阅读说明：本技术 一种活体单细胞阻抗快速检测方法、系统、装置及介质 (Method, system, device and medium for rapidly detecting impedance of living single cell ) 是由 朱滨 邹丽丽 伊翔 于 2021-07-26 设计创作，主要内容包括：本发明提供的一种活体单细胞阻抗快速检测方法、系统、装置及介质,方法包括以下步骤：施加正弦交流激励,通过氧化铟锡电极从包含有活细胞和死细胞的细胞悬液与高渗溶液的混合溶液中获取细胞阻抗变化信号；将细胞阻抗变化信号通过I/V转化得到第一中间信号,将第一中间信号进行选频滤波得到第二中间信号；将第二中间信号与参考信号进行锁相放大处理,得到被锁相处理的细胞阻抗信号；根据细胞阻抗信号形成数据文件,根据数据文件获得活体单细胞的数量,确定细胞的存活率；方法有效获得微弱的细胞阻抗检测信号,提高了信噪比,有利于更准确快速地自动检测细胞数量和存活率,可广泛应用于生物医学检测预测技术领域。(The invention provides a method, a system, a device and a medium for rapidly detecting the impedance of a living single cell, wherein the method comprises the following steps: applying sinusoidal alternating current excitation, and acquiring a cell impedance change signal from a mixed solution of a cell suspension containing live cells and dead cells and a hypertonic solution through an indium tin oxide electrode; carrying out I/V conversion on the cell impedance change signal to obtain a first intermediate signal, and carrying out frequency-selective filtering on the first intermediate signal to obtain a second intermediate signal; performing phase-locked amplification processing on the second intermediate signal and the reference signal to obtain a cell impedance signal subjected to phase-locked processing; forming a data file according to the cell impedance signal, obtaining the number of living single cells according to the data file, and determining the survival rate of the cells; the method effectively obtains weak cell impedance detection signals, improves the signal-to-noise ratio, is beneficial to more accurately and rapidly automatically detecting the cell number and the survival rate, and can be widely applied to the technical field of biomedical detection and prediction.)

1. A method for rapidly detecting the impedance of a living single cell is characterized by comprising the following steps:

applying sinusoidal alternating current excitation, and acquiring a cell impedance change signal from a mixed solution of a cell suspension containing live cells and dead cells and a hypertonic solution through an indium tin oxide electrode;

carrying out I/V conversion on the cell impedance change signal to obtain a first intermediate signal, and carrying out frequency-selective filtering on the first intermediate signal to obtain a second intermediate signal;

performing phase locking processing on the second intermediate signal and a reference signal to obtain a cell impedance signal subjected to phase locking processing;

and forming a data file according to the cell impedance signal, obtaining the number of living single cells according to the data file, and determining the survival rate of the cells.

2. The method as claimed in claim 1, wherein the step of obtaining the first intermediate signal by I/V converting the cell impedance variation signal, and obtaining the second intermediate signal by frequency-selective filtering the first intermediate signal comprises:

and converting the cell impedance change signal into a voltage signal through a trans-impedance amplifier to obtain the first intermediate signal.

3. The method as claimed in claim 1, wherein the step of obtaining the first intermediate signal by I/V converting the cell impedance variation signal, and obtaining the second intermediate signal by frequency-selective filtering the first intermediate signal further comprises:

and carrying out frequency-selecting filtering on the first intermediate signal through a band-pass filter to obtain the second intermediate signal.

4. The method as claimed in claim 1, wherein the step of performing phase-locking processing on the second intermediate signal and a reference signal to obtain a cell impedance signal subjected to phase-locking processing comprises:

lock-in amplifying the second intermediate signal with the reference signal to lock the second intermediate signal in a frequency range of the reference signal based on synchronous phase-sensitive detection;

and filtering out signal components which are different in frequency from the reference signal in the amplified signal through low-pass filtering, so as to obtain the cell impedance signal which is the same in frequency as the reference signal.

5. The method as claimed in claim 1, wherein the step of forming a data file according to the cell impedance signal, obtaining the number of living single cells according to the data file, and determining the survival rate of the cells comprises:

and performing wavelet analysis denoising and multi-scale peak position detection on the cell impedance signals in the data file, extracting to obtain a pulse peak position, and determining the number of living cells and the cell survival rate according to the pulse peak value of the pulse peak position.

6. The method for rapid impedance detection of single cells in vivo according to any one of claims 1-5, wherein the method further comprises the steps of:

and visually displaying the number of the living single cells and the survival rate of the cells.

7. A system for rapidly detecting the impedance of a single cell in a living body, which is characterized by comprising:

the microfluidic pump is used for respectively injecting a cell solution containing live cells and dead cells and a hypertonic solution into a channel of the microfluidic chip;

a signal generator for providing a sinusoidal ac excitation and a reference signal;

the micro-fluidic chip is used for acquiring cell impedance change from a mixed solution of a cell suspension containing live cells and dead cells and a hypertonic solution by applying sinusoidal alternating current excitation;

the cell impedance phase-locking detection module is used for receiving the cell impedance change signal, converting the cell impedance change signal into a first voltage signal through I/V (input/output) conversion, and performing frequency-selective filtering on the first voltage signal to obtain a second voltage signal;

performing phase locking processing on the second voltage signal and a reference signal to obtain a cell impedance signal subjected to phase locking processing;

the data acquisition card is used for acquiring the cell impedance signals and forming the cell impedance signals into data files through LabVIEW software;

and the MATLAB processing module is used for acquiring and storing the data file, determining the number of living single cells from the data file, determining the survival rate of the cells and performing visual display.

8. The system as claimed in claim 2, wherein the cell impedance phase-locked detection module comprises a preamplifier unit, a band-pass filtering and amplifying unit, a phase-locked amplifier unit, a low-pass filtering unit and an amplifier;

the output end of the preamplifier unit is connected to the input end of the band-pass filtering amplification unit, the output end of the band-pass filtering amplification unit is connected to the input end of the phase-locked amplifier unit, the output end of the phase-locked amplifier unit is connected to the input end of the low-pass filtering unit, and the output end of the low-pass filtering unit is connected to the input end of the amplifier.

9. A living body single cell impedance rapid detection device is characterized by comprising:

at least one processor;

at least one memory for storing at least one program;

when the at least one program is executed by the at least one processor, the at least one processor is caused to execute a method for rapid detection of impedance of a single cell in a living body according to any one of claims 1 to 7.

10. A storage medium for fast impedance detection of single cells in a living body, wherein a program executable by a processor is stored, and when the program is executed by the processor, the program is used for executing a method for fast impedance detection of single cells in a living body according to any one of claims 1 to 7.

Technical Field

The invention relates to the technical field of biomedical detection, in particular to a method, a system, a device and a medium for rapidly detecting impedance of a living body single cell.

Background

Cell survival rate is an important index for evaluating cell state and monitoring diseases, and is widely applied to the field of biological and medical research and detection.

At present, the difference between dead cells and living cells is reflected in the difference of physiological functions and properties, and in the common method for detecting the cell survival rate, although the staining method is simple, the staining method wastes time and labor, the precision is uncertain, and the cells are easy to pollute so as to be not beneficial to continuous use; although the flow method has high accuracy, the detection apparatus is expensive, and the labeled cells cannot be used continuously.

The cell impedance measurement is label-free, fast and non-invasive and can be quantitatively analyzed, especially on a chip. In hypertonic solutions, living cells have osmotic capacity and the volume of living cells is reduced while the volume of dead cells is unchanged. By utilizing the characteristic, cell suspension containing live cells and dead cells is mixed with hypertonic solution and is injected into a channel of the microfluidic chip through a microfluidic pump. Because the living cells shrink under the stimulation of hypertonic solution, and the dead cell volume is unchanged. The dead cells have unchanged electrical properties between the channel detection well electrodes due to unchanged volume. When the volume of the living cells changes to cause the impedance between the detection holes of the connecting channel to change, the electrodes connected with the detection holes of the channel detect pulse signals generated by the electrodes and transmit the pulse signals to the cell impedance detection circuit. The cell impedance signal is detected and the data of the signal is analyzed to obtain the number of living cells in the cell solution, so that the survival rate of the cells is determined.

Because the signal detected by the cell impedance is extremely weak, the outside brings large noise interference. Making detection of weak cell impedance signals very difficult in the presence of large noise.

Disclosure of Invention

In view of the above, to at least partially solve one of the above technical problems, embodiments of the present invention provide a method for rapidly detecting impedance of a single cell in a living body with higher accuracy, and a system, an apparatus and a computer-readable storage medium for implementing the method.

In a first aspect, the present application provides a method for rapidly detecting impedance of a single cell in a living body, comprising the steps of:

applying sinusoidal alternating current excitation, and acquiring a cell impedance change signal from a mixed solution of a cell suspension containing live cells and dead cells and a hypertonic solution through an indium tin oxide electrode;

carrying out I/V conversion on the cell impedance change signal to obtain a first intermediate signal, and carrying out frequency-selective filtering on the first intermediate signal to obtain a second intermediate signal;

performing phase locking processing on the second intermediate signal and a reference signal to obtain a cell impedance signal subjected to phase locking processing;

and forming a data file according to the cell impedance signal, obtaining the number of living single cells according to the data file, and determining the survival rate of the cells.

In a possible embodiment of the present disclosure, the step of obtaining a first intermediate signal by I/V converting the cell impedance change signal, and obtaining a second intermediate signal by frequency-selective filtering the first intermediate signal includes:

and converting the cell impedance change signal into a voltage signal through a trans-impedance amplifier to obtain the first intermediate signal.

In a possible embodiment of the present disclosure, the step of obtaining a first intermediate signal by I/V converting the cell impedance change signal, and obtaining a second intermediate signal by frequency-selective filtering the first intermediate signal further includes:

and carrying out frequency-selecting filtering on the first intermediate signal through a band-pass filter to obtain the second intermediate signal.

In a possible embodiment of the present disclosure, the step of performing a phase-locking process on the second intermediate signal and a reference signal to obtain a cell impedance signal subjected to the phase-locking process includes:

based on synchronous phase-sensitive detection, performing phase-locked amplification on the second intermediate signal and the reference signal, and locking the second intermediate signal in a reference frequency range;

and filtering out signal components with different frequencies from the reference signal in the signals subjected to the phase locking processing through low-pass filtering to obtain the cell impedance signals with the same frequencies as the reference signal.

In a possible embodiment of the present disclosure, the step of forming a data file according to the cell impedance signal, obtaining the number of living single cells according to the data file, and determining the survival rate of the cells includes:

and performing wavelet analysis denoising and multi-scale peak position detection on the cell impedance signals in the data file, extracting to obtain a pulse peak position, and determining the number of living cells and the cell survival rate according to the pulse peak value of the pulse peak position.

In a possible embodiment of the present disclosure, the detection method further includes the following steps: and visually displaying the number of the living single cells and the survival rate of the cells.

In a second aspect, the present invention further provides a system for rapidly detecting impedance of a living single cell, comprising:

the microfluidic pump is used for respectively injecting a cell solution containing live cells and dead cells and a hypertonic solution into a channel of the microfluidic chip;

a signal generator for providing a sinusoidal ac excitation and a reference signal;

the micro-fluidic chip is used for acquiring cell impedance change from a mixed solution of a cell suspension containing live cells and dead cells and a hypertonic solution by applying sinusoidal alternating current excitation;

the cell impedance phase-locking detection module is used for receiving the cell impedance change signal, converting the cell impedance change signal into a first voltage signal through I/V (input/output) conversion, and performing frequency-selective filtering on the first voltage signal to obtain a second voltage signal; performing phase locking processing on the second voltage signal and a reference signal to obtain a cell impedance signal subjected to phase locking processing;

the data acquisition card is used for acquiring the cell impedance signals and forming the cell impedance signals into data files through LabVIEW software;

and the MATLAB processing module is used for acquiring and storing the data file, determining the number of living single cells from the data file, determining the survival rate of the cells and performing visual display.

In a possible embodiment of the present disclosure, the cell impedance phase-locked detection module in the detection system includes a preamplifier unit, a band-pass filtering and amplifying unit, a phase-locked amplifier unit, a low-pass filtering unit, and an amplifier;

the output end of the preamplifier unit is connected to the input end of the band-pass filtering amplification unit, the output end of the band-pass filtering amplification unit is connected to the input end of the phase-locked amplifier unit, the output end of the phase-locked amplifier unit is connected to the input end of the low-pass filtering unit, and the output end of the low-pass filtering unit is connected to the input end of the amplifier.

In a third aspect, the present invention further provides a device for rapidly detecting impedance of a living single cell, comprising:

at least one processor;

at least one memory for storing at least one program;

when the at least one program is executed by the at least one processor, the at least one processor is enabled to execute the method for rapid detection of impedance of a living single cell in the first aspect.

In a fourth aspect, the present invention also provides a storage medium, in which a processor-executable program is stored, and the processor-executable program is used for executing the method in the first aspect when being executed by a processor.

Advantages and benefits of the present invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention:

according to the technical scheme, weak cell impedance signals to be detected can be effectively extracted from noise through frequency mixing phase locking processing, and cell impedance detection signals with high signal-to-noise ratio are obtained through further conditioning the cell impedance signals; then, the parameters of the living body number, the survival rate and the like of the cells are determined through later-stage data processing; the scheme can effectively obtain weak cell impedance detection signals, improves the signal-to-noise ratio, and is favorable for more accurately and rapidly automatically detecting the cell number and the survival rate.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.

FIG. 1 is a schematic structural diagram of a system for rapid impedance detection of single cells in a living body according to an embodiment of the present invention;

FIG. 2 is a schematic structural diagram of a cell impedance phase-locking detection module according to an embodiment of the present invention;

FIG. 3 is a flowchart illustrating a method for rapidly detecting impedance of a single cell in a living body according to an embodiment of the present invention;

FIG. 4 is a schematic circuit diagram of a preamplifier unit according to an embodiment of the invention;

FIG. 5 is a schematic circuit diagram of a band-pass filtering amplifying unit according to an embodiment of the present invention;

FIG. 6 is a schematic circuit diagram of a lock-in amplifier unit according to an embodiment of the present invention;

fig. 7 is a schematic data processing flow diagram based on LabVIEW data acquisition processing software in the embodiment of the present invention.

Detailed Description

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the accompanying drawings are illustrative only for the purpose of explaining the present invention, and are not to be construed as limiting the present invention. The step numbers in the following embodiments are provided only for convenience of illustration, the order between the steps is not limited at all, and the execution order of each step in the embodiments can be adapted according to the understanding of those skilled in the art.

The scheme of the application aims to solve the problem that living single cells can be judged quickly and accurately when the number of the cells and the survival rate are detected, and provides a scheme concept of quick detection of living single cell impedance; the scheme can adopt a synchronous phase-sensitive detection technology, weak cell impedance signals to be detected can be effectively extracted from noise through frequency-mixing phase-locking processing of an LIA (lock-in-amplifier), and cell impedance signals are further conditioned, so that cell impedance detection signals with high signal-to-noise ratio are obtained, the signal-to-noise ratio of the signals is improved, and the cell quantity and the survival rate can be automatically detected more accurately and rapidly.

Based on the above theoretical basis, in a first aspect, the application provides a living body single cell impedance rapid detection system, which mainly comprises the following functional modules:

the two micro-flow pumps are used for respectively injecting cell solution containing live cells and dead cells and hypertonic solution into the channels of the micro-fluidic chip;

a signal generator for providing a sinusoidal ac excitation and a reference signal;

the micro-fluidic chip is used for acquiring cell impedance change from a mixed solution of a cell suspension containing live cells and dead cells and a hypertonic solution by applying sinusoidal alternating current excitation;

the cell impedance phase-locking detection module is used for receiving the cell impedance change signal, converting the cell impedance change signal into a first voltage signal through I/V (input/output) conversion, and performing frequency-selective filtering on the first voltage signal to obtain a second voltage signal; performing phase locking processing on the second voltage signal and the reference signal to obtain a cell impedance signal subjected to phase locking processing;

the data acquisition card is used for acquiring cell impedance signals and forming the cell impedance signals into a data file in a TDMS format through LabVIEW software;

and the MATLAB processing module is used for acquiring and storing data files, determining the number of living single cells from the data files, determining the survival rate of the cells and carrying out visual display.

Specifically, as shown in fig. 1, in the single-cell rapid detection system based on the impedance detection technology provided in this embodiment, the cell impedance phase-locked detection device detects a cell impedance change signal passing through the detection hole of the microfluidic chip channel through the ITO detection electrode, and the cell impedance phase-locked detection device detects and outputs cell impedance signal data processed by phase locking; collecting Data through a Data Acquisition card (DAQ) according to Data collection conditions set by the LabVIEW, transmitting the Data to a LabVIEW software Data collection processing unit, and displaying and generating TDMS Data; and then obtaining TDMS data generated by a LabVIEW software data acquisition and processing unit through an MATLAB data processing terminal, and performing later-stage wavelet denoising and peak searching processing on the data to obtain the number of living single cells, thereby determining the survival rate of the cells.

In some possible embodiments, embodiments provide a single cell rapid detection system, as shown in fig. 2, the cell impedance phase-locked detection module includes a preamplifier unit (TIA unit), a band-pass filtering and amplifying unit, a phase-locked amplifier unit (LIA unit), a low-pass filtering unit, and an amplifier;

the preamplifier unit in the module is a trans-impedance amplifier (TIA) unit and is used for converting a cell impedance change signal obtained by an ITO (indium tin oxide) detection electrode connected with a detection hole of a microfluidic chip channel into a voltage signal. A Lock-in Amplifier (LIA) unit is connected with the band-pass filtering amplification unit; the band-pass filtering amplification unit obtains the voltage value output from the TIA unit, performs frequency-selective amplification on the voltage value, suppresses noise except the center frequency and outputs the noise to the LIA unit. The band-pass filtering amplification unit is connected with a synchronous phase-sensitive detection lock-in amplifier (namely an LIA unit), and a low-pass filtering unit is connected behind the LIA unit. The phase-locked amplifier unit is based on a synchronous phase-sensitive detection technology and is used for separating weak impedance detection signals from noise and transmitting the signals to the low-pass filtering unit. The low-pass filtering unit in the module performs low-pass filtering on the cell impedance detection signal extracted by the phase-locked amplifier to filter high-frequency components, so as to obtain a signal with a high signal-to-noise ratio; finally, the amplifier unit in the module finally amplifies and outputs the cell impedance detection signal processed by the phase-locked amplification and low-pass filtering unit.

It can be understood that the system of the embodiment may further be provided with a power supply unit for providing a matched power supply for each unit of the system. Specifically, in an embodiment, the power supply unit includes a power charging module, a power management module, and an ldo (low drop out regulator) power output module, which are connected in sequence.

Based on the living single cell impedance rapid detection system provided in the first aspect, in a second aspect, as shown in fig. 3, the technical solution of the present application further provides a living single cell impedance rapid detection method, which includes steps S100-S400:

s100, applying sinusoidal alternating current excitation, and obtaining a cell impedance change signal from a mixed solution of a cell suspension containing live cells and dead cells and a hypertonic solution through an indium tin oxide electrode;

specifically, in the embodiment system, a cell solution containing live cells and dead cells is mixed with a hypertonic solution through a micro-flow pump, and the volume of the cell solution is reduced because the live cells shrink under the action of the hypertonic solution; at the same time, the dead cells are not changed in volume because they have lost activity. By detecting the volume of the cells, the number of live cells and dead cells can be effectively distinguished. In the system of the embodiment, the microfluidic chip channel is provided with detection holes, ITO (indium tin oxide) detection electrodes are arranged and connected on two sides of the detection holes, and when a cell passes through the detection holes between the electrodes, the impedance between the electrodes changes. A sinusoidal (f 450kHz) ac excitation is applied to the detection electrodes to generate a pulsed signal of varying cell impedance. The system is connected with two ends of a channel detection hole of the microfluidic chip through an ITO detection electrode, and when cells pass through the detection hole of the microfluidic chip channel, the cells are transmitted to a cell impedance phase-locking detection module through a radio frequency interface. And carrying out data acquisition processing on the impedance change condition between the detection electrodes through subsequent processing steps so as to determine the number of living single cells and the cell survival rate.

S200, obtaining a first intermediate signal by I/V conversion (converting a current signal into a voltage signal) of the cell impedance change signal, and performing frequency-selective filtering on the first intermediate signal to obtain a second intermediate signal;

specifically, the cell impedance phase-locked detection module in the embodiment converts a current signal into a voltage signal through a low-noise preamplifier, reduces signal noise, improves the signal-to-noise ratio of the signal, and converts a cell impedance change signal detected by an ITO detection electrode and passing through a microfluidic chip channel detection hole into a voltage value, namely a first intermediate signal; and then, a cell impedance signal output from the TIA unit is obtained through the band-pass filtering amplification unit, and frequency-selective filtering amplification is carried out on the cell impedance signal, so that noise outside the central frequency is suppressed, and the signal data after filtering and denoising, namely a second intermediate signal, is output to the LIA unit.

S300, performing phase locking processing on the second intermediate signal and the reference signal to obtain a cell impedance signal subjected to phase locking processing;

specifically, because the cell volume is very small, the detection circuit is very easily interfered by the surrounding environment, which causes great noise even exceeding the signal of the cell itself, and it is difficult to effectively distinguish the information of the real cell. Therefore, the cell impedance signal is amplified and processed by the embodiment system based on the phase lock of the synchronous phase-sensitive detection technology, and a weak cell impedance detection signal can be effectively extracted from noise with great interference.

In the embodiment, pulse signal data of cell impedance change is obtained in step S200, converted into a voltage signal through ITA, amplified through band-pass filtering and frequency-selecting filtering, and noise except the center frequency is removed; and then the voltage pulse direct current component related to the size of the cell passing through the detection electrode is obtained by performing phase-locked amplification processing on the reference signal and the phase-locked amplifier. Finally, the signal is further amplified by an amplifier, and the cell impedance signal is output to a data acquisition card (DAQ).

S400, forming a data file according to the cell impedance signal, obtaining the number of living single cells according to the data file, and determining the survival rate of the cells;

specifically, the data processing module in the embodiment system may include a LabVIEW software data acquisition processing unit and a MATLAB data processing terminal. According to data acquisition requirement parameters set by LabVIEW, the cell impedance signal data is acquired through DAQ and transmitted to a LabVIEW software data acquisition processing unit, cell impedance change signal data is obtained, and a TDMS data file is generated. And the MATLAB data processing terminal obtains a TDMS data file generated by a LabVIEW software data acquisition and processing unit, performs data processing such as noise reduction and peak searching in the later period on the cell impedance signal data, obtains the number of living single cells, and accordingly determines the survival rate of the cells.

In some possible embodiments, the step S200 of converting the cell impedance variation signal by I/V conversion to obtain a first intermediate signal and performing frequency-selective filtering on the first intermediate signal to obtain a second intermediate signal may include the step S210 of converting the cell impedance variation signal into a voltage signal by a transimpedance amplifier to obtain the first intermediate signal.

Specifically, in the embodiment, as shown in fig. 4, the preamplifier of the cell impedance phase-lock detection module adopts a Low Noise Amplifier (LNA) OPA656 as the preamplifier, and the feedback resistance is set to 200k Ω; the input impedance is large, the signal noise can be reduced, the signal to noise ratio of the signal is improved, and the cell impedance change signal which is detected by the ITO detection electrode and passes through the detection hole of the microfluidic chip channel is converted into a voltage value. The OPA656 chip is used as a low-noise amplifier, adopts FET input power amplification, has the characteristics of high input impedance, low noise, ultralow bias current and the like, improves the signal-to-noise ratio of weak input signals, converts cell impedance input signals into voltage signals, and well solves the problem of high load effect of the output impedance of the microfluidic chip.

In some possible embodiments, the step S200 of converting the cell impedance variation signal by I/V to obtain a first intermediate signal of a voltage value, and performing frequency-selective filtering on the first intermediate signal to obtain a second intermediate signal may further include the step S220 of performing frequency-selective filtering on the first intermediate signal by a band-pass filter to obtain the second intermediate signal.

Specifically, as shown in fig. 5, the band-pass filtering and amplifying unit in the cell impedance phase-locked detection module according to the embodiment is mainly a band-pass filtering amplifier, and performs frequency-selective amplification on the cell impedance signal from the TIA, and first suppresses noise outside the center frequency (450kHz) through a band-pass filter, and then amplifies, drives and outputs the filtered signal through a power amplifier chip LT1224, and transmits the amplified signal to the LIA unit.

In some optional embodiments, the step S300 of performing a phase-lock amplification process on the second intermediate signal and the reference signal to obtain a cell impedance signal subjected to the phase-lock amplification process may include steps S310-S320;

s310, based on synchronous phase-sensitive detection, performing phase-locked amplification on the second intermediate signal and the reference signal, and locking the second intermediate signal in a reference frequency range;

and S320, filtering out signal components which are different in frequency from the reference signal in the phase-locked signal through low-pass filtering to obtain a cell impedance signal which is the same in frequency as the reference signal.

In the embodiment, the LIA unit selects a broadband high-precision analog multiplier chip MPY634, and based on a synchronous phase-sensitive detection technology, a weak cell impedance detection signal is subjected to frequency mixing with a reference signal, phase-locked amplification, a signal to be detected is separated from noise, and then the detection signal is transmitted to a low-pass filtering unit, and the low-pass filtering unit performs low-pass filtering on the signal processed by the LIA, so that the cell impedance detection signal with a high signal-to-noise ratio is obtained.

Specifically, as shown in fig. 6, the LIA unit is an analog multiplier based on a synchronous phase-sensitive detection technology as a mixer, a weak impedance detection signal is multiplied by a reference signal in the LIA, a sinusoidal signal with the same frequency (450kHz) as the signal to be detected is given to the LIA through a signal generator as the reference signal, and meanwhile, the impedance signal of the cell to be detected is accessed. Because the signal to be detected is very weak and is very easy to be interfered by external noise, the signal to be detected A sin (omega)₁t + α) and a reference signal B sin (ω)₂t + β) according to the sine function multiplication formula: a sin (omega)₁t+α)×B sin(ω₂t+β)＝1/2AB{cos[(ω₁-ω₂)t+(α-β)]-cos[(ω₁+ω₂)t+(α+β)]When the frequencies are the same, i.e. ω₁＝ω₂ω; the result is 1/2AB { cos (. alpha. -beta) -cos [ 2. omega. t + (α + β)]}. The lock-in amplifier is followed by a low-pass filter to filter out cos [2 ω t + (α + β)]And in part, 1/2ABCos (alpha-beta) direct current parts with the same frequency as the reference signal are left, so that a signal with a high signal-to-noise ratio is obtained.

In addition, the amplifier unit in the embodiment is a high-gain inverting amplifier, the feedback resistance is 20k Ω, and the gain is 20. And finally amplifying the cell impedance detection signal subjected to the phase-locked amplification and low-pass filtering and outputting the amplified cell impedance detection signal.

In some optional embodiments, the step S400 of forming a data file according to the cell impedance signal, obtaining the number of living single cells according to the data file, and determining the survival rate of the cells may further be specifically: wavelet analysis denoising and multi-scale peak position detection are carried out on cell impedance signals in the data file, pulse peak positions are extracted to obtain pulse peak positions, and the number of single living cells and the cell survival rate are determined according to the pulse peak values of the pulse peak positions.

Specifically, as shown in fig. 7, in the embodiment, the data acquisition card (DAQ) employs a USB-6361 multifunctional I/O device to perform data acquisition on the cell impedance signal, and is configured to receive the cell impedance signal detected by the cell impedance phase-locked detection device; the LabVIEW software unit can set data acquisition function parameters, and the sampling frequency is at least twice of the frequency to be measured according to the Nyquist sampling theorem. The sampling frequency of the DAQ data acquisition card can reach 2MS/s, and the actual sampling rate is 1.5-2 MS/s. The wiring terminal is configured and displays cell impedance detection data in real time through the image display unit, and then the detection data is converted into TDMS-form large-capacity data; the MATLAB data processing terminal comprises TDMS data reading, TDMS data opening, wavelet analysis denoising and multi-scale peak position detection algorithm for cell impedance detection data; and the MATLAB data processing terminal is used for carrying out data processing on the data of the cell impedance detection signals collected by LabVIEW, carrying out wavelet denoising, detecting pulse peak positions and extracting pulse peak values, so that the size and the number of cells are determined, and the cell detection and the survival rate are determined.

In addition, in the embodiment, the LabVIEW software unit can set the data acquisition function parameters and display the cell impedance detection data in real time through the image display unit.

In a third aspect, the technical solution of the present application further provides a device for rapidly detecting impedance of a living single cell, which includes at least one processor; at least one memory for storing at least one program; when the at least one program is executed by the at least one processor, the at least one processor is caused to execute a method for rapid detection of impedance of a living single cell as in the first aspect.

Specifically, the device comprises a cell impedance phase-locking detection device, a data acquisition and LabVIEW software data acquisition and processing unit and an MATLAB data processing terminal; the cell impedance phase-locking detection device detects cell impedance change signals passing through the detection holes of the microfluidic chip channel through the ITO detection electrodes, and the cell impedance phase-locking detection device detects and outputs cell impedance signal data subjected to phase-locking processing; collecting data through a data acquisition card (DAQ) according to data acquisition conditions set by the LabVIEW, transmitting the data to a LabVIEW software data acquisition processing unit, and displaying and generating TDMS data; and the MATLAB data processing terminal obtains TDMS data generated by the LabVIEW software data acquisition and processing unit and performs later-stage wavelet denoising and peak searching processing on the data. Thereby determining the size and the number of the cells, and realizing the results of living single cell detection, survival rate determination and the like.

An embodiment of the present invention further provides a storage medium storing a program, where the program is executed by a processor to implement the method in the first aspect.

In summary, it can be concluded from the foregoing specific embodiments and implementation processes that the technical solution provided by the present invention has the following advantages or advantages compared with the prior art:

(1) according to the technical scheme, the number of live cells and dead cells in a cell solution is indirectly determined through the change of cell impedance caused by the change of the volume of single cells under the action of a hypertonic solution, so that the parameters such as the survival rate of the cells are determined;

(2) according to the technical scheme, the single cell impedance phase-locked detection device is adopted, and the synchronous phase-sensitive phase-locked detection technology is combined, so that the required weak cell impedance signal can be detected from noise, and the accuracy and the high efficiency of cell impedance signal detection can be improved to a great extent;

(3) according to the single-cell living body impedance rapid detection system based on the phase locking technology, a preamplifier of a cell impedance phase locking detection device adopts an OPA656 chip of TI company as a low-noise amplifier, and adopts an FET input power amplifier, so that the system has the characteristics of high input impedance, low noise, ultralow bias current and the like, improves the signal-to-noise ratio of weak input signals, converts the cell impedance input signals into voltage signals, and well solves the problem of high load effect of the output impedance of a microfluidic chip;

(4) according to the technical scheme, the four-quadrant analog multiplier chip MPY634 is used for mixing cell impedance signals to be detected with reference signals, converting cell impedance signal components with the same frequency as the reference signals into direct currents after phase-locked filtering, and filtering other high-frequency noises through low-pass filtering processing of a low-pass filter connected in series. The common commercial lock-in amplifier equipment is expensive and large in size. The multiplier chip can simplify the hardware design of the detection device and greatly reduce the system design cost;

(5) according to the technical scheme, the LabVIEW data acquisition system acquires cell impedance signal data according to acquisition function parameters and stores the cell impedance signal data as a TDMS-form high-capacity data file. For the condition of high sampling rate, the storage form can obtain data with large data volume in a longer time period, is suitable for storing mass-level data, and is high in speed, convenient and easy to access;

(6) according to the technical scheme, wavelet analysis denoising and peak searching algorithm is carried out on the single cell impedance signal through MATLAB software, signals and high-frequency noise are effectively distinguished, transient signals and transient signal noise are removed, the number of cells is obtained through determination of peak values, and therefore the number of single cells and the survival rate are determined and further analyzed.

In alternative embodiments, the functions/acts noted in the block diagrams may occur out of the order noted in the operational illustrations. For example, two blocks shown in succession may, in fact, be executed substantially concurrently, or the blocks may sometimes be executed in the reverse order, depending upon the functionality/acts involved. Furthermore, the embodiments presented and described in the flow charts of the present invention are provided by way of example in order to provide a more thorough understanding of the technology. The disclosed methods are not limited to the operations and logic flows presented herein. Alternative embodiments are contemplated in which the order of various operations is changed and in which sub-operations described as part of larger operations are performed independently.

Furthermore, although the present invention is described in the context of functional modules, it should be understood that, unless otherwise stated to the contrary, one or more of the functions and/or features may be integrated in a single physical device and/or software module, or one or more of the functions and/or features may be implemented in a separate physical device or software module. It will also be appreciated that a detailed discussion of the actual implementation of each module is not necessary for an understanding of the present invention. Rather, the actual implementation of the various functional modules in the apparatus disclosed herein will be understood within the ordinary skill of an engineer, given the nature, function, and internal relationship of the modules. Accordingly, those skilled in the art can, using ordinary skill, practice the invention as set forth in the claims without undue experimentation. It is also to be understood that the specific concepts disclosed are merely illustrative of and not intended to limit the scope of the invention, which is defined by the appended claims and their full scope of equivalents.

The logic and/or steps represented in the flowcharts or otherwise described herein, e.g., an ordered listing of executable instructions that can be considered to implement logical functions, can be embodied in any computer-readable medium for use by or in connection with an instruction execution system, apparatus, or device, such as a computer-based system, processor-containing system, or other system that can fetch the instructions from the instruction execution system, apparatus, or device and execute the instructions.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

While embodiments of the invention have been shown and described, it will be understood by those of ordinary skill in the art that: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.

While the preferred embodiments of the present invention have been illustrated and described, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.