CN112461751B - Cancer cell activity detection and evaluation device and method based on multi-adhesion strength fusion - Google Patents
Cancer cell activity detection and evaluation device and method based on multi-adhesion strength fusion Download PDFInfo
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Abstract
The invention discloses a cancer cell activity detection evaluation device and method based on multi-adhesion strength fusion.A transparent cover of a centrifuge is used as a load of an electrode leading-out end, an impedance analyzer is connected to the transparent cover, when the transparent cover is closed, the electrode leading-out end on the transparent cover can be contacted with the electrode leading-out end on a microfluidic chip to directly measure an impedance value in the microfluidic chip, an annular channel is arranged in the microfluidic chip, and when the microfluidic chip is centrifuged, the stress difference of cells in the annular channel is small, so that the adhesion strength of the cells can be well screened; the cell adhesion strength is used as a physical index for quantitatively evaluating the heterogeneity of cell activity, a plurality of cell adhesion strength parameters are used as input through a RBF neural network, and the IC of an anticancer drug reaction experiment is used50The value of (A) is output, and a cell activity evaluation prediction model is established, so that the method is more accurate than a method for evaluating cells by using a single parameter.
Description
Technical Field
The invention relates to the technical field of detection and evaluation of cell activity, in particular to a cancer cell activity detection and evaluation device and method based on multi-adhesion strength information fusion.
Background
Inaccurate cell activity assessment can lead to serious consequences such as failure of anticancer drug tests and inaccurate cytotoxicology studies, so that how to accurately detect cell activity is very important. The patent document with the Chinese patent application number of 201910858707.9 discloses a fluorescent cell counting method, which includes the steps of obtaining a fluorescent image to be counted, carrying out binarization processing on the image to obtain a binarized image, identifying a cell area in the image according to preset cell area parameters, counting the number of fluorescent cells, and evaluating cell activity according to the number of the fluorescent cells, wherein the cell is dyed by the counting method to cause certain damage and toxicity to the cells, so that the physiological function of the cells is changed, deviation is easily caused, and the image is easily influenced by impurities in the shooting process to interfere detection. The document of chinese patent application No. 201911173367.2 proposes an apparatus and method for evaluating cell activity based on adhesion strength, which considers the heterogeneity of cell activity, but evaluates cell activity using a single parameter with large error.
Disclosure of Invention
Aiming at the defects of low accuracy and error in manual operation when the activity of cells is evaluated by a single parameter at present, the invention provides a cancer cell activity detection and evaluation device and method based on multi-adhesion strength fusion,
and (3) centrifuging the centrifugal microfluidic chip by using a centrifuge, extracting multi-parameter information of cell adhesion strength, establishing a cell activity evaluation prediction model, and accurately and comprehensively evaluating the cell activity.
The cancer cell activity detection and evaluation device based on multi-adhesion strength fusion realizes the technical purpose through the following technical scheme: including a centrifuge, an MCU controller, an impedance analysis appearance and a PC end host computer, characterized by: the outer part of the centrifugal machine is formed by connecting a transparent cover and a base, a rotating motor, an elastic buckle and a centrifugal micro-fluidic chip are respectively arranged in the base from bottom to top, the rotating motor is vertically arranged in the middle of the inner part of the base, an output shaft is vertically upward and is connected with the centrifugal micro-fluidic chip which is horizontally arranged through the elastic buckle; the lower half part of the transparent cover is in a circular ring shape, a semicircular counter electrode lead-out metal layer and a semicircular working electrode lead-out metal layer are tightly attached to the surface of the circular lower ring, and the counter electrode lead-out metal layer and the working electrode lead-out metal layer on the transparent cover are respectively connected with the input end of an impedance analyzer; the outer surface of the top of the base is fixedly connected with a steering engine, and the steering engine can drive the transparent cover to open and close by rotating clockwise and anticlockwise; the centrifugal micro-fluidic chip is formed by coaxially and tightly attaching a disc-shaped PDMS cover plate and a PDMS substrate up and down, wherein the PDMS substrate protrudes out of an edge area of a circular ring relative to the PDMS cover plate along the diameter direction, a counter electrode lead-out metal layer and a working electrode lead-out metal layer are tightly attached to the upper surface of the edge area, when the transparent cover is closed, the counter electrode lead-out metal layer on the edge area is contacted with the counter electrode lead-out metal layer on the transparent cover, and the working electrode lead-out metal layer on the edge area is contacted with the working electrode lead-out metal layer on the transparent cover; an annular channel is surrounded on the periphery of the middle of the PDMS substrate, a plurality of separation channels which are all arranged along the diameter direction are uniformly surrounded on the periphery of the annular channel, the radial outer end of each separation channel is communicated with a circular outlet, the radial inner end of each separation channel is communicated with the annular channel, annular interdigital electrodes are etched in the annular channel, and one end of a counter electrode lead-out metal layer and one end of a working electrode lead-out metal layer on the edge area are respectively connected with the annular interdigital electrodes; the PC end upper computer is associated with the MCU controller through the WIFI module, and the MCU controller is connected with the input end of the impedance analyzer through a serial port and is also connected with the rotating motor and the steering engine through a serial port.
The detection and evaluation method of the cancer cell activity detection and evaluation device based on multi-adhesion strength fusion realizes the technical purpose through the following technical scheme: the method comprises the following steps:
step 1: culturing cancer cells in the same culture dish for the first time, wherein a part of cancer cell population A is used for half-inhibition rate IC50To obtain IC50Value X of1And the rest part of the cancer cell group B is reserved;
step 2: the PC end upper computer controls the transparent cover to be opened, a cell-free culture medium is dripped into the centrifugal micro-fluidic chip, the rotating motor is controlled to drive the centrifugal micro-fluidic chip to rotate at the maximum speed, the transparent cover is controlled to be closed, and the impedance analyzer obtains a cell impedance threshold value Z0And uploading to a PC end upper computer;
and step 3: opening the transparent cover, dripping the cancer cell group B into the centrifugal microfluidic chip, closing the transparent cover, and obtaining an initial cancer cell impedance value N by the impedance analyzer0And uploading to a PC end upper computer;
and 4, step 4: opening the transparent cover and rotating the motor at an initial speed omega0Driving the centrifugal microfluidic chip to rotate for a set time;
and 5: controlling the rotating motor to stop running, closing the transparent cover, and obtaining a cell impedance value N by the impedance analyzer1And uploading to a PC end upper computer; the PC end upper computer calculates the cell index CI ═ CI1(ii) a Then the transparent cover is opened again, and the rotating motor is controlled to be omega1＝ω0+ delta omega is accelerated, and delta omega is the rotation speed difference of one acceleration;
step 6: repeating the step 5 for n times, and obtaining n cell indexes CI (CI) by the PC end upper computer1,CI2,…,CIn]N rotation speeds ω ═ ω0,ω2,…,ωn-1]And N cell impedance values [ N1，N2……，Nn]N cell impedance values [ N ]1，N2……，Nn]And the cell impedance threshold value Z0By comparison, when less than or equal to the impedance threshold Z0Stopping repeated execution;
and 7: respectively calculating n rotating speeds omega by the PC-end upper computer to obtain n centrifugal forces tau, and respectively changing n cell indexes CI to [ CI ═ CI1,CI2,…,CIn]Fitting the calculated values with n centrifugal forces to obtain a sigmoid function CI ═ f (tau), and calculating a first set of cell adhesion strengths [ tau ] according to the inverse function of the sigmoid function CI ═ f (tau)0,τ10,...,τ90]，τNRepresents the centrifugal force to which (100-N)% of cells are detached, N is 0,10, …, 90;
and 8: repeating steps 1-7 to obtain a second group of cell adhesion strengths [ tau ]0,τ10,...,τ90]And a second half-inhibition ratio IC50Value X2；
And step 9: repeating the step 8 to obtain n groups of cell adhesion strength [ tau ]0,τ10,...,τ90]And n ICs50Value of (2) ([ X ]1,X2,...,Xn]The adhesion strength [ tau ] of n groups of cells0,τ10,...,τ90]As input, n ICs50Value of (2) ([ X ]1,X2,...,Xn]Establishing a cancer cell activity RBF neural network prediction model X ═ f (tau) as output;
step 10: taking out the cancer cell group to be detected from the new culture dish, dripping the cancer cell group into the centrifugal microfluidic chip, and respectively and repeatedly executing the step 4, the step 5, the step 6 and the step 7 to obtain the cell adhesion strength [ tau'0,τ′10,...,τ′90]，τ′NCentrifugal force N ═ 0,10, …,90, indicating that (100-N)% of cells detached; cell adhesion Strength [ tau'0,τ′10,...,τ′90]Inputting the obtained data into the prediction model X ═ f (tau) to obtain corresponding IC50Value X' of (A), and IC50Value X' of and cell viabilityThe cellular activity was assessed by sex correlation.
Compared with the prior art, the method has the following advantages:
1. generally, an impedance analyzer is required to be connected to a microfluidic chip for measuring an impedance value, but when a centrifugal machine rotates, a wire needs to be manually taken down, otherwise the microfluidic chip can throw the wire away when rotating, for the condition, the invention designs a full-automatic device, a transparent cover of the centrifugal machine is used as a load of an electrode leading-out end, the impedance analyzer is connected to the transparent cover, and when the transparent cover is closed, the electrode leading-out end on the transparent cover can be contacted with the electrode leading-out end on the microfluidic chip, so that the impedance value in the microfluidic chip can be directly measured. The transparent cover of the centrifuge is used as a carrier of the electrode leading-out end, the impedance analyzer is directly connected to the transparent cover to measure impedance, manual wiring is not needed after centrifugation is finished every time, and automation is realized.
2. The invention designs a centrifugal micro-fluidic chip, wherein an annular channel is arranged in the micro-fluidic chip, when the centrifugal micro-fluidic chip is used for centrifugation, the stress difference of cells in the annular channel is small, the cells are easy to count, and the adhesion strength of the cells can be well screened out.
3. For the situation that the error of the cell activity is large by using a single parameter to evaluate the cell activity, the invention considers the heterogeneity of the cell activity, adopts the cell adhesion strength as a physical index for quantitatively evaluating the heterogeneity of the cell activity, takes a plurality of cell adhesion strength parameters as input through an RBF neural network, and takes the IC of an anticancer drug reaction experiment as the input50The cell activity evaluation prediction model is established, a plurality of cell adhesion strength parameters are fused, the multi-parameter information fusion evaluation method is used, the cell activity is comprehensively evaluated by giving a larger weight to strong adhesion cells and a smaller weight to weak adhesion cells in a cell population, and the method is more accurate than the method for evaluating the cells by a single parameter.
4. According to the invention, no additional reagent is required to be added in the process of measuring the cell activity, so that the nondestructive detection of the cell is realized.
Drawings
FIG. 1 is a schematic diagram of the overall structure of the device for detecting and evaluating the activity of cancer cells based on fusion of multiple adhesion strengths according to the present invention;
fig. 2 is an enlarged view of the external structure of the centrifuge 1 when the transparent cover 2 of fig. 1 is closed;
fig. 3 is a bottom view of the transparent cover 2 of fig. 1;
fig. 4 is an enlarged view of the centrifugal microfluidic chip 7 in fig. 1;
FIG. 5 is a top view of the PDMS substrate 9 of FIG. 4;
fig. 6 is a schematic view of a connection structure of the annular interdigital electrode 23 shown in fig. 5;
fig. 7 is an enlarged front view of the elastic buckle 6 in fig. 1;
FIG. 8 is a flow chart of the detection evaluation method of the cancer cell activity detection evaluation device based on multi-adhesion strength fusion according to the present invention.
The serial numbers and names of the various components in the drawings: 1. the device comprises a centrifugal machine, a 2 part, a transparent cover, a 3 part, a base, a 4 part, a liquid crystal screen, a 5 part, a rotating motor, a 6 part, an elastic buckle, a 7 part, a centrifugal micro-fluidic chip, a 8 part, a PDMS cover plate, a 9 part, a PDMS substrate, a 10 part, an inlet, an 11 part, a counter electrode lead-out metal layer of an interdigital electrode on the micro-fluidic chip, a 12 part, a working electrode lead-out metal layer of the interdigital electrode on the micro-fluidic chip, a 13 part, a counter electrode lead-out metal layer on the transparent cover, a 14 part, a working electrode lead-out metal layer on the transparent cover, a 15 part, an MCU controller, a 16 part, an impedance analyzer, a 17 part, a WIFI module, an 18 part, a PC end upper computer, a 19 part, a fixed port, a 20 part, a ring-shaped channel, a 21 part, an outlet, a 22 part, a separation channel, a 23 part, a ring-shaped interdigital electrode, a 24 part, a 25 part, a first detection hole, a 26 part, a second detection hole, a 27 part, an edge area, a 28 part, a small hook, a 29 part, a groove area, a 30 part, a steering engine, a 31 part, a steering wheel, a cover, a, A connecting arm.
Detailed Description
Referring to fig. 1, the cancer cell activity detection and evaluation device based on multi-adhesion strength fusion of the present invention comprises a centrifuge 1, a MCU controller 15, an impedance analyzer 16 and a PC host computer 18. Centrifuge 1 includes rotating electrical machines 5, elasticity are buckled 6, transparent lid 2 and base 3 etc. and centrifuge 1's outside is from last down to be connected by transparent lid 2 and base 3 and is constituteed, has a LCD screen 4 in the middle of the outer wall of base 3 is positive, shows centrifuge 1's centrifugation time and rotational speed on LCD screen 4. The inside of the base 3 is provided with a rotating motor 5, an elastic buckle 6 and a centrifugal micro-fluidic chip 7 from bottom to top respectively. The rotating motor 5 is vertically arranged in the middle of the inside of the base 3, an output shaft is vertically upward, an elastic buckle 6 is vertically fixed at the top end of the output shaft of the rotating motor 5, and the rotating motor is connected with a centrifugal microfluidic chip 7 which is horizontally arranged through the elastic buckle 6.
Referring to fig. 2, the transparent cover 2 of the centrifuge 1 is composed of two parts, the upper half part is a hollow hemisphere, the lower half part is a ring 24, the inner diameter of the ring 24 is R1, and the outer diameter is R2. The outer surface of the top of the base 3 is fixedly connected with a steering engine 30, an output shaft of the steering engine 30 is coaxially sleeved with a round rudder disc 31, the steering engine 30 and the rudder disc 31 move synchronously, one end of a connecting arm 32 is fixed on the outer surface of the rudder disc 31, the other end of the connecting arm 32 is fixedly connected to the outer surface of the bottom of the circular ring 24 of the transparent cover 2, and the steering engine 30 can drive the connecting arm 32 to rotate through clockwise and anticlockwise rotation by 90 degrees, so that the transparent cover 2 can be opened and closed.
Referring to fig. 3, two electrode lead-out metal layers, namely, a counter electrode lead-out metal layer 13 and a working electrode lead-out metal layer 14, are tightly adhered to the surface of the lower ring of the ring-shaped ring 24 of the transparent cover 2, the two electrode lead-out metal layers are both semicircular, and the ring widths of the two electrode lead-out metal layers are both d 1. The semicircular openings of the two electrode lead-out metal layers are arranged in a face-to-face mode, the inner diameter of the counter electrode lead-out metal layer 13 is smaller than that of the working electrode lead-out metal layer 14, the inner diameter of the counter electrode lead-out metal layer 13 is larger than the inner diameter R1 of the circular ring 24, and the outer diameter of the working electrode lead-out metal layer 14 is smaller than the outer diameter R2 of the circular ring 24. The two electrode lead-out metal layers are respectively provided with two ends, and one end of each electrode lead-out metal layer is provided with a through hole which is vertical up and down and is communicated with the outside of the transparent cover 2. The through hole opened at one end of the counter electrode lead-out metal layer 13 is a first detection hole 25, and the through hole opened at one end of the working electrode lead-out metal layer 14 is a second detection hole 26. The counter electrode lead-out metal layer 13 and the working electrode lead-out metal layer 14 are led out to the outside of the transparent cover 2 through the first detection hole 25 and the second detection hole 26, respectively, and then are connected to the input end of the impedance analyzer 16, see fig. 1.
Referring to fig. 4, the centrifugal microfluidic chip 7 is composed of two parts of discs, the upper half part is a disc-shaped PDMS cover plate 8, the lower half part is a disc-shaped PDMS substrate 9, and the two parts are coaxial and tightly attached up and down. In the middle area of the microfluidic chip 7 is a square fixing port 19 with a cross-sectional area of S1, and on the upper surface of the PDMS cover plate 8 is an inlet 10, from which the culture medium and cells are dropped. The outer diameter of the PDMS cover plate 8 is R3, which is smaller than the outer diameter R4 of the PDMS substrate 9, so that the PDMS substrate 9 protrudes a ring region along the diameter direction relative to the PDMS cover plate 8, the part of the ring region of the PDMS substrate 9 that is larger than the PDMS cover plate 8 is called as an edge region 27, a counter electrode lead-out metal layer 11 and a working electrode lead-out metal layer 12 are tightly attached to the upper surface of the edge region 27, the ring width of the two metal layers is d1, and the two electrode lead-out metal layers are face-to-face openings. The inner diameter of the counter electrode lead-out metal layer 11 on the chip is the same as the inner diameter of the counter electrode lead-out metal layer 13 on the transparent cover 2, and the inner diameter of the working electrode lead-out metal layer 12 on the chip is the same as the inner diameter of the working electrode lead-out metal layer 14 on the transparent cover 2. The outer diameter R3 of the PDMS cover sheet 8 is smaller than the inner diameter R1 of the upper ring 24, and the outer diameter R4 of the PDMS substrate 9 is smaller than the outer diameter R2 of the upper ring 24, so as to ensure that the edge region 27 fits perfectly with the ring 24 on the transparent cover 2 when the transparent cover 2 is closed, at this time, the counter electrode lead-out metal layer 11 on the edge region 27 contacts with the counter electrode lead-out metal layer 13 on the transparent cover 2, and the working electrode lead-out metal layer 12 on the edge region 27 contacts with the working electrode lead-out metal layer 14 on the transparent cover 2.
Referring to fig. 5, the middle region of the PDMS substrate 9 is a square fixing port 19 having a cross-sectional area of S1, and a circular channel 20 is surrounded at the periphery of the fixing port 19. The annular channel 20 is surrounded by a plurality of funnel-shaped separation channels 22 (15 separation channels 22 are shown in fig. 5), the separation channels 22 are arranged in a diameter direction, the radial outer end of each separation channel 22 is communicated with a circular outlet 21, and the radial inner end of each separation channel 22 is communicated with the annular channel 20. When the centrifugal microfluidic chip 7 is rotated and centrifuged, the less active cells will be separated from the annular channel 20 and will pass through the separation channel 22 to the outlet 21, and the more active cells will still adhere to the annular channel 20. The plurality of separation channels 22 is designed to allow the exfoliated cells to enter the outlet 21 more quickly and efficiently, so as not to cause unnecessary errors in the counting of the cells in the annular channel 20. An annular interdigital electrode 23 is etched within the annular channel 20. The outermost periphery of the PDMS substrate 9 is the rim region 27. One end of the counter electrode lead-out metal layer 11 on the edge region 27 is led out from the annular channel 20, and one end of the working electrode lead-out metal layer 12 is led out from the annular channel 20.
Referring to fig. 6, the annular interdigital electrode 23 is arc-shaped, and is uniformly etched on the upper surface of the annular channel 20 for detecting the cell impedance value in the annular channel 20, the inner diameter of the innermost ring of the annular interdigital electrode 23 is greater than the inner diameter of the annular channel 20, the outer diameter of the annular channel 20 is greater than the outer diameter of the outermost ring of the annular interdigital electrode 23, the radial distance between two adjacent rings of interdigital electrodes is d2, the interdigital width is d3, that is, the distance between two end points of the end where the counter electrode lead-out metal layer 11 and the working electrode lead-out metal layer 12 are respectively connected with the annular interdigital electrode 23 is d3, and d3 is greater than zero. The semicircular ring counter electrode lead-out metal layer 11 with the ring width of d1 is connected with the annular interdigital electrode 23, is led out from the annular channel 20 and is tightly attached to a half area of the upper surface of the edge area 27, and the semicircular ring working electrode lead-out metal layer 12 with the ring width of d1 is connected with the annular interdigital electrode 23 and is led out from the annular channel 20 and is tightly attached to the other half area of the upper surface of the edge area 27. The two electrode lead-out metal layers are open face to face, the adjacent end points of the counter electrode lead-out metal layer 11 and the working electrode lead-out metal layer 12 are separated by d3, and the inner diameters and the ring widths of the counter electrode lead-out metal layer 11, the working electrode lead-out metal layer 12 and the two electrode lead-out metal layers on the transparent cover 2 are respectively the same. When the transparent cover 2 is covered, the counter electrode lead-out metal layer 13 on the transparent cover 2 is contacted with the counter electrode lead-out metal layer 11 on the centrifugal micro-fluidic chip 7, the working electrode lead-out metal layer 14 on the transparent cover 2 is contacted with the working electrode lead-out metal layer 12 on the centrifugal micro-fluidic chip 7, and because the width d3 exists in the interdigital, the d3 is more than 0, so the electrode lead-out metal layer on the transparent cover 2 can be always contacted with the electrode lead-out metal layer on the micro-fluidic chip 7 no matter what angle the micro-fluidic chip 7 rotates horizontally.
Referring to fig. 7, the elastic buckle 6 is composed of three parts, the upper part is two forked small hooks 28, the middle part is a groove area 29, the bottom part is a cuboid, and the outline width of the cuboid is equal to the total outline width of the two small hooks 28. When the centrifugal micro-fluidic chip 7 is installed, two small hooks 28 penetrate through the fixing port 19 at the bottom of the centrifugal micro-fluidic chip 7 and penetrate out from the top of the centrifugal micro-fluidic chip 7 to be positioned above the centrifugal micro-fluidic chip 7, so that the centrifugal micro-fluidic chip 7 is clamped and fixed in the groove area 29 and is perpendicular to the elastic buckle 6. The cuboid has the external dimension larger than the fixed port 19 and can not pass through the centrifugal micro-fluidic chip 7, and the cuboid is arranged below the centrifugal micro-fluidic chip 7. The elastic buckle 6 is vertically and fixedly connected to an output shaft of the rotating motor 5 through a cuboid at the bottom of the elastic buckle, and the output shaft of the rotating motor 5 is vertically upward, so that the centrifugal micro-fluidic chip 7 is horizontally arranged above the top of the rotating motor 5.
Referring to fig. 1 and 3, the counter electrode lead-out metal layer 13 and the working electrode lead-out metal layer 14 on the transparent cover 2 are respectively led out to the outside of the transparent cover 2 through the first detection hole 25 and the second detection hole 26 to be connected to the input end of the impedance analyzer 16, and are responsible for transmitting the impedance value to the impedance analyzer 16. The PC end upper computer 18 is associated with the MCU controller 15 through the WIFI module 17, and sends a starting or closing command to the MCU controller 15. The MCU controller 15 is connected to the input terminal of the impedance analyzer 16 via a serial port, and is responsible for controlling the on/off of the impedance analyzer 16. The MCU controller 15 is also connected to the input end of the centrifuge 1 through a serial port, and is responsible for controlling the start and stop of the rotating motor 5 in the centrifuge 1. The MCU controller 15 is connected to the input end of the steering engine 30 through a serial port and is responsible for controlling the counterclockwise rotation and the clockwise rotation of the steering engine 30. When the MCU controller 15 receives a starting command, the steering engine 30 is firstly controlled to start, the output shaft of the steering engine 30 rotates 90 degrees anticlockwise, the transparent cover 2 is opened, and then the rotating motor 5 is started, so that the centrifugal microfluidic chip 7 is driven to rotate, and the impedance analyzer 16 is controlled to be in a closed state; when the MCU controller 15 receives a closing command, it first controls the rotating motor 5 to stop rotating, so that the centrifugal microfluidic chip 7 remains stationary, then controls the steering engine 30 to rotate 90 ° clockwise, so that the transparent cover 2 is closed, and controls the impedance analyzer 16 to be in an open state, and starts to acquire the impedance value in the annular channel 20 in the microfluidic chip 7. The output end of the impedance analyzer 16 is connected to the PC end upper computer 18 and is responsible for transmitting the acquired data back to the PC end upper computer 18 for processing and storing.
Referring to fig. 1-7 and fig. 8, the cancer cell activity detection and evaluation device based on multi-adhesive strength fusion of the present invention works in two stages: the method comprises a building stage of a cancer cell activity RBF neural network prediction model based on multi-adhesion strength fusion and a cancer cell activity detection evaluation stage based on multi-adhesion strength fusion. Since the impedance value is approximately proportional to the number of cells, the change of the cell index is monitored by the impedance value, and the cell index CI is calculated by the formula:
N1as the impedance value of adherent cancer cells remaining after centrifugation, N0Is the initial cancer cell impedance value within the annular channel 20.
The centrifugal force tau suffered by the cells is calculated by the rotating speed omega of the centrifugal microfluidic chip 7:
wherein r is the distance between the cell and the center of the centrifugal microfluidic chip 7, rho is the density of the culture medium, and mu is the viscosity of the culture medium.
In the first stage, the establishment of a cancer cell activity RBF neural network prediction model based on multi-adhesion strength fusion comprises the following specific steps:
step 1: the cancer cells are cultured in the same culture dish for the first time, and then a part of the cancer cell population A is taken for half inhibition rate (half inhibition) IC50The assay of (1) using cisplatinThe reagent is subjected to anti-cancer drug reaction experiment to obtain IC50Value X of1(ii) a The remaining part of the cancer cell population B was used to extract the cell adhesion strength. Since the cells were removed from the same culture dish, the activity status of cancer cell group A and cancer cell group B was the same.
Step 2: the PC end upper computer 18 sends a starting command to the MCU controller 15 through the WIFI module 17, firstly, the steering engine 30 is controlled to rotate 90 degrees anticlockwise to open the transparent cover 2, a cell-free culture medium is dripped into the centrifugal micro-fluidic chip 7, after the transparent cover 2 is opened for a set T1 minutes, the PC end upper computer 18 controls the rotating motor 5 to start at a maximum speed V to drive the centrifugal micro-fluidic chip 7 to rotate for a set T2 minutes, after the T2 minutes, the PC end upper computer 18 controls the transparent cover 2 to close, at the moment, the working electrode lead-out metal layer 14 on the transparent cover 2 is in contact with the working electrode lead-out metal layer 12 on the centrifugal micro-fluidic chip 7, the counter electrode lead-out metal layer 13 on the transparent cover 2 is in contact with the counter electrode lead-out metal layer 11 on the centrifugal micro-fluidic chip 7, and the annular interdigital electrode 23 detects an electric signal in an annular channel 20 in the centrifugal micro-fluidic chip 7, and input into the impedance analyzer 16 through the contacted leading-out metal layer, and the impedance analyzer 16 obtains the cell impedance value Z in the annular channel 20 at this time0. The PC end upper computer 18 is provided with a cell impedance value Z when the cell-free culture medium is dripped into the centrifugal micro-fluidic chip 70Setting the number of detection runs to n for the impedance threshold, and the initial rotation speed omega of the rotary motor 5 in the centrifuge 1 to omega0The acceleration is performed once for each rotation speed increase by a rotation speed difference Δ ω.
And step 3: and (3) controlling the steering engine 30 to rotate 90 degrees anticlockwise by the PC-end upper computer 18, opening the transparent cover 2 again, and dropping the cancer cell group B prepared in the step (1) into the centrifugal microfluidic chip 7, wherein the centrifugal microfluidic chip 7 does not rotate at the moment. After the transparent cover 2 is opened for T1 minutes, the PC-end upper computer 18 controls the steering engine 30 to rotate 90 degrees clockwise, so that the transparent cover 2 is closed again, the impedance analyzer 16 obtains the cell impedance value in the annular channel 20 in the centrifugal micro-fluidic chip 7 at the moment, the cell impedance value is the cell impedance value when the centrifugal micro-fluidic chip 7 does not rotate, and the cell impedance value is set as the initial cancer cell impedance value N0Initiating cancer cellsImpedance value N0And uploading to the PC end upper computer 18.
And 4, step 4: the PC end upper computer 18 controls the steering engine 30 to rotate 90 degrees anticlockwise, so that the transparent cover 2 is opened, and after the transparent cover 2 is opened for T1 minutes, the PC end upper computer 18 controls the rotating motor 5 to rotate at the initial rotating speed omega 0 to drive the centrifugal micro-fluidic chip 7 to rotate for T2 minutes.
And 5: after T2 minutes, the PC-end upper computer 18 sends a closing command to the MCU controller 15 through the WIFI module 17, firstly controls the rotating motor 5 to stop running, secondly controls the steering engine 30 to rotate 90 degrees clockwise, so that the transparent cover 2 is closed, and at the moment, the impedance analyzer 16 measures a cell impedance value N1 in the annular channel 20 in the centrifugal micro-fluidic chip 7 and transmits the cell impedance value to the PC-end upper computer 18. The PC upper computer 18 calculates the cell index CI ═ CI using the formula (1), and obtains the cell index CI ═ CI at that time1And storing. After T1 minutes when the transparent cover 2 is closed, the transparent cover 2 is opened again, the rotating motor 5 is controlled to run at an accelerated speed for one time, and the rotating motor 5 is enabled to run at omega1＝ω0+ Δ ω run and run at this speed for T2 minutes.
Step 6: the step 5 is repeatedly executed n times, and the PC-side upper computer 18 obtains n cell indexes CI ═ GI1,GI2,…,GIn]N rotation speeds ω ═ ω0,ω2,…,ωn-1]And N cell impedance values [ N1，N2……，Nn]N cell impedance values [ N ]1，N2……，Nn]And the cell impedance threshold value Z in step 20By comparison, when N cell impedance values [ N ]1，N2……，Nn]Less than or equal to an impedance threshold value Z0And stopping repeatedly executing the steps.
And 7: the PC-end upper computer 18 calculates n rotation speeds ω using a formula (2) to obtain n centrifugal forces τ, and converts n cell indices CI ═ CI1,CI2,…,CIn]And fitting a Logistic function (sigmoid curve) with the n centrifugal forces tau, wherein the n centrifugal forces tau are used as an x axis, the n cell indexes CI are used as a y axis, and the fitting refers to obtaining the sigmoid function CI (f (tau) with the centrifugal forces tau as the x axis and the cell indexes CI as the y axis. Then according toThe inverse of the sigmoid function CI ═ f (τ) calculated the first set of cell adhesion intensities [ τ [ tau ] ]0,τ10,...,τ90]，τNIndicates the centrifugal force (N-0, 10, …,90) to which (100-N)% of the cells are subjected.
And 8: repeating steps 1-7, i.e. repeating steps from culturing cancer cells in the same culture dish for the second time, and repeating step 7 to obtain the second group of cell adhesion strengths [ tau ]0,τ10,...,τ90]And a second half-inhibition ratio IC50Value X2。
And step 9: repeating step 8, i.e. co-culturing cancer cells n times in the same culture dish, to obtain n groups of cell adhesion strength [ tau ]0,τ10,...,τ90]And n different ICs50Value X ═ X1,X2,...,Xn]. Adhesion strength [ tau ] of n groups of cells0,τ10,...,τ90]As input, n different ICs50Value X ═ X1,X2,...,Xn]And establishing a cancer cell activity RBF neural network prediction model X ═ f (tau) as output. The RBF neural network prediction model is divided into an input layer, a hidden layer and an output layer, and weight parameters in the hidden layer are calculated through given data of the input layer and the output layer, so that a relational expression between input and output is obtained.
And a second stage, cancer cell activity detection evaluation based on multi-adhesion strength fusion. The standard method for evaluating the activity of cells is to semi-inhibit the rate IC of cancer cells with anticancer drugs50The invention adopts a method of extracting adhesive strength to realize IC50The prediction method comprises the following specific steps:
step A): the PC end upper computer 18 sends a starting command, controls the steering engine 30 to rotate 90 degrees anticlockwise, opens the transparent cover 2, and then takes out the cancer cell group to be detected from a new culture dish and drops the cancer cell group into the microfluidic chip 7. After the transparent cover 2 is opened for T1 minutes, the steering engine 30 is controlled to rotate clockwise by 90 degrees, so that the transparent cover 2 is closed.
Step B): same as step 4 of the first stage.
Step C): same as step 5 of the first stage.
Step D): repeating the step C) n times to obtain n cell indices CI '═ C1'1,CI′2,…,GI′n]And n rotation speeds ω '═ ω'1,ω′2,…,ω′n]Until the cell impedance value in the annular channel 20 is less than or equal to the impedance threshold value Z0。
Step E): the PC-end upper computer 18 processes the n rotation speeds ω 'using a formula (2) to obtain n centrifugal forces τ', and performs Logistic function (S-curve) fitting on the n cell indices CI 'and the n centrifugal forces τ', to obtain an S-function CI '═ h (τ') with the centrifugal force as the x-axis and the cell index as the y-axis. Cell adhesion strength [ tau ] was then calculated from the inverse of the sigmoid function CI '═ h (tau'0,τ′10,...,τ′90]，τ′NIndicates the centrifugal force (N-0, 10, …,90) to which (100-N)% of the cells are subjected.
Step F): the cell adhesion strength [ tau ] obtained in step E)'0,τ′10,...,τ′90]Inputting into the established cancer cell activity RBF neural network prediction model X ═ f (tau), and obtaining corresponding IC50Value X' of (A), and IC50The value of (b) X' corresponds to the cell activity, thereby evaluating the cell activity.
Claims (6)
1. A cancer cell activity detection and evaluation device based on multi-adhesion strength fusion comprises a centrifuge (1), an MCU controller (15), an impedance analyzer (16) and a PC end upper computer (18), and is characterized in that: the outer part of the centrifugal machine (1) is formed by connecting a transparent cover (2) and a base (3), the inner part of the base (3) is respectively provided with a rotating motor (5), an elastic buckle (6) and a centrifugal microfluidic chip (7) from bottom to top, the rotating motor (5) is vertically arranged in the middle of the inner part of the base (3), an output shaft is vertically upward and is connected with the centrifugal microfluidic chip (7) which is horizontally arranged through the elastic buckle (6); the lower half part of the transparent cover (2) is a circular ring (24), a semicircular counter electrode lead-out metal layer (13) and a working electrode lead-out metal layer (14) are tightly attached to the surface of the lower ring of the circular ring (24), and the counter electrode lead-out metal layer (13) and the working electrode lead-out metal layer (14) on the transparent cover (2) are respectively connected with the input end of an impedance analyzer (16); the outer surface of the top of the base (3) is fixedly connected with a steering engine (30), and the steering engine (30) can rotate clockwise and anticlockwise to drive the transparent cover (2) to open and close; the centrifugal microfluidic chip (7) is formed by coaxially and tightly attaching a disc-shaped PDMS cover plate (8) and a PDMS substrate (9) up and down, the PDMS substrate (9) protrudes out of an edge area (27) of a ring along the diameter direction relative to the PDMS cover plate (8), a counter electrode lead-out metal layer (11) and a working electrode lead-out metal layer (12) are tightly attached to the upper surface of the edge area (27), when the transparent cover (2) is closed, the counter electrode lead-out metal layer (11) on the edge area (27) is contacted with a counter electrode lead-out metal layer (13) on the transparent cover (2), and the working electrode lead-out metal layer (12) on the edge area (27) is contacted with a working electrode lead-out metal layer (14) on the transparent cover (2); an annular channel (20) is surrounded at the middle periphery of the PDMS substrate (9), a plurality of separation channels (22) which are all arranged along the diameter direction are uniformly surrounded at the periphery of the annular channel (20), the radial outer end of each separation channel (22) is communicated with a circular outlet (21), the radial inner end of each separation channel (22) is communicated with the annular channel (20), annular interdigital electrodes (23) are etched in the annular channel (20), and one ends of a counter electrode lead-out metal layer (11) and a working electrode lead-out metal layer (12) on an edge area (27) are respectively connected with the annular interdigital electrodes (23); PC end host computer (18) are correlated with MCU controller (15) through WIFI module (17), and MCU controller (15) pass through serial ports connection impedance analysis appearance (16) input and still pass through serial ports connection rotating electrical machines (5) and steering wheel (30).
2. The device for detecting and evaluating the activity of cancer cells based on multi-adhesive strength fusion according to claim 1, wherein: the upper surface of the elastic buckle (6) is provided with two forked small hooks (28), the middle part is provided with a groove area (29), the bottom part is provided with a cuboid, the centrifugal micro-fluidic chip (7) is fixed on the groove area (29), the cuboid is arranged below the centrifugal micro-fluidic chip (7), and the two small hooks (28) are arranged above the centrifugal micro-fluidic chip (7).
3. The device for detecting and evaluating the activity of cancer cells based on multi-adhesive strength fusion according to claim 1, wherein: the counter electrode lead-out metal layer (13) and the working electrode lead-out metal layer (14) on the transparent cover (2) are both in a semicircular shape, the ring width is d1, the openings of the semicircular rings are arranged in a face-to-face manner, and one end of each of the semicircular rings is provided with a detection hole which is vertically communicated with the outside of the transparent cover (2); the counter electrode lead-out metal layer (11) and the working electrode lead-out metal layer (12) on the edge area (27) are both semicircular, the ring widths are both d1, and the openings of the semicircular rings are arranged in a face-to-face mode.
4. The method for detecting and evaluating the activity of the cancer cells based on the fusion of multiple adhesion strengths according to claim 1, comprising the steps of:
step 1: culturing cancer cells in the same culture dish for the first time, wherein a part of cancer cell population A is used for half-inhibition rate IC50To obtain IC50Value X of1And the rest part of the cancer cell group B is reserved;
step 2: the PC end upper computer (18) controls the transparent cover (2) to be opened, a cell-free culture medium is dripped into the centrifugal micro-fluidic chip (7), the rotating motor (5) is controlled to drive the centrifugal micro-fluidic chip (7) to rotate at the maximum speed, the transparent cover (2) is controlled to be closed, and the impedance analyzer (16) obtains a cell impedance threshold value Z0And uploading to a PC end upper computer (18);
and step 3: the transparent cover (2) is opened, the cancer cell group B is dripped into the centrifugal micro-fluidic chip (7), the transparent cover (2) is closed, and the impedance analyzer (16) obtains the initial cancer cell impedance value N0And uploading to a PC end upper computer (18);
and 4, step 4: the transparent cover (2) is opened, and the rotating motor (5) rotates at the initial rotating speed omega0Driving the centrifugal micro-fluidic chip (7) to rotate for a set time;
and 5: the rotating motor (5) is controlled to stop running, the transparent cover (2) is closed, and the impedance analyzer (16) obtains the cell impedance value N1And uploading to a PC end upper computer (18); the PC end upper computer (18) calculates the cell index CI ═ CI1(ii) a Then the transparent cover (2) is opened again, and the rotating motor (5) is controlled to omega1＝ω0+ Δ ω for acceleration, Δ ω for one accelerationA difference value of the rotating speeds;
step 6: repeating the step 5 n times, and obtaining n cell indexes CI (CI) by a PC (18) end upper computer1,CI2,…,CIn]N rotation speeds ω ═ ω0,ω2,…,ωn-1]And N cell impedance values [ N1，N2……，Nn]N cell impedance values [ N ]1，N2……，Nn]And the cell impedance threshold value Z0By comparison, when less than or equal to the impedance threshold Z0Stopping repeated execution;
and 7: the PC end upper computer (18) respectively calculates n rotating speeds omega to obtain n centrifugal forces tau, and n cell indexes CI ═ CI1,CI2,…,CIn]Fitting the obtained S-shaped function CI-f (tau) with n centrifugal forces tau, and calculating a first group of cell adhesion strengths [ tau ] according to the inverse function of the S-shaped function CI-f (tau)0,τ10,...,τ90]，τNRepresents the centrifugal force to which (100-N)% of cells are detached, N is 0,10, …, 90;
and 8: repeating steps 1-7 to obtain a second group of cell adhesion strengths [ tau ]0,τ10,...,τ90]And a second half-inhibition ratio IC50Value X2；
And step 9: repeating the step 8 to obtain n groups of cell adhesion strength [ tau ]0,τ10,...,τ90]And n ICs50Value of (2) ([ X ]1,X2,...,Xn]The adhesion strength [ tau ] of n groups of cells0,τ10,...,τ90]As input, n ICs50Value of (2) ([ X ]1,X2,...,Xn]Establishing a cancer cell activity RBF neural network prediction model X ═ f (tau) as output;
step 10: taking out the cancer cell group to be detected from the new culture dish, dripping the cancer cell group into a centrifugal microfluidic chip (7), and respectively and repeatedly executing the step 4, the step 5, the step 6 and the step 7 to obtain the cell adhesion strength [ tau'0,τ′10,...,τ′90]，τ′NCentrifugal force N ═ 0,10, …,90, indicating that (100-N)% of cells detached; will be provided withCell adhesion Strength [ tau'0,τ′10,...,τ′90]Inputting the obtained data into the prediction model X ═ f (tau) to obtain corresponding IC50Value X' of (A), and IC50The value of (A) corresponds to the activity of the cell, and the activity of the cell is evaluated.
5. The test evaluation method of claim 4, wherein: in step 5, the calculation formula of the cell index CI is as follows:
N1impedance value of adherent cancer cell remaining after centrifugation, N0Is the initial cancer cell impedance value within the annular channel 20.
6. The test evaluation method of claim 4, wherein: in step 7, the centrifugal force
r is the distance between the cell and the center of the centrifugal microfluidic chip (7), rho is the density of the culture medium, and mu is the viscosity of the culture medium.
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