CN117538382A - Non-marking electrical impedance flow cytometer for circulating tumor cells and detection method - Google Patents
Non-marking electrical impedance flow cytometer for circulating tumor cells and detection method Download PDFInfo
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Abstract
The invention discloses a non-marking electrical impedance flow cytometer for circulating tumor cells and a detection method, comprising a sample injection system, an impedance spectrometer, a sample injection control system and a computer, wherein the sample injection system comprises a sample injection pump, a switching valve connected with the sample injection pump, a first centrifuge tube filled with a cleaning solution and connected with a first liquid inlet of the switching valve, a second centrifuge tube filled with a sample to be detected and connected with a second liquid inlet of the switching valve, a third centrifuge tube filled with a standard sample and connected with a third liquid inlet of the switching valve, a microfluidic chip connected with a liquid outlet of the switching valve and a waste liquid collecting bottle connected with the microfluidic chip, the sample injection control system comprises a stepping motor driving plate and a control plate, the control plate is respectively connected with the switching valve and the stepping motor driving plate, the impedance spectrometer applies a multi-frequency excitation signal to the microfluidic chip, the microfluidic chip outputs a feedback current signal to the impedance spectrometer, and the impedance spectrometer outputs an original signal to the computer for signal processing and identifying cell types.
Description
Technical Field
The invention relates to the field of non-labeling detection of cells, in particular to a non-labeling electrical impedance flow cytometer for circulating tumor cells and a detection method.
Background
As a "seed" for cancer metastasis and invasion, circulating tumor cells have important scientific and clinical research value. The sorting, counting and analyzing means of circulating tumor cells widely used at present often use an immune labeling method, and the circulating tumor cells with activity cannot be obtained, so that the biophysical characteristics of the circulating tumor cells such as drug resistance, environmental suitability and the like are further analyzed. Therefore, to obtain active circulating tumor cells, it is an important basis to implement non-labeling detection and species identification techniques of cells. The dielectric properties of cells, one of the important biophysical properties, can be an important parameter for cell count and species identification.
The prior art CN113866074A discloses a white blood cell classifying and counting micro-fluidic chip based on an electrical impedance method of position compensation, which comprises a micro-fluidic chip, wherein the micro-fluidic chip comprises a fluid channel, electrolyte solution is filled in the fluid channel, seven electrodes are symmetrically distributed at equal intervals at the bottom of the fluid channel, and the micro-fluidic chip adopts a double-differential structure. The micro-fluidic chip for classifying and counting the white blood cells based on the electrical impedance method of position compensation integrates double differential structure electrodes, has the characteristics of high flux, micro-sample, no-label, high sensitivity and the like, can rapidly and accurately count and classify the white blood cells, has small size and high integration level, can be light and convenient to carry, is simple to operate, takes short time without professional skills or additional medical instruments, can realize timely detection in daily diagnosis even in poor areas with deficient medical conditions, eliminates errors of manual counting, and reduces personnel infection risks. However, the above-mentioned prior art cannot meet more various detection requirements, and has poor versatility. In addition, the condition that the sample solution pollutes the sample solution for the next detection in the last detection is easy to occur in the use process of the conventional cytometer, so that the detection precision and the detection result are affected.
Disclosure of Invention
The invention aims to: a first object of the present invention is to provide a non-labeled electrical impedance flow cytometer for identifying cell types using flow ac impedance signals of cells at multiple frequencies.
The second object of the invention is to provide a detection method of a non-labeled electrical impedance flow cytometer oriented to circulating tumor cells.
The technical scheme is as follows: the invention discloses a non-marking electrical impedance flow cytometer for circulating tumor cells, which comprises a sample injection system, an impedance spectrometer, a sample injection control system and a computer, wherein the sample injection system comprises a sample injection pump, a switching valve connected with the sample injection pump, a first centrifuge tube filled with cleaning solution and connected with a first liquid inlet of the switching valve, a second centrifuge tube filled with a sample to be detected and connected with a second liquid inlet of the switching valve, a third centrifuge tube filled with a standard sample and connected with a third liquid inlet of the switching valve, a microfluidic chip connected with a liquid outlet of the switching valve and a waste liquid collecting bottle connected with the microfluidic chip, the sample injection control system comprises a stepping motor driving plate and a control plate for driving the sample injection pump, the control plate is respectively connected with the switching valve and the stepping motor driving plate, the impedance spectrometer applies a frequency excitation signal to the microfluidic chip, the microfluidic chip outputs a feedback current signal to the impedance spectrometer, and the impedance spectrometer outputs an original signal to the computer for signal processing and identifying cell types.
Furthermore, the multi-frequency excitation signal is a mixed multi-frequency alternating current signal, the frequency range of the mixed multi-frequency alternating current signal is 500 kHz-50 MHz, and the number of the selected frequencies is not more than 8.
Further, the microfluidic chip includes a glass substrate, a first electrode, a second electrode, a third electrode, a fourth electrode, and a fifth electrode integrated on the glass substrate and coplanar.
Preferably, the microfluidic chip is provided with a three-electrode detection mode and a five-electrode detection mode, when in the three-electrode detection mode, a multi-frequency excitation signal is applied to the third electrode, the second electrode and the fourth electrode output feedback current signals to the impedance spectrometer, and the first electrode and the fifth electrode are not externally connected with a signal source; in the five-electrode detection mode, a multi-frequency excitation signal is applied to the third electrode, the second electrode and the fourth electrode output feedback current signals to the impedance spectrometer, and the first electrode and the fifth electrode are externally connected with an electric signal with opposite sign to the third electrode.
Furthermore, the microfluidic chip further comprises a microfluidic channel, the microfluidic channel is an asymmetric sinusoidal channel, the length L of a detection channel in the asymmetric sinusoidal channel is larger than the electrode distribution width at the bottom of the microfluidic channel, the width W of the detection channel is equal to the width of a channel with small curvature in the asymmetric sinusoidal channel, and the height H of the detection channel is larger than the maximum diameter of the detected cells.
Further, the control board has a cleaning mode, a calibration mode and a detection mode,
during a cleaning mode, the control board controls the switching valve to be switched to the first sample inlet, the control board controls the sample injection pump to pump cleaning solution in the first centrifuge tube through the stepping motor driving board and pumps the cleaning solution to the switching valve, the switching valve is switched to the liquid outlet, and the cleaning solution flows to the microfluidic chip through the liquid path pipeline to clean the microfluidic chip;
during a calibration mode, the control board controls the switching valve to be switched to a third sample inlet, the control board controls the sample pump to pump standard solution in the third centrifuge tube through the stepping motor driving board and pumps the standard solution to the switching valve, the switching valve is switched to a liquid outlet, the standard solution flows to the microfluidic chip through the liquid path pipeline, a multi-frequency excitation signal is applied to the microfluidic chip, the microfluidic chip outputs a feedback current signal to the impedance spectrometer, and the impedance spectrometer outputs an original signal to the computer to serve as a reference value of a cell electrical impedance signal;
during the detection mode, the control board controls the switching valve to be switched to the second sample inlet, the control board controls the sample injection pump to pump the solution to be detected in the second centrifuge tube through the stepping motor driving board and pumps the solution to the switching valve, the switching valve is switched to the liquid outlet, the solution to be detected flows to the microfluidic chip through the liquid path pipeline, the multi-frequency excitation signal is applied to the microfluidic chip, the microfluidic chip outputs the feedback current signal to the impedance spectrometer, and the impedance spectrometer outputs the original signal to the computer for signal processing and identification of the cell type.
And the power module is respectively connected with the computer, the impedance spectrometer and the direct-current conversion module, and the direct-current conversion module is respectively connected with the control board, the stepping motor driving board, the sample injection pump and the switching valve.
The invention discloses a detection method based on a non-marking electrical impedance flow cytometer for circulating tumor cells, which comprises the following steps:
in the case of the three-electrode detection mode,
the control board controls the switching valve to switch to the second sample inlet, the control board controls the sample pump to pump the solution to be tested in the second centrifuge tube through the stepping motor driving board and pumps the solution to the switching valve, the switching valve is switched to the liquid outlet, and the solution to be tested flows to the microfluidic chip through the liquid path pipeline;
applying a multi-frequency excitation signal to the third electrode, outputting feedback current signals to the impedance spectrometer by the second electrode and the fourth electrode, wherein the first electrode and the fifth electrode are not externally connected with a signal source; the impedance spectrometer outputs an original signal to a computer, the computer firstly carries out noise reduction treatment on the original signal, the signal base line is leveled, the cell electrical impedance signal is extracted, and the cell type is identified based on a convolutional neural network model;
the number of alternating current frequencies applied to the third electrode is set as k in the convolutional neural network model, single cells undergo n times of signal sampling during the period that single cells pass through an electrical impedance detection area at a fixed flow rate, and cell electrical impedance signals intercepted at each frequency form a matrix of 2k rows and n columns; the real part of the cell alternating current electrical impedance signal under the 1 st action and the 1 st frequency in the matrix, and the imaginary part of the cell alternating current electrical impedance signal under the 1 st action and the 2 nd action in the matrix; by analogy, the imaginary part of the cell alternating current electrical impedance signal at the kth frequency of the kth behavior of 2; and (3) after carrying out convolution calculation on the matrix, sending the result into a maximum pooling layer, sending the result into a fully-connected neural network, and finally outputting cell type information.
The invention discloses a detection method based on a non-marking electrical impedance flow cytometer for circulating tumor cells, which comprises the following steps:
in the case of the five-electrode detection mode,
the control board controls the switching valve to switch to the second sample inlet, the control board controls the sample pump to pump the solution to be tested in the second centrifuge tube through the stepping motor driving board and pumps the solution to the switching valve, the switching valve is switched to the liquid outlet, and the solution to be tested flows to the microfluidic chip through the liquid path pipeline;
applying a multi-frequency excitation signal to the third electrode, outputting feedback current signals to the impedance spectrometer by the second electrode and the fourth electrode, and externally connecting an electric signal with opposite sign with the third electrode by the first electrode and the fifth electrode; the impedance spectrometer outputs an original signal to a computer, the computer firstly carries out noise reduction treatment on the original signal, the signal base line is leveled, the cell electrical impedance signal is extracted, and the cell type is identified based on a convolutional neural network model;
the number of alternating current frequencies applied to the third electrode is set as k in the convolutional neural network model, single cells undergo n times of signal sampling during the period that single cells pass through an electrical impedance detection area at a fixed flow rate, and cell electrical impedance signals intercepted at each frequency form a matrix of 2k rows and n columns; the real part of the cell alternating current electrical impedance signal under the 1 st action and the 1 st frequency in the matrix, and the imaginary part of the cell alternating current electrical impedance signal under the 1 st action and the 2 nd action in the matrix; by analogy, the imaginary part of the cell alternating current electrical impedance signal at the kth frequency of the kth behavior of 2; and (3) after carrying out convolution calculation on the matrix, sending the result into a maximum pooling layer, sending the result into a fully-connected neural network, and finally outputting cell type information.
The beneficial effects are that: compared with the prior art, the invention has the following remarkable advantages:
(1) The invention does not need to apply immunofluorescence labeling technology to identify the cell types, and can easily obtain active cells after detection so as to further scientific research;
(2) The cell electrical impedance detection microfluidic chip can realize single differential electrical impedance signal detection of cells based on three electrodes or secondary differential electrical impedance signal detection based on five electrodes. The two detection modes can be switched freely conveniently. The single differential detection based on three electrodes is suitable for high-flux and high-cell concentration sample detection. The detection requirement of the cell sample with high sensitivity, high signal-to-noise ratio and low concentration is based on the secondary differential detection of five electrodes;
(3) The cell impedance detection micro-fluidic chip based on the asymmetric sinusoidal flow channel can realize high-flux single-row passive focusing of cells in the flow channel so as to improve the detection precision of the cell impedance signals;
(4) The cell type identification algorithm based on the cell electrical impedance detection signal can effectively improve the generalization capability of a cell type identification model; the influence of the processing error of the electrical impedance detection chip is small.
Drawings
FIG. 1 (a) is a front view of the present invention;
FIG. 1 (b) is a schematic diagram of the internal structure of the present invention;
FIG. 1 (c) is a schematic diagram of the internal structure of the second embodiment of the present invention;
FIG. 2 is a schematic diagram of the present invention;
FIG. 3 (a) is a schematic structural diagram of a microfluidic chip and a fixture according to the present invention;
FIG. 3 (b) is a schematic diagram showing the assembly of a microfluidic chip and a fixture according to the present invention;
FIG. 4 is a schematic illustration of an electrode arrangement of an electrical impedance detection region according to the present invention;
FIG. 5 is a schematic illustration of an asymmetric sinusoidal flow path in accordance with the present invention;
FIG. 6 is a schematic diagram of the working principle of the liquid path of the sample injection system according to the present invention;
FIG. 7 is a graph showing the comparison of detection performance of coplanar three-electrode and five-electrode structures of the present invention;
FIG. 8 is a schematic representation of the electrical impedance signal of a single cell of the present invention;
FIG. 9 is a block diagram of an artificial neural network for cell type identification based on a convolution algorithm in the present invention;
FIG. 10 is a schematic diagram showing the performance of the artificial neural network-based cell type recognition model according to the present invention.
Detailed Description
The technical scheme of the invention is further described below with reference to the accompanying drawings.
The invention relates to a non-marking electrical impedance flow cytometer for circulating tumor cells, which comprises an instrument shell 5, a sample injection system, an impedance spectrometer 6, a transimpedance amplifier 7, a sample injection control system and a computer 8, wherein the sample injection system comprises the shell 5, a sample injection pump 10, a switching valve 11, a first centrifuge tube 2, a second centrifuge tube 3, a third centrifuge tube 4, microfluidic chips 1-3 and a waste liquid collecting bottle 9, and the sample injection control system comprises a stepping motor driving plate 13, a control plate 15, a power supply module 12 and a direct current conversion module 14. The switching valve 11 is connected with the sample injection pump 10, the first centrifuge tube 2 is filled with a cleaning solution, the cleaning solution can be phosphate buffer salt solution, the first centrifuge tube 2 is connected with a first liquid inlet P1 of the switching valve 11, the second centrifuge tube 3 is filled with a sample to be tested, the second centrifuge tube 3 is connected with a second liquid inlet P3 of the switching valve 11, the third centrifuge tube 4 is filled with a standard sample, and the standard sample can be phosphate buffer salt solution of hyaluronic acid dissolved with suspended standard micron particles; the third centrifuge tube 4 is connected with a third liquid inlet P2 of the switching valve, a liquid outlet P4 of the switching valve 11 is connected with the microfluidic chip 1-3, and the microfluidic chip 1-3 is also connected with a waste liquid collecting bottle 9. The control board 15 is connected with the switching valve 11 and the stepping motor driving board 13 respectively, and the stepping motor driving board 13 is connected with the sample injection pump 10 for driving the sample injection pump. The power module is respectively connected with the computer 8, the impedance spectrometer 6 and the direct current conversion module 14, the direct current conversion module 14 is respectively connected with the control board 15, the stepping motor driving board 13, the sample injection pump 10 and the switching valve 11, the power module is a total power interface, and the whole instrument is uniformly powered by the total power interface. The positive pressure or the negative pressure is provided for the liquid path of the sample injection system by the sample injection pump 10 to push or suck the liquid sample, and the switching of the liquid path is completed by the rotation of the valve body in the switching valve 11. Because the sample injection pump and the switching valve are internally provided with a narrow gap which is difficult to clean and a dead volume which is difficult to empty, in order to avoid contacting with a sample containing cells, the sample liquid storage pipeline is a section of liquid storage pipeline with the inner volume of the pipe being larger than the maximum sample pumping capacity. When the liquid sample in the pumping liquid storage pipeline enters the microfluidic chip, the switching valve is switched to the position of the liquid outlet P4, so that the sample injection pump, the switching valve and the microfluidic chip form a fluid passage, and other valve ports are in a closed and open state, so that the pumping action is not influenced. For example, in the purge mode, the operation logic of the sample pump and the switching valve is as follows: the switching valve is switched to the first liquid inlet P1- & gt, (2) the sampling pump pumps quantitative cleaning solution, the switching valve is switched to the liquid outlet P4- & gt, (4) the sampling pump pumps the cleaning solution at a set speed. Other patterns follow similar action logic and are not described in detail herein.
The impedance spectrometer 6 applies a multi-frequency excitation signal to the microfluidic chip 1-3, the microfluidic chip 1-3 outputs a feedback current signal to the impedance spectrometer 6, and the impedance spectrometer 6 outputs an original signal to the computer 8 for signal processing to identify the cell type. The multi-frequency excitation signal is a mixed multi-frequency alternating current signal, the frequency range of the mixed multi-frequency alternating current signal is 500 kHz-50 MHz, the number of the selected frequencies is not more than 8, and the cell type is identified by using the flow type alternating current impedance signal of the cell under the multi-frequency. The microfluidic chip 1-3 is fixed by a clamp 1-1 and is loaded on an upper cover plate of the instrument shell 5, and is connected with a detection circuit inside the instrument through a spring pin 1-2, wherein the detection circuit comprises an impedance spectrometer and a differential amplifier, and the microfluidic chip 1-3 at the upper cover plate of the instrument and the clamp 1-1 can be opened in a detachable manner so as to replace the microfluidic chip 1-3 therein, as shown in fig. 3. The microfluidic chip 1-3 comprises a glass substrate, a first electrode 1-3-1, a second electrode 1-3-2, a third electrode 1-3-3, a fourth electrode 1-3-4 and a fifth electrode 1-3-5, wherein the first electrode 1-3-1, the second electrode 1-3-2, the third electrode 1-3, the fourth electrode 1-3-4 and the fifth electrode 1-3-5 are integrated on the glass substrate and are coplanar.
The electrode arrangement schematic diagram of an electrical impedance detection area close to an outlet in a micro-fluidic chip for detecting micro-cell electrical impedance is shown in fig. 4, five coplanar microelectrodes are integrated on a glass substrate of a micro-fluidic chip 1-3, so that a cell electrical impedance signal has two detection modes, namely, the micro-fluidic chip 1-3 has a three-electrode detection mode and a five-electrode detection mode, a multi-frequency excitation signal is applied to a third electrode 1-3-3 in the three-electrode detection mode, a feedback current signal is output by a second electrode 1-3-2 and a fourth electrode 1-3-4 through a differential amplifier 7, a signal with specific frequency is extracted through phase locking of an impedance spectrometer, and then the signal is amplified and input into the impedance spectrometer 6, and the first electrode 1-3-1 and the fifth electrode 1-3-5 are not externally connected with a signal source; the three-electrode detection mode is suitable for high-flux and high-cell-concentration sample detection. High flux means more than 100. Mu.L/min, high cell concentration means more than 10 5 cells/mL. The second electrode 1-3-2 and the fourth electrode 1-3-4 output feedback current signals are amplified and then are differentiated, so that the cell electrical impedance detection signals have higher signal to noise ratio. In the detection mode, the electrical impedance detection area of the cells is small in volume, and the length occupied in the flow channel is short, so that the detection method is suitable for detecting samples with high flux and high cell concentration.
Five-electrode detection dieWhen in formula, a multi-frequency excitation signal is applied to the third electrode 1-3-3, the second electrode 1-3-2 and the fourth electrode 1-3-4 output feedback current signals to the impedance spectrometer 6, and the first electrode 1-3-1 and the fifth electrode 1-3-5 are externally connected with an electric signal with opposite sign to the third electrode 1-3-3; the five-electrode detection mode has higher detection signal-to-noise ratio, and is suitable for the detection requirements of high sensitivity, high signal-to-noise ratio and low concentration cell samples, wherein the high sensitivity refers to the volume fraction of cells in a detection area>320ppm, low concentration cell sample is less than 10 5 cells/mL. The alternating current signals with opposite signs on the third electrode 1-3-3 and the first electrode 1-3-1 and the fifth electrode 1-3-5 are externally connected through a special passive inverter. The five-electrode detection mode is mainly characterized in that current signals are subjected to passive secondary difference, namely the five-electrode detection mode is characterized in that secondary difference is realized without other electrical elements, and cell electrical impedance signals with higher signal to noise ratio are obtained, so that the detection requirements of high-sensitivity and low-concentration cell samples are met.
The invention selects the same microfluidic chip with a five-electrode structure, and the detection circuit is connected. As shown in fig. 7 (a), the reverse phase signal that should be applied to the two side edge electrodes is cut off, and the detection circuit is equivalent to a single differential electrical impedance detection method based on a three-electrode structure. A sinusoidal AC signal having a frequency of 500kHz and a voltage peak-to-peak value of 1V was applied to the intermediate electrode. Standard polystyrene particles with a diameter of 4 μm were introduced into the microfluidic chip at a flow rate of 80. Mu.L/min. After a certain amount of original data is saved and recorded, under the condition that other conditions are unchanged, the inversion signal on the edge electrode is turned on, as shown in (b) of fig. 7, and the original data of the electrical impedance signal is saved and recorded as another group of data, and the ordinate of the data diagram in fig. 7 is converted into an arbitrary unit a.u. according to the same method to compare the difference of the two detection methods.
As is clear from the signal diagram of the 4 μm particle in fig. 7 (a), the single differential detection method of the particle electrical impedance based on the three-electrode structure cannot detect a significant peak. While only signals within 75ms are randomly intercepted in the figure, the invention examines signals within about 30s time span under the detection condition, and no signal peak which obviously accords with the characteristics of 4 mu m particles is found. In this case, the signal noise range is about ±300a.u, and therefore it can be concluded that the particle signal having a diameter of not more than 4 μm cannot be detected well by the particle single differential electrical impedance detection method based on the three-electrode structure. From fig. 7 (b), the signal peak of the 4 μm particle can be clearly recognized in the particle impedance secondary differential detection mode based on the five-electrode structure.
In summary, the instrument provided by the invention can realize free switching between the three-electrode and five-electrode detection modes, and the signal-to-noise ratio of the secondary differential electrical impedance detection based on the five-electrode structure is higher than that of the single differential electrical impedance detection mode based on the three-electrode structure. The length of the detection area in the two detection modes determines that the five-electrode structure is only suitable for detecting samples with low cell concentration, otherwise, the condition that a plurality of cells enter the detection area simultaneously is easy to occur, and the decoupling and analysis of data are not facilitated.
The microfluidic chip 1-3 further comprises a microfluidic channel, the microfluidic channel is an asymmetric sinusoidal channel, the length L of a detection channel in the asymmetric sinusoidal channel is larger than the electrode distribution width at the bottom of the microfluidic channel, the width W of the detection channel is equal to the width of a channel with small curvature in the asymmetric sinusoidal channel, and the height H of the detection channel is larger than the maximum diameter of a cell to be detected. According to the invention, the cells 1-3-6 form single-column focusing in the micro-channel by utilizing the coupling effect of the inertial force and the elastic force in the asymmetric sinusoidal channel, so that the detection precision of the cell electrical impedance signals is improved. The suspension of the cell sample is phosphate buffer salt solution for dissolving Hyaluronic Acid (HA for short), and is a non-Newtonian fluid. Under the coupling effect of inertial force and elastic force in the flow field in the flow channel, the cells can realize accurate single-column focusing in the flow channel, and the electrical impedance signals of the standard polystyrene microspheres have good uniformity, so that necessary conditions are provided for the accurate acquisition of the subsequent electrical impedance signals of the cells. The size and structure characteristics of the asymmetric sinusoidal flow channel and the acting force of the internal flow field on cells can be referred to the literature [1,2] . The length L of the detection flow channel is larger than the distribution width of the electrode at the bottom of the detection flow channel, the width W is equivalent to the flow channel width of the smaller curvature part in the asymmetric sinusoidal flow channel, and the height H is as high as possibleIt may be slightly larger than the largest diameter of the cell being measured, typically 30-40 microns.
In the invention, the control board 15 is provided with a cleaning mode, a calibration mode and a detection mode, and the control board 15 based on the singlechip is responsible for receiving instructions given by the computer and reporting the action proceeding state to the host computer. The computer transmits the mode (cleaning mode, calibration mode or detection mode) to be executed to the control board 15, and then the specific instruction is converted and then transmitted to the stepping motor driving board 13 or the switching valve 11, and the stepping motor driving board 13 controls the rotating speed and the rotating quantity of the sample injection pump 10.
During a cleaning mode, the control board controls the switching valve to be switched to the first sample inlet, the control board controls the sample injection pump to pump cleaning solution in the first centrifuge tube through the stepping motor driving board and pumps the cleaning solution to the switching valve, the switching valve is switched to the liquid outlet, and the cleaning solution flows to the microfluidic chip through the liquid path pipeline to clean the microfluidic chip;
during a calibration mode, the control board controls the switching valve to be switched to a third sample inlet, the control board controls the sample pump to pump standard solution in the third centrifuge tube through the stepping motor driving board and pumps the standard solution to the switching valve, the switching valve is switched to a liquid outlet, the standard solution flows to the microfluidic chip through the liquid path pipeline, a multi-frequency excitation signal is applied to the microfluidic chip, the microfluidic chip outputs a feedback current signal to the impedance spectrometer, the impedance spectrometer outputs an original signal to the computer, and the original signal is used in the calibration mode as a reference value of a cell electrical impedance signal; in the calibration mode, the specific role of the electrical impedance data of the particles is as follows: under the excitation of low-frequency alternating current (the frequency is not higher than 500 kHz), the fluctuation peak value P of the detection current caused by particles or cells passing over the microelectrode is in a proportional relation of P=k.V with the volume V, and the low-frequency electrical impedance data of the particles obtained in the calibration mode can be used for calculating the positive proportionality coefficient k and using the positive proportionality coefficient k in the subsequent rapid calculation of the cell diameter.
During the detection mode, the control board controls the switching valve to be switched to the second sample inlet, the control board controls the sample injection pump to pump the solution to be detected in the second centrifuge tube through the stepping motor driving board and pumps the solution to the switching valve, the switching valve is switched to the liquid outlet, the solution to be detected flows to the microfluidic chip through the liquid path pipeline, the multi-frequency excitation signal is applied to the microfluidic chip, the microfluidic chip outputs the feedback current signal to the impedance spectrometer, and the impedance spectrometer outputs the original signal to the computer for signal processing and identification of the cell type.
The invention discloses a detection method of a non-marking electrical impedance flow cytometer for circulating tumor cells, which comprises the following steps:
in the case of the three-electrode detection mode,
the control board controls the switching valve to switch to the second sample inlet, the control board controls the sample pump to pump the solution to be tested in the second centrifuge tube through the stepping motor driving board and pumps the solution to the switching valve, the switching valve is switched to the liquid outlet, and the solution to be tested flows to the microfluidic chip through the liquid path pipeline;
applying a multi-frequency excitation signal to the third electrode, outputting feedback current signals to the impedance spectrometer by the second electrode and the fourth electrode, wherein the first electrode and the fifth electrode are not externally connected with a signal source; the impedance spectrometer outputs an original signal to a computer, the computer firstly carries out noise reduction treatment on the original signal, the signal base line is leveled, the cell electrical impedance signal is extracted, and the cell type is identified based on a convolutional neural network model;
the cell type identification algorithm is a deep learning algorithm based on a convolutional neural network, the number of alternating current frequencies applied to a third electrode is set as k in a convolutional neural network model, single cells undergo n times of signal sampling during the period that single cells pass through an electrical impedance detection area at a fixed flow rate, and cell electrical impedance signals intercepted at each frequency form a matrix of 2k rows and n columns; the real part of the cell alternating current electrical impedance signal under the 1 st action and the 1 st frequency in the matrix, and the imaginary part of the cell alternating current electrical impedance signal under the 1 st action and the 2 nd action in the matrix; by analogy, the imaginary part of the cell alternating current electrical impedance signal at the kth frequency of the kth behavior of 2; and (3) after carrying out convolution calculation on the matrix, sending the result into a maximum pooling layer, sending the result into a fully-connected neural network, and finally outputting cell type information.
The invention discloses a detection method of a non-marking electrical impedance flow cytometer for circulating tumor cells, which comprises the following steps:
in the case of the five-electrode detection mode,
the control board controls the switching valve to switch to the second sample inlet, the control board controls the sample pump to pump the solution to be tested in the second centrifuge tube through the stepping motor driving board and pumps the solution to the switching valve, the switching valve is switched to the liquid outlet, and the solution to be tested flows to the microfluidic chip through the liquid path pipeline;
applying a multi-frequency excitation signal to the third electrode, outputting feedback current signals to the impedance spectrometer by the second electrode and the fourth electrode, and externally connecting an electric signal with opposite sign with the third electrode by the first electrode and the fifth electrode; the impedance spectrometer outputs an original signal to a computer, the computer firstly carries out noise reduction treatment on the original signal, a signal base line is leveled, a cell electrical impedance signal is extracted, and noise is reduced: removing the high frequency noise portion (> 100 kHz) of the signal using a wavelet decomposition algorithm; baseline flattening of the signal: to remove jitter of the signal baseline over time, a wavelet decomposition algorithm is used to remove low frequency components (< 1000 Hz) in the signal; cell electrical impedance signal extraction: peak searching is carried out on the signals after denoising and baseline leveling, and the peak value is recorded as an electric signal of the cell; identifying cell types based on a convolutional neural network model;
the number of alternating current frequencies applied to the third electrode is set as k in the convolutional neural network model, single cells undergo n times of signal sampling during the period that single cells pass through an electrical impedance detection area at a fixed flow rate, and cell electrical impedance signals intercepted at each frequency form a matrix of 2k rows and n columns; the real part of the cell alternating current electrical impedance signal under the 1 st action and the 1 st frequency in the matrix, and the imaginary part of the cell alternating current electrical impedance signal under the 1 st action and the 2 nd action in the matrix; by analogy, the imaginary part of the cell alternating current electrical impedance signal at the kth frequency of the kth behavior of 2; and (3) after carrying out convolution calculation on the matrix, sending the result into a maximum pooling layer, sending the result into a fully-connected neural network, and finally outputting cell type information.
The cell type identification algorithm based on the cell electrical impedance detection signal can effectively improve the generalization capability of a cell type identification model. The electrical impedance signals of the cells are matrixed. It is assumed that the alternating current frequencies used in the multi-frequency electrical impedance detection of cells are aMHz, bMHz, cMHz and dhmhz, respectively. Signal lengthVector of n sampling pointsJust comprising the signal fluctuations caused by the passage of individual cells through the electrical impedance detection region, then the individual signals are recorded +.>Andis the magnitude and phase vector of the electrical impedance signal at aMHz. Then the electrical impedance signal of a single cell can be recorded in a matrix form as shown in fig. 8, similar to the manner in which the pixels of the picture form a matrix.
For the convolution operation of matrix C, an embodiment of the present invention selects 8 convolution kernelsAnd the initial value of the element in K is defined as a random number. The step length of the convolution operation in the two directions of the matrix C is set to be 2, and the element value in each convolution kernel K is finally corrected to a proper value according to an optimization algorithm along with the increase of training times. The result is fed into a 2 x 2 max pooling layer after the convolution operation. And then, recombining the maximized result into a one-dimensional vector l, and sending the one-dimensional vector l into a fully-connected neural network. The number of neurons of each layer of the fully connected network is 120, 512, 512, 120 and 8 in sequence, and finally the cell types are output. The loss function of the overall neural network selects a cross entropy classification loss function, an adaptive learning rate method (Root Mean Square Propagation, RMSProp) is adopted as an optimization function, the learning rate lr=0.0005, the activation function of a hidden layer of the neural network is a Relu function, and the activation function of an output layer is a Softmax function. The structure of the entire neural network is shown in fig. 9.
As shown in fig. 10, in this embodiment, the recognition model of cell type with a strong generalization ability is trained by using multichip data, and the target is focused on the discrimination of WBCs from tumor cells. And (3) sending the electrical impedance data of 452634 cells measured by the microfluidic chips from four different manufacturing batches into a neural network for training, and obtaining a cell type identification precision confusion matrix on the test set. As can be seen from the confusion matrix, the neural network has better generalization capability.
The present invention provides a non-marking electrical impedance flow cytometer and detection method for circulating tumor cells, which is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the principles of the present invention, and these improvements and modifications should also be considered as the protection scope of the present invention. The components not explicitly described in this embodiment can be implemented by using the prior art.
Claims (9)
1. The utility model provides a non-mark electrical impedance flow cytometry towards circulation tumour cell, its characterized in that includes sampling system, impedance spectrometer, sampling control system and computer, sampling system includes the sampling pump, the switching valve that links to each other with the sampling pump, the first centrifuging tube that is equipped with the cleaning solution that links to each other with the first inlet of switching valve, the second centrifuging tube that is equipped with the sample that awaits measuring that links to each other with the second inlet of switching valve, the third centrifuging tube that is equipped with the standard sample that links to each other with the third inlet of switching valve, microfluidic chip that links to each other with the liquid outlet of switching valve and the waste liquid collecting bottle that links to each other with microfluidic chip, sampling control system includes step motor drive plate and the control panel that is used for driving the sampling pump, the control panel links to each other with switching valve and step motor drive plate respectively, impedance spectrometer applys the frequency excitation signal to microfluidic chip, microfluidic chip outputs feedback current signal to impedance spectrometer, the original signal of impedance spectrometer output to computer carries out signal processing and discerns the cell type.
2. A non-labeled electrical impedance flow cytometer for treating circulating tumor cells as described in claim 1 wherein: the multi-frequency excitation signal is a mixed multi-frequency alternating current signal, the frequency range of the mixed multi-frequency alternating current signal is 500 kHz-50 MHz, and the number of the selected frequencies is not more than 8.
3. A non-labeled electrical impedance flow cytometer for treating circulating tumor cells as described in claim 1 wherein: the microfluidic chip comprises a glass substrate, a first electrode, a second electrode, a third electrode, a fourth electrode and a fifth electrode, wherein the first electrode, the second electrode, the third electrode, the fourth electrode and the fifth electrode are integrated on the glass substrate and are coplanar.
4. A non-labeled electrical impedance flow cytometer for treating circulating tumor cells as described in claim 3, wherein: the microfluidic chip is provided with a three-electrode detection mode and a five-electrode detection mode, when in the three-electrode detection mode, a multi-frequency excitation signal is applied to the third electrode, the second electrode and the fourth electrode output feedback current signals to the impedance spectrometer, and the first electrode and the fifth electrode are not externally connected with a signal source; in the five-electrode detection mode, a multi-frequency excitation signal is applied to the third electrode, the second electrode and the fourth electrode output feedback current signals to the impedance spectrometer, and the first electrode and the fifth electrode are externally connected with an electric signal with opposite sign to the third electrode.
5. A non-labeled electrical impedance flow cytometer for treating circulating tumor cells as described in claim 3, wherein: the microfluidic chip further comprises a microfluidic channel, the microfluidic channel is an asymmetric sinusoidal channel, the length L of a detection channel in the asymmetric sinusoidal channel is larger than the electrode distribution width at the bottom of the microfluidic channel, the width W of the detection channel is equal to the width of a channel with small curvature in the asymmetric sinusoidal channel, and the height H of the detection channel is larger than the maximum diameter of a cell to be detected.
6. A non-labeled electrical impedance flow cytometer for treating circulating tumor cells as described in claim 1 wherein: the control panel has a cleaning mode, a calibration mode and a detection mode,
during a cleaning mode, the control board controls the switching valve to be switched to the first sample inlet, the control board controls the sample injection pump to pump cleaning solution in the first centrifuge tube through the stepping motor driving board and pumps the cleaning solution to the switching valve, the switching valve is switched to the liquid outlet, and the cleaning solution flows to the microfluidic chip through the liquid path pipeline to clean the microfluidic chip;
during a calibration mode, the control board controls the switching valve to be switched to a third sample inlet, the control board controls the sample pump to pump standard solution in the third centrifuge tube through the stepping motor driving board and pumps the standard solution to the switching valve, the switching valve is switched to a liquid outlet, the standard solution flows to the microfluidic chip through the liquid path pipeline, a multi-frequency excitation signal is applied to the microfluidic chip, the microfluidic chip outputs a feedback current signal to the impedance spectrometer, and the impedance spectrometer outputs an original signal to the computer to serve as a reference value of a cell electrical impedance signal;
during the detection mode, the control board controls the switching valve to be switched to the second sample inlet, the control board controls the sample injection pump to pump the solution to be detected in the second centrifuge tube through the stepping motor driving board and pumps the solution to the switching valve, the switching valve is switched to the liquid outlet, the solution to be detected flows to the microfluidic chip through the liquid path pipeline, the multi-frequency excitation signal is applied to the microfluidic chip, the microfluidic chip outputs the feedback current signal to the impedance spectrometer, and the impedance spectrometer outputs the original signal to the computer for signal processing and identification of the cell type.
7. A non-labeled electrical impedance flow cytometer for treating circulating tumor cells as described in claim 1 wherein: the device also comprises a power module and a direct current conversion module for power conversion, wherein the power module is respectively connected with the computer, the impedance spectrometer and the direct current conversion module, and the direct current conversion module is respectively connected with the control board, the stepping motor driving board, the sample injection pump and the switching valve.
8. A detection method based on the non-labeled electrical impedance flow cytometer for circulating tumor cells according to any of claims 1 to 7, comprising the steps of:
in the case of the three-electrode detection mode,
the control board controls the switching valve to switch to the second sample inlet, the control board controls the sample pump to pump the solution to be tested in the second centrifuge tube through the stepping motor driving board and pumps the solution to the switching valve, the switching valve is switched to the liquid outlet, and the solution to be tested flows to the microfluidic chip through the liquid path pipeline;
applying a multi-frequency excitation signal to the third electrode, outputting feedback current signals to the impedance spectrometer by the second electrode and the fourth electrode, wherein the first electrode and the fifth electrode are not externally connected with a signal source; the impedance spectrometer outputs an original signal to a computer, the computer firstly carries out noise reduction treatment on the original signal, the signal base line is leveled, the cell electrical impedance signal is extracted, and the cell type is identified based on a convolutional neural network model;
the number of alternating current frequencies applied to the third electrode is set as k in the convolutional neural network model, single cells undergo n times of signal sampling during the period that single cells pass through an electrical impedance detection area at a fixed flow rate, and cell electrical impedance signals intercepted at each frequency form a matrix of 2k rows and n columns; the real part of the cell alternating current electrical impedance signal under the 1 st action and the 1 st frequency in the matrix, and the imaginary part of the cell alternating current electrical impedance signal under the 1 st action and the 2 nd action in the matrix; by analogy, the imaginary part of the cell alternating current electrical impedance signal at the kth frequency of the kth behavior of 2; and (3) after carrying out convolution calculation on the matrix, sending the result into a maximum pooling layer, sending the result into a fully-connected neural network, and finally outputting cell type information.
9. A detection method based on the non-labeled electrical impedance flow cytometer for circulating tumor cells according to any of claims 1 to 7, comprising the steps of:
in the case of the five-electrode detection mode,
the control board controls the switching valve to switch to the second sample inlet, the control board controls the sample pump to pump the solution to be tested in the second centrifuge tube through the stepping motor driving board and pumps the solution to the switching valve, the switching valve is switched to the liquid outlet, and the solution to be tested flows to the microfluidic chip through the liquid path pipeline;
applying a multi-frequency excitation signal to the third electrode, outputting feedback current signals to the impedance spectrometer by the second electrode and the fourth electrode, and externally connecting an electric signal with opposite sign with the third electrode by the first electrode and the fifth electrode; the impedance spectrometer outputs an original signal to a computer, the computer firstly carries out noise reduction treatment on the original signal, the signal base line is leveled, the cell electrical impedance signal is extracted, and the cell type is identified based on a convolutional neural network model;
the number of alternating current frequencies applied to the third electrode is set as k in the convolutional neural network model, single cells undergo n times of signal sampling during the period that single cells pass through an electrical impedance detection area at a fixed flow rate, and cell electrical impedance signals intercepted at each frequency form a matrix of 2k rows and n columns; the real part of the cell alternating current electrical impedance signal under the 1 st action and the 1 st frequency in the matrix, and the imaginary part of the cell alternating current electrical impedance signal under the 1 st action and the 2 nd action in the matrix; by analogy, the imaginary part of the cell alternating current electrical impedance signal at the kth frequency of the kth behavior of 2; and (3) after carrying out convolution calculation on the matrix, sending the result into a maximum pooling layer, sending the result into a fully-connected neural network, and finally outputting cell type information.
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