps of applying sinusoidal voltage to an attached membrane in an attached membrane clamp mode and detecting corresponding membrane current; inducing virus or pseudovirus to infect cells by combining the virus or pseudovirus in the electrode internal liquid with a receptor on the attached membrane; phase demodulation is carried out on corresponding membrane current signals by taking the phase of input sinusoidal voltage as a standard, so as to obtain the imaginary part and the real part of the membrane current, and real-time calibration and calibration are carried out; calculating the film capacitance of the attached film on the basis of calibration and calibration according to the current real part signal and the current imaginary part signal; and identifying virus or pseudovirus infected cell events and modes thereof according to the dynamic characteristics and polarity of the capacitance change of the diaphragm, and estimating the scale of the virus or pseudovirus according to the size of the capacitance change of the diaphragm. The invention can realize real-time high-precision detection on various viruses or pseudoviruses infecting host cells in a capsule membrane and cell membrane fusion mode and a virus or pseudovirus particle endocytosis mode on acute separated cells, cultured cells and separated tissue living cells.
Description
Technical Field
The invention relates to the technical field of biological and medical detection, in particular to a method and a system for detecting virus or pseudovirus infected cells.
Background
The research of the virus and the development of the therapeutic drug thereof are started from various links of infection, reverse transcription, replication, self-packaging and the like of the virus. Taking the new coronavirus as an example, the detection and the research of the infection cells of the new coronavirus, which is the first defense line, are very important for preventing and treating the new crown epidemic situation. The diameter of the virus is about tens to hundreds of nanometers, and the process of infecting the cells is completed in millisecond time. The observation technical means for virus detection and cell infection in the current field comprise: electron microscope observation, high-resolution fluorescence microscopic imaging based on protein expression, immunoblotting, nucleic acid detection and the like.
The electron microscope observation method has high spatial precision and intuition, can observe the infection event of a single virus under low probability, but cannot realize the observation of living cells and can only complete the static detection of the cell or subcellular morphology at a certain time point. The method has the limitations that the treatment process is long and tedious, and the sample is easy to be polluted and damaged. And the high-resolution fluorescence microscopic imaging based on protein expression can detect the motion trail of a single virus particle in real time under lower time resolution (sub-second level), but an observation sample is only limited to culture cells, and gene or protein modification must be carried out on viruses. The methods such as immunoblotting and nucleic acid detection have no time-space information and limited signal precision, and it is difficult to accurately monitor the dynamic process of virus infecting living cells in real time.
In summary, these methods are difficult to accurately monitor the kinetics of viral infection into living cells in real time, either because of the technology itself, or because of the genetic or protein modifications that must be made to the virus to alter the virus's characteristics. In addition, the methods can not carry out direct virus infection observation on human living tissues, so that the corresponding observation is far away from the real human pathological phenomenon, and the diagnosis and treatment of virus-related diseases and the research and development process of medicines are limited. In view of the above, it is necessary to develop a method for detecting virus infection of living cells in real time and accurately by innovative means.
Disclosure of Invention
Aiming at the problems in the existing virus infection cell detection technology, the invention realizes the real-time and high-precision detection of various viruses or pseudoviruses with RNA or DNA replaced on living cells in acute isolated cells, cultured cells and isolated tissues of animals and human beings.
The technical scheme of the invention is realized as follows:
according to one aspect of the invention, a method for detecting infection of a cell by a virus or pseudovirus is provided.
The method for detecting the virus or pseudovirus infected cells comprises the following steps:
establishing an attached patch clamp mode to ensure that viruses or pseudo viruses in the electrode internal liquid can be combined with receptors on patches contained in the electrodes to induce the viruses or the pseudo viruses to infect cells;
applying sinusoidal voltage to the cell local attached membrane in the attached membrane clamp mode and detecting corresponding membrane current;
performing phase demodulation on corresponding membrane current signals by taking the phase of input sinusoidal voltage as a standard to obtain imaginary part signals and real part signals of the membrane current, and calibrating in real time;
detecting and calculating the membrane capacitance change caused by virus or pseudovirus infecting cells on the basis of calibration and calibration according to the current real part signal and the current imaginary part signal;
and identifying virus or pseudovirus infected cell events and modes thereof according to the dynamic characteristics and polarity of the capacitance change of the diaphragm, and estimating the scale of the virus or pseudovirus according to the size of the capacitance change of the diaphragm.
In addition, the method for detecting the virus or pseudovirus infected cells further comprises the following steps: on the basis of realizing the recording of the cell attached type patch clamp for achieving giant resistance electrical isolation inside and outside the electrode, a phase demodulation module is configured, and a signal generation function of the phase demodulation module is used for applying high-frequency sine wave voltage to the attached type patch and detecting corresponding membrane current.
The phase demodulation of the corresponding membrane current signal by taking the phase of the input sinusoidal voltage as a standard to obtain the imaginary part and the real part of the membrane current comprises the following steps: and detecting corresponding membrane current after voltage is applied to the cell membrane in an attached patch clamp mode, and carrying out phase demodulation on the membrane current to obtain a current real part signal and a current imaginary part signal of the membrane current.
Wherein detecting and calculating the change in membrane capacitance caused by infection of cells by viruses or pseudoviruses based on the real current signal and the imaginary current signal on a calibration and calibration basis comprises:
from the real and imaginary signals of the current, changes in the imaginary and real signals are directly related to changes in capacitance by calibration, a scaling factor K is determined by scaling, and the membrane capacitance change is calculated by the following calculation:
where Cv is the change in the cell membrane capacitance in the detection region, re is the change in the real current component signal, and Im is the change in the imaginary current component signal.
Wherein, the identification of virus or pseudovirus infected cell event and its mode according to the dynamic characteristics and polarity of the diaphragm capacitance change comprises: if the membrane capacitance is increased in a step mode, identifying the cell infection event in a mode of fusion of a virus or pseudovirus cyst membrane and a cell membrane; if the membrane capacitance step decreases, an invading cellular event is identified by endocytosis of the virion or pseudovirion. The scale of the virus or pseudovirus is estimated from the magnitude of the change in membrane capacitance, and the scale of the virus or pseudovirus (envelope area or endocytic envelope) is estimated from the ratio of the magnitude of the change in membrane capacitance to the capacitance per membrane area.
According to another aspect of the invention, there is provided a system for detecting infection of cells by a virus or pseudovirus.
The detection system for the virus or pseudovirus infected cells comprises:
the infection inducing unit is used for establishing a patch clamp mode so that viruses or pseudoviruses in electrode liquid can be combined with receptors on patches contained in the electrodes to induce the viruses or the pseudoviruses to infect cells;
a membrane current detection unit for applying a sinusoidal voltage to an attachment patch of a local cell in an attachment patch clamp mode and detecting a corresponding membrane current;
the phase demodulation unit is used for carrying out phase demodulation on corresponding membrane current signals by taking the phase of the input sinusoidal voltage as a standard to obtain imaginary part signals and real part signals of the membrane current, and carrying out real-time calibration and calibration;
the membrane capacitance calculation unit is used for detecting and calculating the membrane capacitance change caused by the infection of the cells by the virus or the pseudovirus according to the current real part signal and the current imaginary part signal on the basis of calibration and calibration;
and the event identification unit is used for identifying virus or pseudovirus infected cell events and modes thereof according to the dynamic characteristics and polarity of the diaphragm capacitance change and estimating the scale of the virus or pseudovirus according to the magnitude of the diaphragm capacitance change.
The membrane current detection unit is configured with a phase demodulation module and applies high-frequency sine wave voltage to the attached membrane by using the signal generation function of the phase demodulation module on the basis of realizing the recording of the cell attached membrane clamp for realizing the giant-resistance electrical isolation between the inside and the outside of the electrode and detecting the corresponding membrane current.
The phase demodulation unit is used for carrying out phase demodulation on corresponding membrane current signals by taking the phase of input sinusoidal voltage as a standard to obtain the imaginary part and the real part of the membrane current, detecting the corresponding membrane current after voltage is applied to a cell membrane in an attached patch clamp mode, and carrying out phase demodulation on the membrane current to obtain the real current part signals and the imaginary current part signals of the membrane current.
Wherein, when detecting and calculating the change of the membrane capacitance caused by virus or pseudovirus infecting cells based on the real current part signal and the imaginary current part signal, the membrane capacitance calculating unit makes the change of the imaginary part signal and the real part signal directly related to the change of the capacitance by calibration according to the real current part signal and the imaginary current part signal, determines a scale coefficient K by calibration, and calculates the change of the membrane capacitance by the following calculation formula:
where Cv is a change in the cell membrane capacitance of the detection region, re is a change in the signal of the real current component, and Im is a change in the signal of the imaginary current component.
When the event identification unit identifies virus or pseudovirus infected cell events and modes thereof according to the dynamic characteristics and the polarity of the capacitance change of the diaphragm, if the membrane capacitance step rises, the event is identified as the infected cell event in a mode of fusion of a virus or pseudovirus cyst membrane and a cell membrane; if the membrane capacitance step decreases, an invading cellular event is identified by endocytosis of the virion or pseudovirion. When the scale of the virus or the pseudovirus is estimated according to the size of the change of the membrane capacitance, the scale of the virus or the pseudovirus (the area of the cyst membrane or the endocytic envelope) is estimated according to the ratio of the size of the change of the membrane capacitance to the capacitance per membrane area.
Has the advantages that:
the invention uses an electrical method to realize the real-time monitoring of the dynamic process of wild and various mutant viruses or pseudoviruses infected cells under physiological conditions and pharmacological intervention conditions, and the precision of the dynamic process reaches the observation of single virus or pseudovirus infected cell events; the detection technology can also be used for a high-throughput large-scale rapid drug screening system by expanding a measurement channel and capacity, can also be used for direct and high-precision observation of virus or pseudovirus infected artificial membrane systems, cultured cell systems and living cells of human tissues, and has huge potential in virus prevention and treatment.
Compared with the electron microscope imaging method which can not realize the observation of living cells, the method can only complete the static detection of the cell or subcellular morphology at a certain time point, has the limitations of tedious and tedious processing process, easy pollution and damage of samples and the like, and compared with the high-resolution fluorescence microscopic imaging observation sample based on protein expression which is only limited to cultured cells and must carry out gene or protein modification and the like on viruses, the method has no time-space information and limited signal precision compared with the methods such as immunoblotting, nucleic acid detection and the like, and is difficult to accurately monitor the dynamic process of the virus infection living cells in real time. Is the observation method of the activity of virus infected living cells closest to the actual human pathological conditions.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 is a schematic flow chart of a method for detecting pseudoneocoronaviruse-infected cells according to an embodiment of the present invention;
FIG. 2 is a structural view of a detection system for pseudoneocoronaviruses-infected cells according to an embodiment of the present invention;
FIG. 3 is a flow chart of the detection of a single pseudoneocoronaviral infection event in an ACE2/TMPRSS2 expressing cell line according to an embodiment of the present invention;
FIG. 4 is a flowchart of the detection of single pseudoneocoronaviruse infection events in the transfection of ACE2 and TMPRSS2 by HEK293T, coca-2 and other cell lines according to the embodiment of the present invention;
FIG. 5 is a flowchart of a single pseudoneocoronaviral infection event detection of alveolar cells in human lung tissue, according to an embodiment of the present invention.
Detailed Description
The technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments that can be derived by one of ordinary skill in the art from the embodiments given herein are intended to be within the scope of the present invention.
According to embodiments of the present invention, a method and system for detecting infection of HEK293T cells expressing ACE2 and TMPRSS2 proteins by pseudoneocoronavirus is provided.
As shown in fig. 1, a method for detecting infection of cells by pseudoneocoronaviruses according to an embodiment of the present invention includes:
s101, establishing an attached patch clamp mode to enable pseudo-new coronavirus in electrode internal liquid to be combined with a receptor on a patch contained in an electrode to induce the pseudo-new coronavirus to infect cells;
step S103, applying input sinusoidal voltage to a cell local attached patch in an attached patch clamp mode and detecting corresponding membrane current;
step S105, phase discrimination demodulation is carried out on corresponding membrane current signals by taking the phase of the input sinusoidal voltage as a standard, so as to obtain the imaginary part and the real part of the membrane current, and real-time calibration and calibration are carried out;
step S107, detecting and calculating the membrane capacitance change caused by the infection of the cells by the pseudoneocoronavirus on the basis of calibration and calibration according to the current real part signal and the current imaginary part signal;
and step S109, identifying the cell infection event and the mode of the pseudo-new coronavirus according to the dynamic characteristics and the polarity of the capacitance change of the diaphragm, and estimating the scale of the pseudo-new coronavirus according to the capacitance change of the diaphragm.
In one embodiment, the method for detecting infection of cells by pseudoneocoronaviruses further comprises: by configuring a phase demodulation module, sinusoidal voltage is applied to a diaphragm contained in an electrode by utilizing the signal generation function of a phase-locked amplifier; recording a membrane current induced by applying voltage to a membrane in an attached membrane clamp mode, and performing phase discrimination demodulation on a corresponding membrane current signal by using the phase discrimination function of a phase-locked amplifier and taking the phase of input sinusoidal voltage as a standard to obtain an imaginary part and a real part of the membrane current;
in one embodiment, the changes in the imaginary and real signals are directly related to the capacitance change by calibration, the scaling factor K is determined by scaling, and the membrane capacitance change is calculated from the following calculation:
where Cv is a change in the cell membrane capacitance of the detection region, re is a change in the signal of the real part of current, and Im is a change in the signal of the imaginary part of current.
In one embodiment, the virus or pseudovirus infection event identification and analysis module identifies pseudoneocoronaviruses infected cell events and their modes according to the dynamic characteristics and polarity of the capacitance change of the patch, and estimates the scale of the pseudoneocoronaviruses according to the magnitude of the capacitance change of the patch, including: if the membrane capacitance step rises, identifying the cell infection event in a pseudo-new coronavirus cyst membrane and cell membrane fusion mode; if the membrane capacitance step decreases, an invading cellular event in the manner of endocytosis of pseudoneocoronavirus particles is identified. The pseudoneocoronavirus (envelope area or endocytic envelope) scale was estimated from the ratio of the magnitude of the change in membrane capacitance to the capacitance per membrane area.
In order to facilitate understanding of the above-described technical solutions of the present invention, the above-described technical solutions of the present invention are described in detail below through specific application descriptions and experiments.
In an example of detecting infection of cultured cells expressing the receptor protein ACE2 and the helper protein TMPRSS2 by pseudoneocoronaviruses, the experimental procedure for detecting infection of cultured cells by pseudoneocoronaviruses is shown in FIG. 3: the novel coronavirus receptor protein ACE2 and the accessory protein TMPRSS2 are expressed on HEK293T cells as candidate host cells. In the experiment, candidate host cells (groups) are transferred into a recording groove containing extracellular fluid in a patch clamp electrophysiological platform.
The extracellular fluid comprises the following formula:
| composition (I) | g/L |
| NaCl | 9 |
| KCl | 0.4 |
| MgCl2 | 0.5 |
| CaCl2 | 0.3 |
| HEPES | 2.5 |
| D-Glucose | 2 |
| Distilled water | Adding 1000ml of the solution, and adjusting the pH to 7.4 |
The detection activities were performed in cell-attached patch clamp mode: the glass microelectrode contains a test solution of pseudo-new coronavirus, the diameter of the tip is about 1 mu m, the glass microelectrode is clamped in a probe of a conventional electrophysiological amplifier, negative pressure airflow is applied to tightly seal the tip of the glass microelectrode with the surface of a cell membrane, and the giant resistance electrical isolation inside and outside the electrode is realized so as to greatly reduce background noise and achieve high signal-to-noise ratio measurement. The detection system for the cells infected by the pseudoneocoronaviruses is shown in fig. 2 and comprises an electrophysiological signal acquisition module, a phase discrimination demodulation module, a phase real-time calibration module, a capacitance signal calibration module, a membrane capacitance detection module and a virus or pseudovirus infected cell event identification and analysis module. The method specifically comprises the following steps:
the electrophysiological signal acquisition module: the cell attaching type patch clamp which takes a patch clamp amplifier as a main body and achieves giant resistance electrical isolation inside and outside an electrode is recorded, and attached patch current is collected in a high-frequency sine wave voltage clamp mode of direct current level modulation and is output to a phase demodulation module after high-gain amplification.
The phase demodulation module: outputting high-frequency sine wave (non-radio frequency sine wave above 5 kHz) voltage serving as a modulation signal to an electrophysiological signal acquisition module, and performing phase discrimination demodulation on a current signal input from the electrophysiological signal acquisition module by taking the modulation signal as a phase-locked standard to obtain an imaginary part and a real part of the signal;
the phase real-time calibration module: the real-time compensation method is used for realizing the real-time compensation of the distributed conductance and the capacitance of the system, so that the change of imaginary part and real part signals is directly related to the change of membrane capacitance, and the optimal signal-to-noise ratio is kept;
a membrane capacitance signal scaling module: for scaling the detected membrane capacitance signal. The scaling factor K is determined by scaling.
A membrane capacitance detection module: the high-speed analog/digital converter is used for carrying out analog-digital conversion on imaginary part signals and real part signals obtained by phase demodulation on the attached diaphragm current signals output by the phase demodulation module, software and hardware are combined to realize attached diaphragm capacitance and conductance signal detection based on real-time phase calibration and signal calibration, and corresponding information is stored in a computer.
A virus or pseudovirus infected cell event recognition and analysis module: and (4) post-processing the attached membrane capacitance and the conductance signal stored in a computer by software. Identifying a pseudo-new coronavirus infected cell event and a mode thereof according to the dynamic characteristics and polarity of the capacitance change of the diaphragm, and identifying the cell event infected by the pseudo-new coronavirus in a fusion mode of a cyst membrane and a cell membrane if the membrane capacitance is increased in a step manner; if the membrane capacitance step decreases, an invasive cell event in the form of pseudoneocoronaviral particle endocytosis is identified. When the scale of the pseudoneocoronaviruses is estimated according to the magnitude of the change in the membrane capacitance, the scale of the pseudoneocoronaviruses (envelope area or endocytic envelope) is estimated according to the ratio of the magnitude of the change in the membrane capacitance to the capacitance per membrane area. Therefore, the size, infection mode, kinetic process and time sequence characteristics of the pseudoneocoronavirus are analyzed.
When the detection system is specifically applied to different scenes, taking the pseudo-new coronavirus as an example, the experimental process for detecting the cells infected by the pseudo-new coronavirus by using the detection system provided by the invention is as follows:
1) Artificial cell membrane
As shown in FIG. 3, the event of infecting cells with pseudoneocoronaviruses was measured by a cell-attached lower membrane capacitance measurement technique for liposomes embedded in ACE2/TMPRSS2 membrane proteins.
2) Culturing cell lines
As shown in FIG. 4, ACE2 and TMPRSS2 were transfected in HEK293T, coca-2 and other cell lines, and the cell invasion event of pseudoneocoronaviruses was detected by the membrane-attached lower capacitance measurement technique.
Preparing a human lung tissue biopsy, and detecting infection of the pseudoneocoronavirus.
As shown in fig. 5, the living pathological tissue excised from the lung of the patient by surgery is used as a sample, the sample is prepared into a living lung tissue section with the thickness of 150-400 microns by using a vibrating microtome, and then the cell attaching type membrane capacitance detection is carried out on the alveolar epithelial cells, and the single pseudoneocoronaviruse infected cell event is detected by using the membrane capacitance measurement technology.
Therefore, by means of the technical scheme, the invention realizes real-time detection on the infected host cells of the pseudoneocoronaviruses in a cyst membrane and cell membrane fusion mode and a pseudoneocoronaviruse particle endocytosis mode at the levels of artificial cell membranes, isolated cells, cultured cells and living tissues of animals and humans by an electric method, and the precision of the detection reaches the observation of a single pseudoneocoronaviruse infected cell event.
The detection technology can also be used for direct and high-precision observation of other viruses or pseudoviruses infecting an artificial membrane system, a cultured cell system and living cells of human tissues, can also be used for high-flux large-scale rapid drug screening by expanding a measurement channel and capacity, and has huge potential in virus prevention and treatment.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and should not be taken as limiting the scope of the present invention, which is intended to cover any modifications, equivalents, improvements, etc. within the spirit and scope of the present invention.
Claims (10)
1. A method for detecting infection of a cell by a virus or pseudovirus, comprising:
establishing an attached patch clamp mode to ensure that viruses or pseudo viruses in the electrode internal liquid can be combined with receptors on patches contained in the electrodes to induce the viruses or the pseudo viruses to infect cells;
applying sine voltage to a local attached patch of a cell in an attached patch clamp mode and detecting corresponding membrane current;
performing phase demodulation on corresponding membrane current signals by taking the phase of input sinusoidal voltage as a standard to obtain imaginary part signals and real part signals of the membrane current, and calibrating in real time;
detecting and calculating the membrane capacitance change caused by virus or pseudovirus infecting cells on the basis of calibration and calibration according to the current real part signal and the current imaginary part signal;
and identifying virus or pseudovirus infected cell events and modes thereof according to the dynamic characteristics and polarity of the capacitance change of the diaphragm, and estimating the scale of the virus or pseudovirus according to the size of the capacitance change of the diaphragm.
2. The method of claim 1, further comprising: on the basis of realizing the recording of the cell attached type patch clamp for achieving giant resistance electrical isolation inside and outside the electrode, a phase demodulation module is configured, and a signal generation function of the phase demodulation module is used for applying high-frequency sine wave voltage to the attached type patch and detecting corresponding membrane current.
3. The method of claim 1, wherein the phase demodulation of the corresponding membrane current signal using the phase of the input sinusoidal voltage as a standard to obtain the imaginary and real parts of the membrane current comprises: and detecting corresponding membrane current after voltage is applied to the cell membrane in an attached patch clamp mode, and carrying out phase demodulation on the membrane current to obtain a current real part signal and a current imaginary part signal of the membrane current.
4. The method of claim 1, wherein detecting and calculating changes in membrane capacitance caused by infection of cells by a virus or pseudovirus based on the real current signal and the imaginary current signal on a calibration and calibration basis comprises:
from the real and imaginary signals of the current, changes in the imaginary and real signals are directly related to changes in capacitance by calibration, a scaling factor K is determined by scaling, and the membrane capacitance change is calculated by the following calculation:
wherein Cv is the change of the cell membrane capacitance of the detection area, re is the change of the current real part signal, and Im is the change of the current imaginary part signal.
5. The method for detecting virus or pseudovirus infected cells according to claim 1, wherein the step of identifying the virus or pseudovirus infected cell event and the manner thereof based on the kinetic characteristics and polarity of the capacitance change of the patch and estimating the scale of the virus or pseudovirus based on the magnitude of the capacitance change of the patch comprises:
if the membrane capacitance is increased in a step mode, identifying the cell infection event in a mode of fusion of a virus or pseudovirus cyst membrane and a cell membrane;
if the membrane capacitance step decreases, identifying an invading cell event in the manner of endocytosis of the virion or pseudovirion;
the scale of the virus or pseudovirus (envelope area or endocytic envelope) is estimated from the ratio of the magnitude of the change in membrane capacitance to the capacitance per membrane area.
6. A system for detecting infection of a cell by a virus or pseudovirus, comprising:
the infection inducing unit is used for establishing an attached patch clamp mode so that viruses or pseudo viruses in electrode internal liquid can be combined with receptors on patches contained in the electrodes to induce the viruses or the pseudo viruses to infect cells;
a membrane current detection unit for applying a sinusoidal voltage to an attachment patch of a local cell in an attachment patch clamp mode and detecting a corresponding membrane current;
the phase demodulation unit is used for carrying out phase demodulation on corresponding membrane current signals by taking the phase of the input sinusoidal voltage as a standard to obtain imaginary part signals and real part signals of the membrane current, and carrying out real-time calibration and calibration;
the membrane capacitance calculation unit is used for detecting and calculating the membrane capacitance change caused by the infection of the cells by the virus or the pseudovirus according to the current real part signal and the current imaginary part signal on the basis of calibration and calibration;
and the event identification unit is used for identifying virus or pseudovirus infected cell events and modes thereof according to the dynamic characteristics and polarity of the diaphragm capacitance change and estimating the scale of the virus or pseudovirus according to the magnitude of the diaphragm capacitance change.
7. The system of claim 6, wherein the membrane current detection unit is configured with a phase demodulation module and applies a high-frequency sine wave voltage to the attached membrane by using the signal generation function of the phase demodulation module on the basis of cell attached membrane clamp record that the inside and outside of the electrode achieve giant resistance electrical isolation, and detects the corresponding membrane current.
8. The system of claim 6, wherein the phase demodulation unit detects the corresponding membrane current after the voltage is applied to the cell membrane in the patch clamp mode, and performs phase demodulation on the membrane current to obtain the real current signal and the imaginary current signal of the membrane current, when the phase of the input sinusoidal voltage is used as a standard to perform phase demodulation on the corresponding membrane current signal to obtain the imaginary part and the real part of the membrane current.
9. The system for detecting virus-or pseudovirus-infected cells according to claim 6, wherein the membrane capacitance calculating unit calculates the membrane capacitance change by the following calculation formula by calibrating the change of the imaginary signal and the real signal to the capacitance change according to the real and imaginary current signals, and determining the scale factor K by calibration, when detecting and calculating the change of the membrane capacitance caused by virus-or pseudovirus-infected cells based on the real and imaginary current signals and the calibration and calibration:
wherein Cv is the change of the cell membrane capacitance of the detection area, re is the change of the current real part signal, and Im is the change of the current imaginary part signal.
10. A system for detecting infection of cells by a virus or pseudovirus according to claim 6, wherein: when the event identification unit identifies virus or pseudovirus infected cell events and modes thereof according to the dynamic characteristics and the polarity of the capacitance change of the diaphragm, if the membrane capacitance step rises, the event is identified as the infected cell event in a mode of fusion of the envelope and the cell membrane of the virus or pseudovirus; if the membrane capacitance is decreased in a step, an event infecting cells is identified as the endocytosis mode of the virus particles or the pseudovirus particles, and when the scale of the virus or the pseudovirus is estimated according to the size of the change of the membrane capacitance, the scale of the virus or the pseudovirus (the area of the envelope of the cyst membrane or the endocytic envelope) is estimated according to the ratio of the size of the change of the membrane capacitance to the capacitance of the unit membrane area.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211362714.8A CN115856271A (en) | 2022-11-02 | 2022-11-02 | Method and system for detecting virus or pseudovirus infected cells |
| PCT/CN2022/133108 WO2024092891A1 (en) | 2022-11-02 | 2022-11-21 | Method and system for detecting virus or pseudovirus infecting cell |
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| CN202211362714.8A CN115856271A (en) | 2022-11-02 | 2022-11-02 | Method and system for detecting virus or pseudovirus infected cells |
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| US20110294155A1 (en) * | 2008-11-27 | 2011-12-01 | Howard Florey Institute | Modulation of an ion channel or receptor |
| US9057734B2 (en) * | 2010-08-23 | 2015-06-16 | President And Fellows Of Harvard College | Optogenetic probes for measuring membrane potential |
| CN106033069B (en) * | 2015-03-10 | 2019-05-28 | 中国科学院生物物理研究所 | Single Vesicle fusion and recycling and its patch-clamp detection device coupled with calcium channel |
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