A multifunctional electrical detection chip for detecting cell parameters

Abstract

The invention discloses a multifunctional electrical detection chip for detecting cell parameters, comprising various levels of flow channels connected in sequence through connecting flow channels; a pair of first-level conformable stereoscopic electrodes are symmetrically arranged on both sides of the end of the input flow channel close to the connecting flow channel; a pair of second-level conformable stereoscopic electrodes are symmetrically arranged on both sides of the end of the first-level contraction flow channel close to the connecting flow channel; a third-level conformable stereoscopic electrode is arranged on one side of the end of the first-level contraction flow channel close to the connecting flow channel; a fourth-level conformable stereoscopic electrode is arranged on one side of the middle of the second-level contraction flow channel; a fifth-level conformable stereoscopic electrode is arranged on one side of the end of the third-level contraction flow channel close to the connecting flow channel; a sixth-level conformable stereoscopic electrode pair is symmetrically arranged on both sides of the end of the third-level contraction flow channel close to the connecting flow channel; a seventh-level conformable stereoscopic electrode pair is symmetrically arranged on both sides of the end of the output flow channel close to the connecting flow channel; the ends of the conformable stereoscopic electrodes of each level are conformal to the wall of the corresponding flow channel, and the ends of the conformable stereoscopic electrodes of each level serve as the wall of the corresponding flow channel.

Description

Technical Field

[0001] The invention relates to the field of microfluidics, in particular to a multifunctional electrical detection chip for detecting cell parameters. Background Art

[0002] Live single-cell analysis is a core tool for revealing the behavior, function and microscopic heterogeneity of cells. It can effectively clarify the basic structure, functional information and pathological state of cells, and reveal the individual differences between cells. It is a research hotspot in the fields of cell biology, developmental biology, disease pathology, cell quality control analysis, drug development, medical diagnosis and treatment, etc. It is of great significance for revealing the laws of life and exploring the mechanisms of major diseases. By constructing micron-scale channels that match the size of cells, microfluidics technology provides extremely convenient conditions for achieving efficient manipulation and precise detection of cells.

[0003] The patent with application number 201910858617.X discloses a design method for a microfluidic chip, which can capture two types of single cells to achieve further analysis of cell characteristics. The patent with application number 202010226539.4 discloses a microfluidic chip that can accurately obtain the intrinsic size information of cells and cell nuclei through four T-shaped compression channels. Due to the limitations of the detection chip structure and impedance measurement strategy, the existing microfluidic impedance cytometer can usually only analyze and process some characteristic parameters, which affects the reliability of cell detection. In addition, most of the characteristic parameters extracted from the cell impedance peak can only indirectly characterize the overall dielectric properties and mechanical properties of the cell, but cannot provide insight into the local information of the cell. Therefore, at this stage, there is still a lack of an electrical detection chip that can characterize multiple parameters of the cell to be tested at one time. Summary of the invention

[0004] Purpose of the invention: The technical problem to be solved by the present invention is to provide a multifunctional electrical detection chip that can characterize multiple parameters of cells at one time in view of the deficiencies in the prior art.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0006] A multifunctional electrical detection chip for detecting cell parameters, comprising an input channel, a primary contraction channel, a primary recovery channel, a secondary contraction channel, a secondary recovery channel, a tertiary contraction channel, and an output channel, which are sequentially connected through a connecting channel;

[0007] A pair of first-level conformal three-dimensional electrodes are symmetrically arranged on both sides of the end of the input flow channel close to the rear connecting flow channel;

[0008] The first-level contraction channel is symmetrically provided with a pair of second-level conformal three-dimensional electrodes on both sides of the end portion close to the front connection channel;

[0009] A three-stage conformal stereoscopic electrode is provided on one side of the end of the first-stage contraction channel close to the rear connection channel;

[0010] A four-stage conformal three-dimensional electrode is provided on one side of the middle portion of the secondary contraction flow channel;

[0011] A five-level conformal three-dimensional electrode is provided on one side of the end of the three-level contraction flow channel close to the front connecting flow channel;

[0012] Six-stage conformal stereoscopic electrode pairs are symmetrically arranged on both sides of the end of the three-stage contraction flow channel close to the rear connection flow channel;

[0013] The output flow channel is symmetrically provided with seven levels of conformal stereoscopic electrode pairs on both sides of the end close to the front connecting flow channel;

[0014] The ends of the conformal three-dimensional electrodes at each level are conformal to the wall surface of the corresponding flow channel, and the ends of the conformal three-dimensional electrodes at each level serve as the wall surface of the corresponding flow channel.

[0015] Furthermore, the cross-sections of the input flow channel, the primary contraction flow channel, the primary recovery flow channel, the secondary contraction flow channel, the secondary recovery flow channel, the tertiary contraction flow channel, the output flow channel, and the connecting flow channels therebetween are all circular;

[0016] The flow channel diameters of the input flow channel, the first-level recovery flow channel, the second-level recovery flow channel and the output flow channel are the same and slightly larger than the diameter of the cells to be tested; the flow channel diameters of the first-level contraction flow channel, the second-level contraction flow channel and the third-level contraction flow channel are the same and slightly smaller than the diameter of the cells to be tested.

[0017] Preferably, the three-level conformal stereoscopic electrode and the five-level conformal stereoscopic electrode are symmetrically distributed with the four-level conformal stereoscopic electrode as the symmetry axis;

[0018] The three-level conformal stereo electrode and the five-level conformal stereo electrode are located on the same side, and the four-level conformal stereo electrode is located on the other side.

[0019] Specifically, the first-level conformable stereo electrode pair and the seventh-level conformable stereo electrode pair constitute a group of electrode pairs; the second-level conformable stereo electrode pair and the sixth-level conformable stereo electrode pair constitute a group of electrode pairs; the third-level conformable stereo electrode, the fourth-level conformable stereo electrode and the fifth-level conformable stereo electrode constitute a group of electrode pairs. After injecting multi-frequency voltage signals into the upper electrodes of the chip in the three groups of electrode pairs, the current signals collected by the lower electrodes of the chip are processed by the differential circuit to obtain positive and negative wave signals. After filtering, signal segmentation and impedance peak extraction and analysis of the positive and negative wave signals, the cell position, cell diameter, cell shape parameters, impedance amplitude, impedance phase parameters, cell impedance opacity (ratio of high-frequency impedance amplitude to low-frequency impedance amplitude) parameters and relaxation index of the cells in different states can be obtained.

[0020] Preferably, the sealing material of each level of flow channel is selected from any one of transparent materials such as PDMS, glass or PMMA; the material of each level of electrode is selected from any one of conductive materials such as gold, ITO or nanosilver.

[0021] Beneficial effects:

[0022] The circular cross-section flow channel of the multifunctional electrical detection chip of the present invention is conducive to the transportation and extrusion deformation of cells, and the symmetry of the circular cross-section of the flow channel is more convenient for theoretical analysis and modeling simulation calculation. The conformal three-dimensional electrode structure that conforms to the circumferential wall of the flow channel increases the volume fraction of cells in the detection area and reduces the leakage of the measured electrical signal, thereby effectively improving the sensitivity of cell impedance measurement and enabling one-time characterization of multiple parameters, which can play an important role in the development and clinical application of single-cell analysis technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0024] Figure 1 It is a schematic diagram of the overall structure of the multifunctional electrical detection chip of the present invention.

[0025] Figure 2 It is a partial schematic diagram of the input flow channel and the first-level conformal three-dimensional electrode pair at the end in the multifunctional electrical detection chip of the present invention.

[0026] Figure 3 It is the signal detection result when the cell to be tested enters the position of the first-level conformable three-dimensional electrode pair.

[0027] Figure 4 It is the signal detection result when the cell to be tested enters the position of the secondary conformal stereo electrode pair.

[0028] Figure 5 It is the signal detection result when the cell to be tested enters the position of the three-level conformal stereo electrode.

[0029] Wherein, each reference numeral represents:

[0030] 11. Input channel, 12. Primary contraction channel, 13. Primary recovery channel, 14. Secondary contraction channel, 15. Secondary recovery channel, 16. Thirdary contraction channel, 17. Output channel, 21. Primary conformable stereoscopic electrode pair, 22. Secondary conformable stereoscopic electrode pair, 23. Thirdary conformable stereoscopic electrode, 24. Fourth conformable stereoscopic electrode, 25. Fifth conformable stereoscopic electrode, 26. Sixth conformable stereoscopic electrode pair, 27. Seventh conformable stereoscopic electrode pair, 31. First connecting channel, 32. Second connecting channel, 33. Third connecting channel, 34. Fourth connecting channel, 35. Fifth connecting channel, 36. Sixth connecting channel. DETAILED DESCRIPTION

[0031] The present invention will be described in detail below in conjunction with the accompanying drawings.

[0032] like Figure 1 As shown, the multifunctional electrical detection chip includes an input channel 11, a primary contraction channel 12, a primary recovery channel 13, a secondary contraction channel 14, a secondary recovery channel 15, a tertiary contraction channel 16, and an output channel 17 which are sequentially connected through connecting channels.

[0033] Among them, the input channel 11 is connected to the first-level contraction channel 12 through the first connecting channel 31; the first-level contraction channel 12 is connected to the first-level recovery channel 13 through the second connecting channel 32; the first-level recovery channel 13 and the second-level contraction channel 14 are connected through the third connecting channel 33; the second-level contraction channel 14 and the second-level recovery channel 15 are connected through the fourth connecting channel 34; the second-level recovery channel 15 and the third-level contraction channel 16 are connected through the fifth connecting channel 35; the third-level contraction channel 16 and the output channel 17 are connected through the sixth connecting channel 36.

[0034] A first-level conformal stereoscopic electrode pair 21 is symmetrically provided on both sides of the end of the input flow channel 11 close to the first connecting flow channel 31;

[0035] A secondary conformal stereoscopic electrode pair 22 is symmetrically provided on both sides of the end of the primary contraction channel 12 close to the first connecting channel 31;

[0036] A three-stage conformal stereoscopic electrode 23 is provided on one side of the end of the first-stage contraction channel 12 close to the second connecting channel 32;

[0037] A four-stage conformal three-dimensional electrode 24 is provided on one side of the middle portion of the secondary contraction flow channel 14;

[0038] A five-level conformal three-dimensional electrode 25 is provided on one side of the end of the three-level contraction channel 16 close to the fifth connecting channel 35;

[0039] Six-stage conformal stereoscopic electrode pairs 26 are symmetrically arranged on both sides of the end of the three-stage contraction channel 16 close to the sixth connecting channel 36;

[0040] Seven levels of conformal three-dimensional electrode pairs 27 are symmetrically provided on both sides of the end of the output channel 17 close to the sixth connecting channel 36 .

[0041] like Figure 2 As shown, the ends of the first-stage conformal stereoscopic electrode pair 21 conform to the corresponding wall surface of the input flow channel 11, and the ends of the first-stage conformal stereoscopic electrode pair 21 serve as the wall surface of the input flow channel 11. The ends of the other stages of conformal stereoscopic electrodes also conform to the corresponding flow channel wall surface, and the ends of the conformal stereoscopic electrodes also serve as the corresponding flow channel wall surface.

[0042] Among them, the cross-sections of the input flow channel 11, the first-level contraction flow channel 12, the first-level recovery flow channel 13, the second-level contraction flow channel 14, the second-level recovery flow channel 15, the third-level contraction flow channel 16, the output flow channel 17, and the connecting flow channels therebetween are all circular; the flow channel diameters of the input flow channel 11, the first-level recovery flow channel 13, the second-level recovery flow channel 15, and the output flow channel 17 are the same and larger than the diameter of the cells to be measured; the flow channel diameters of the first-level contraction flow channel 12, the second-level contraction flow channel 14, and the third-level contraction flow channel 16 are the same and smaller than the diameter of the cells to be measured.

[0043] The three-level conformal 3D electrode 23 and the five-level conformal 3D electrode 25 are symmetrically distributed with the four-level conformal 3D electrode 24 as the symmetry axis; the three-level conformal 3D electrode 23 and the five-level conformal 3D electrode 25 are located on the same side, and the four-level conformal 3D electrode 24 is located on the other side.

[0044] The first-level conformable stereo electrode pair 21 and the seventh-level conformable stereo electrode pair 27 form a group of electrode pairs to detect the signal of the cell in the entire detection area; the second-level conformable stereo electrode pair 22 and the sixth-level conformable stereo electrode pair 26 form a group of electrode pairs to detect the signal of the cell in the entire contraction channel; the third-level conformable stereo electrode 23, the fourth-level conformable stereo electrode 24 and the fifth-level conformable stereo electrode 25 form a group of electrode pairs to detect the signal of the cell in the stage of restoring deformation. After injecting multi-frequency voltage signals into the upper electrodes of the chip in the three groups of electrode pairs, the current signals collected by the lower electrodes of the chip are processed by the differential circuit to obtain positive and negative wave signals. After filtering, signal segmentation and impedance peak extraction and analysis of the positive and negative wave signals, the cell position, cell diameter, cell shape parameters, impedance amplitude, impedance phase parameters, cell impedance opacity (ratio of high-frequency impedance amplitude to low-frequency impedance amplitude) parameters and relaxation index of the cells in different states can be characterized at one time.

[0045] The sealing materials of the flow channels at each level are all selected from PDMS transparent materials; and the materials of the electrodes at each level are all selected from gold conductive materials.

[0046] In this embodiment, the cross-sectional circular diameter of the input channel 11, the first-level recovery channel 13, the second-level recovery channel 15 and the output channel 17 of the multifunctional electrical detection chip is 30 μm, the cross-sectional circular diameter of the first-level contraction channel 12, the second-level contraction channel 14 and the third-level contraction channel 16 is 5 μm, and the cell suspension is selected as MCF-7 suspension, and the MCF-7 suspension is injected into the multifunctional electrical detection chip through the input channel. After the MCF-7 suspension fills the entire chip chamber, the multifunctional electrical detection chip is connected to a pressure injector, and a constant pressure is given by the pressure injector. Breast cancer cells are placed from the input channel, and the breast cancer cells move with the MCF-7 suspension. Adjust the function signal generator to generate an AC excitation voltage with an amplitude of 1V and a frequency of 2MHz. When the breast cancer cells move in the input channel 11, an AC excitation voltage is applied to the first-level conformable stereo electrode pair 21 and the seventh-level conformable stereo electrode pair 27 to obtain a current response signal I 1 (Signal1) and I 6 (Signal6), such as Figure 3 When the breast cancer cells move to the head of the primary contraction channel 13, an excitation voltage is applied to the secondary conformable stereo electrode pair 22 and the sixth conformable stereo electrode pair 26 to obtain a current response signal I 2 (Signal2) and I 5 (Signal5), such as Figure 4 When breast cancer cells move in the primary recovery channel 13, the secondary contraction channel 14 and the secondary recovery channel 15, an excitation voltage is applied to the four-stage conformal stereo electrode 24, and a current response signal I is obtained through the three-stage conformal stereo electrode 23. 3 (Signal3), the current response signal I is obtained through the five-level conformal stereo electrodes 25 4 (Signal4), such as Figure 5 As shown, the collected I 1 ~I 6After the signal is processed by the differential circuit, positive and negative wave signals are obtained, and then it enters the signal conditioning stage. In the signal conditioning stage, the moving average filter is used to perform baseline correction and denoising on the original electrical impedance characteristic signal; in the signal segmentation stage, the conditioned signal is segmented by setting a threshold or reversely identifying the idle time to obtain the corresponding valid data when the cell passes through the sensing area; in the feature extraction stage, Gaussian fitting is used to extract characteristic parameters such as peak value, peak width and passing time from the segmented signal; finally, in the feature processing stage, the influence of different cell positions and fluid fluctuations is considered, and the extracted characteristic parameters are further processed to improve the accuracy of the measurement. After analyzing Signal1 and Signal6 separately and comprehensively, the cell position, cell diameter and cell shape parameters of breast cancer cells at this time can be extracted; after analyzing Signal2 and Signal5 separately and comprehensively, the time parameters of the last contraction and the first contraction of breast cancer cells can be extracted, and the time ratio of the last contraction and the first contraction is the relaxation parameter of the quantified cell; after analyzing Signal3 and Signal4 separately and comprehensively, the impedance amplitude and impedance phase parameters of breast cancer cells at this time can be extracted; at the same time, multi-frequency voltage signals can be injected to obtain cell impedance opacity parameters (the ratio of high-frequency impedance amplitude to low-frequency impedance amplitude).

[0047] The present invention provides a multifunctional electrical detection chip for detecting cell parameters. There are many methods and approaches to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the protection scope of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. A multifunctional electrical detection chip for detecting cell parameters, characterized in that: It comprises an input flow channel (11), a primary contraction flow channel (12), a primary recovery flow channel (13), a secondary contraction flow channel (14), a secondary recovery flow channel (15), a tertiary contraction flow channel (16), and an output flow channel (17) which are sequentially connected via a connecting flow channel; A pair of first-level conformal three-dimensional electrodes (21) are symmetrically provided on both sides of the end of the input flow channel (11) close to the rear connecting flow channel; The first-stage contraction flow channel (12) is symmetrically provided with a second-stage conformal three-dimensional electrode pair (22) on both sides of the end portion close to the front connecting flow channel; A three-stage conformal three-dimensional electrode (23) is provided on one side of the end of the first-stage contraction flow channel (12) close to the rear connection flow channel; A four-stage conformable three-dimensional electrode (24) is provided on one side of the middle portion of the secondary contraction flow channel (14); The three-stage contraction flow channel (16) is provided with a five-stage conformal three-dimensional electrode (25) on one side of the end portion close to the front connecting flow channel; Six-stage conformal stereoscopic electrode pairs (26) are symmetrically arranged on both sides of the end of the three-stage contraction flow channel (16) close to the rear connecting flow channel; Seven-stage conformal three-dimensional electrode pairs (27) are symmetrically arranged on both sides of the end of the output flow channel (17) close to the front connecting flow channel; The ends of the conformal three-dimensional electrodes at each level are conformal to the wall of the corresponding flow channel, and the ends of the conformal three-dimensional electrodes at each level serve as the wall of the corresponding flow channel; The input flow channel (11), the primary contraction flow channel (12), the primary recovery flow channel (13), the secondary contraction flow channel (14), the secondary recovery flow channel (15), the tertiary contraction flow channel (16), the output flow channel (17), and the connecting flow channels therebetween are all circular in cross section; The flow channel diameters of the input flow channel (11), the first-level recovery flow channel (13), the second-level recovery flow channel (15), and the output flow channel (17) are the same and larger than the diameter of the cells to be tested; the flow channel diameters of the first-level contraction flow channel (12), the second-level contraction flow channel (14), and the third-level contraction flow channel (16) are the same and smaller than the diameter of the cells to be tested; The three-level conformable three-dimensional electrode (23) and the five-level conformable three-dimensional electrode (25) are symmetrically distributed with the four-level conformable three-dimensional electrode (24) as a symmetry axis; The three-level conformal stereo electrode (23) and the five-level conformal stereo electrode (25) are located on the same side, and the four-level conformal stereo electrode (24) is located on the other side; The first-level conformable stereo electrode pair (21) and the seventh-level conformable stereo electrode pair (27) form a group of electrode pairs; the second-level conformable stereo electrode pair (22) and the sixth-level conformable stereo electrode pair (26) form a group of electrode pairs; the third-level conformable stereo electrode (23), the fourth-level conformable stereo electrode (24) and the fifth-level conformable stereo electrode (25) form a group of electrode pairs; The sealing material of each level of flow channel is selected from any one of PDMS, glass or PMMA; the material of each level of electrode is selected from any one of gold, ITO or nano silver; When the breast cancer cells move to the head of the primary contraction channel (12), an excitation voltage is applied to the secondary conformal stereo electrode pair (22) and the sixth conformal stereo electrode pair (26) to obtain current response signals (Signal2) and (Signal5); when the breast cancer cells move in the primary recovery channel (13), the secondary contraction channel (14) and the secondary recovery channel (15), an excitation voltage is applied to the fourth conformal stereo electrode (24), and a current response signal (Signal3) is obtained through the third conformal stereo electrode (23), and a current response signal (Signal4) is obtained through the fifth conformal stereo electrode (25); the collected ~ signal is processed by a differential circuit to obtain positive and negative wave signals, and then enters the signal conditioning stage; in the signal conditioning stage, a moving average filter is used to perform baseline correction and de-interference on the original electrical impedance characteristic signal. Noise processing; in the signal segmentation stage, the conditioned signal is segmented by setting a threshold or reversely identifying the idle time to obtain the valid data corresponding to the cell passing through the sensing area; in the feature extraction stage, Gaussian fitting is used to extract characteristic parameters such as peak value, peak width and passing time from the segmented signal; finally, in the feature processing stage, the influence of different cell positions and fluid fluctuations is considered, and the extracted characteristic parameters are further processed to improve the measurement accuracy; after separate and comprehensive analysis of Signal1 and Signal6, the cell position, cell diameter and cell shape parameters of breast cancer cells at this time can be extracted; after separate and comprehensive analysis of Signal2 and Signal5, the time parameters of the last contraction and the first contraction of breast cancer cells can be extracted, and the time ratio of the last contraction to the first contraction is the relaxation parameter of the quantified cell; After analyzing Signal3 and Signal4 individually and comprehensively, the impedance amplitude and impedance phase parameters of breast cancer cells at this time can be extracted; at the same time, multi-frequency voltage signals are injected to obtain cell impedance opacity parameters.

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