CN108037159B - A kind of cytoactive detection method and device based on impedance spectrum integral feature - Google Patents
A kind of cytoactive detection method and device based on impedance spectrum integral feature Download PDFInfo
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Abstract
The present invention discloses a kind of cytoactive detection method and device based on impedance spectrum integral feature for detecting cell activity indirectly using electric cell-substrate impedance sensor, the cell culture fluid containing cell to be detected is injected in first detection hole, the cell culture fluid of same volume of the injection without cell to be detected in second detection hole, respectively obtain in two detection holes culture solution corresponding impedance magnitude at each frequency, it is poor first to calculate impedance magnitude, impedance magnitude difference and preset impedance contrast threshold value comparison are determined into integral frequency range, fitting obtains the impedance spectral function of the cell culture fluid containing cell to be detected and the cell culture fluid without containing cell to be detected in the frequency range again, impedance spectrum integrated value is obtained to impedance spectrum functional integration, finally calculate cell activity index；The present invention indicates current cell activity value using the spectrum integration value in the higher frequency range of cell impedance measurement sensitivity, effectively increases the accuracy and sensitivity of cytoactive detection.
Description
Technical field
The invention belongs to cell detection field, it is related to a kind of detecting cell activity indirectly using electric cell-substrate impedance sensor
Technology.
Background technique
Cell activity is to evaluate the basis of cell culture, cytotoxicity and physiological Study, and cell activity is in cell research, medicine
Play a significant role in object effect, cell engineering and microbial fermentation engineering, health care and environmental monitoring.Current cell is living
Property detection method mainly includes various chemical dyeing methods, immunization, cell impedance indirect method of measurement etc..Wherein, between cell impedance
When connecing mensuration detection cell activity, because its is unmarked, continuity, on-line checking are widely used due to the characteristics of, master
If cell impedance amplitude under single-frequency point is measured with electric cell-substrate impedance sensor, according between impedance magnitude and cell activity
Corresponding relationship to detect cell activity indirectly.But cell impedance amplitude is measured under single-frequency point, it is used for cell activity
Have the defects that detection accuracy is not high when detection, and carries out cell impedance using expensive impedance analyzer mostly
Measurement.In order to improve the detection sensitivity of cell activity, with sensor when carrying out cell impedance measurement, impedance is found first
The most sensitive frequency point of measurement, i.e., obtained under the measurement frequency currently when having cell the impedance magnitude of sensor with it is cell-free
When the impedance magnitude difference measured it is maximum, the cell impedance measurement under subsequent different activities is then carried out under the Frequency point.But
In cell activity change procedure, equivalent capacity, equivalent resistance of cell etc. change, and cell impedance measures most sensitive frequency
Point can also occur to change accordingly.If carrying out cell impedance measurement with the same most sensitive frequency point, impedance measurement mistake will increase
Difference, and then the accuracy of cytoactive detection is reduced indirectly.
Summary of the invention
Detection accuracy is not high when for the cell impedance measurement at present under single-frequency point to evaluate cell activity, uses
Impedance instrument measures the high disadvantage of testing cost, and the present invention proposes a kind of cytoactive detection side based on impedance spectrum integral feature
Method and device carry out cell impedance spectrometry without using high-cost impedance instrument, with cell impedance measurement sensitivity higher frequency band
Interior spectrum integration value indicates current cell activity value, effectively improves the accuracy of cytoactive detection.
A kind of cytoactive detection device based on impedance spectrum integral feature of the present invention adopts the following technical scheme that: packet
The electric cell-substrate impedance sensor with the first, second two detection holes is included, an interdigital electrode is respectively arranged in each detection hole bottom, pitches
Refer to that electrode connects host computer through MCU control circuit, MCU control circuit includes high-speed microprocessor, DDS signal generator, detects and lead to
Road gating circuit, superimposed signal circuit, high-pass filtering circuit, the output end of high-speed microprocessor be separately connected DDS signal generator,
The output end of sense channel gating circuit and superimposed signal circuit, DDS signal generator is superimposed electricity through attenuator circuit connection signal
Road, superimposed signal circuit sequentially connect interdigital electrode, sensor connecting pin through sense channel gating circuit, sensor connector port
The output end of mouth sequentially connects the input terminal of single-chip microcontroller through current/voltage converter circuit, amplifying circuit, high-pass filtering circuit.
A kind of detection method of cytoactive detection device based on impedance spectrum integral feature uses following skill
Art scheme is: the cell culture fluid containing cell to be detected is injected in the first detection hole, injection is without to be checked in the second detection hole
The cell culture fluid of the same volume of cell is surveyed, further comprising the steps of:
Step A: single-chip microcontroller first controls detection hole gating circuit and gates the first detection hole, sequentially controls DDS signal generator
Generate increasing frequency f1,f2,……,fn-1,fnSignal, and respectively acquisition interdigital electrode output to induction signal, calculate first
The corresponding impedance magnitude Z of cell culture fluid in detection hole1,Z2,……,Zn-1,Zn；Then control detection hole gating circuit gating
Second detection hole obtains in the second detection hole cell culture fluid in each frequency f1,f2,……,fn-1,fnUnder corresponding resistance
Anti- amplitude β1,β2,……,βn-1,βn；Each frequency and impedance magnitude are finally uploaded into host computer；
Step B: host computer first presses formula Δ Zi=Zi-βiCalculate impedance magnitude difference Δ Zi, i=1,2 ..., n-1, n,
By impedance magnitude difference Δ ZiCompared with preset impedance contrast threshold value Δ Z, integral frequency range [f is determinedlow,fhigh], flowIt is impedance spectrum
Lower limit of integral frequency flow, fhighIt is impedance spectrum upper limit of integral frequency；It is fitted in the frequency range again containing cell to be detected
The impedance spectral function Z (f) and β (f) of cell culture fluid and the cell culture fluid without containing cell to be detected, to impedance spectral function product
Get impedance spectrum integrated valueWithFinally calculate integrated value Δ I=
Ix-I0With cell activity index Vindex=Δ I/Imax。
Further, in step B, host computer is from initial frequency f1The impedance magnitude started under more each frequency one by one is poor
ΔZiWith impedance contrast threshold value Δ Z, when occurring impedance magnitude difference Δ Z for the first timeiWhen > Δ Z, corresponding frequency is impedance spectrum product
Divide lower frequency limit flow；Later from end frequency fnStart the more each frequency impedance difference in magnitude Δ Z of backwardiWith impedance contrast threshold value Δ
Z, when occurring impedance magnitude difference Δ Z for the first timeiWhen > Δ Z, corresponding frequency is impedance spectrum upper limit of integral frequency fhigh。
The present invention has the advantages that compared with existing methods and techniques
1, the present invention indicates current thin using the spectrum integration value in the higher frequency range of cell impedance measurement sensitivity
Cytoactive value, this method overcome when to detect cell activity indirectly with impedance under single-frequency point by systematic error, idol
The big defects of influences such as right error, outside noise, also overcome and measure most sensitive frequency in cell activity change procedure middle impedance
The problem of point changes and cell impedance measurement sensitivity is caused to reduce, effectively improves the accuracy of cytoactive detection
And sensitivity.
2, the present invention completes pumping signal using the high-speed microprocessor of low cost when acquiring the cell impedance of sensor
It generates and the acquisition of returned signal, and calculates using discrete fourier transform algorithm the impedance of each measurement frequency point
Amplitude, compared with using high-cost impedance instrument, with advantage at low cost, measurement method is easy.
3, in the electrical connection of the copper post of the electrode and external circuit of establishing sensor, using between conductive silver glue enhancing two
Contact area, reduce contact impedance, while facilitating removing and installing for sensor.
Detailed description of the invention
Fig. 1 is a kind of structural schematic diagram of the cytoactive detection device based on impedance spectrum integral feature of the present invention；
Fig. 2 is the structural block diagram of MCU control circuit circuit in Fig. 1；
Fig. 3 is the flow chart of Fig. 1 cytoactive detection method of the present invention；
The serial number and title of each component in attached drawing: 1, electric cell-substrate impedance sensor；2, the first detection hole；3, the second detection hole；
4, interdigital electrode；5, working electrode draws metal layer；6, metal layer is drawn to electrode；7, conductive silver glue；8, pcb board；9, thin copper
Column；10, needle is arranged；11, cushion block；12, fastening screw；13, collect line terminals；14, MCU control circuit；15, sensor connector port；
16, serial communication interface；17, host computer.
Specific embodiment
Referring to Fig. 1, a kind of cytoactive detection device based on impedance spectrum integral feature of the present invention includes a cell
Impedance transducer 1, the tool of electric cell-substrate impedance sensor 1 are the first detection hole 2 and the second detection hole 3 respectively there are two detection hole, each
Detection hole bottom respectively arranges that an interdigital electrode 4, two interdigital electrodes 4 share one and draw metal layer 6 from detection hole to electrode
Middle extraction, each interdigital electrode 4 are drawn metal layer 5 with a working electrode and are drawn from detection hole.One is drawn gold to electrode
Belong to layer 6 and two working electrodes draw respectively one 9 one end of thin copper post of connection of metal layer 5, thin 9 other end of copper post connects through row's needle 10
Connect collection line terminals 13.Collect line terminals 13 to be connected again through the connection MCU control circuit 14 of sensor connector port 15, MCU control circuit
14 are connected on host computer 17 by serial communication interface 16.In this way, making interdigital electrode 4 and the MCU in electric cell-substrate impedance sensor 1
Control circuit 14 establishes connection relationship, and MCU control circuit 14 completes signal acquisition and device controls, and result is transmitted to upper
Machine 17.
Electric cell-substrate impedance sensor 1 is horizontally disposed, and three thin copper post 9 is arranged vertically, and draws metal layer 6 and work electricity to electrode
The top that metal layer 5 is all connected with thin copper post 9 is drawn in pole, and the bottom end of thin copper post 9 is fixed on pcb board 8, and row's needle 10 is also secured at
On pcb board 8, establishes and be electrically connected between thin copper post 9 and row's needle 10.Metal layer 6 and work are drawn with to electrode in each thin copper post 9
Make to inject a certain amount of conductive silver glue 7 in the hole for the junction that electrode draws metal layer 5, thin copper post 9 can be reduced and to electrode
The contact impedance between metal layer 6 and working electrode extraction metal layer 5 is drawn, electric cell-substrate impedance sensor 1 is also facilitated to tear open here
It unloads and installs.In the opposite side of thin copper post 9, electric cell-substrate impedance sensor 1 is connected to pcb board by fastening screw 12 and cushion block 11
8, the effect of cushion block 11 be 1 side of electric cell-substrate impedance sensor is padded, keep electric cell-substrate impedance sensor 1 horizontal.
Referring to fig. 2, MCU control circuit 14 includes high-speed microprocessor, DDS signal generator, sense channel gating circuit, letter
Number supercircuit, high-pass filtering circuit etc..The output end of high-speed microprocessor is separately connected DDS signal generator, sense channel choosing
Circuit passband and superimposed signal circuit, the output end signal of DDS signal generator is through attenuator circuit connection signal supercircuit, signal
Supercircuit sequentially connects the interdigital electricity in electric cell-substrate impedance sensor 1 through sense channel gating circuit, sensor connector port 15
Pole 4.It is two-way between interdigital electrode 4 and MCU control circuit 14 to connect, the output end of sensor connector port 15 sequentially through electric current/
Voltage conversion circuit, amplifying circuit, high-pass filtering circuit connect the input terminal of single-chip microcontroller.Single-chip microcontroller and serial communication interface 16 it
Between be bi-directionally connected.
It is built-in with D/A converter inside single-chip microcontroller, exports DC voltage to superimposed signal circuit, direct current through D/A converter
The effect of pressure is the open-circuit voltage of counter electrode and cell culture fluid.Single-chip microcontroller controls DDS signal generator and exports corresponding frequencies
It otherwise can be destroyed thin with the sinusoidal voltage pumping signal of amplitude due to the amplitude very little for the voltage excitation signals that cell can be born
The normal physiological condition of born of the same parents increases the systematic error of cytoactive detection, so the sinusoidal voltage that DDS signal generator is generated
Pumping signal is first transported in attenuator circuit and decays to 50mV；In this way, 50mV sinusoidal voltage pumping signal and monolithic after decaying
The DC voltage of D/A converter output in machine is input in superimposed signal circuit jointly.From superimposed signal circuit output with straight
The sinusoidal excitation signal of biasing is flowed, hole gating circuit is applied to sensor to the sinusoidal excitation signal with direct current biasing after testing
On connectivity port 15.Control of the detection hole gating circuit by single-chip microcontroller, the first detection hole 2 in gated cells impedance transducer 1
Perhaps the second detection hole 3 work selects the interdigital electrode 4 of the first detection hole 2 or the second detection hole 3 to work, the first detection
The interdigital electrode 4 of 3 bottom of hole 2 or the second detection hole sends the current signal of acquisition to sensor connector port 15, sensing
Current signal is input to current/voltage converter circuit by device connectivity port 15, is converted into voltage by current/voltage converter circuit
Signal, voltage signal amplify using amplifying circuit, and amplified voltage signal is sent to single-chip microcontroller after passing through high-pass filtering circuit,
Signal acquisition is completed as the converter built in single-chip microcontroller.Single-chip microcontroller is completed according to collected signal to cell impedance amplitude
Calculating.Final single-chip microcontroller will be calculated cell impedance amplitude and corresponding frequency and be uploaded to by serial communication interface 16
Position machine 17.
Referring to 1-3, when a kind of cytoactive detection device based on impedance spectrum integral feature of the present invention works, to cell
The Whole Work Flow figure of activity test method.Specific step is as follows:
Step 1: the cell culture fluid of the certain volume containing cell to be detected is injected first in the first detection hole 2, then
The cell culture fluid that the same volume without cell to be detected is injected in the second detection hole 3, stands a period of time.
Step 2: single-chip microcontroller first controls first detection hole 2 of the detection hole gating circuit gating containing cell to be detected, then controls
It is f that DDS signal generator processed, which generates frequency,1(f1For original frequency) sinusoidal excitation signal.DDS signal generator generates just
String pumping signal decays to virtual value 50mV by signal attenuation circuit, and defeated together with the DC bias signal of single-chip microcontroller output
Enter into superimposed signal circuit, the sinusoidal voltage pumping signal containing direct current biasing exported from superimposed signal circuit passes through detection
Hole gating circuit, sensor connector port 15 are applied to the corresponding working electrode of the first detection hole 2 and draw metal layer 5 and to electrode
It draws on metal layer 6, is finally applied in interdigital electrode 4.The current signal that interdigital electrode 4 exports passes through sensor connector port
After 15 sequentially after current/voltage conversion circuit, amplifying circuit and high-pass filtering circuit, by the converter inside single-chip microcontroller
It is acquired.Finally, single-chip microcontroller according to collected signal, calculates in the first detection hole 2 cell culture fluid in initial frequency f1
Under impedance magnitude Z1。
Step 3: in initial frequency f1Impedance magnitude Z1After the completion of calculating, single-chip microcontroller first controls DDS signal generator output
Second frequency f2Sinusoidal excitation signal, i.e., the frequency of sinusoidal excitation signal is become into f2, second frequency f2It is to be risen currently
Beginning frequency f1On the basis of increase a step-length△F, i.e. f2=f1+△f.Later, single-chip microcontroller is according to the detection described in step 2
Journey obtains in the first detection hole 2 cell culture fluid in second frequency f2Under impedance magnitude Z2。
So circulation, single-chip microcontroller sequentially control the signal that DDS signal generator generates increasing frequency, the every secondary control of single-chip microcontroller
The frequency of DDS signal generator output is to increase identical step-length on the basis of previous output frequency△f.So cyclically
N times are detected, until obtaining terminating frequency fnUnder impedance magnitude ZnUntil.Cell culture fluid in the first detection hole 2 is finally obtained to exist
Each frequency f1,f2,……,fn-1,fnUnder corresponding impedance magnitude Z1,Z2,……,Zn-1,Zn.So complete the first detection
The cell impedance value detection of cell culture fluid in hole 2.
Step 4: after the completion of the cell impedance of cell culture fluid in the first detection hole 2 is surveyed, single-chip microcontroller controls detection hole choosing
Second detection hole 3 of the cell culture fluid without containing cell to be detected in circuit passband gated cells impedance transducer 1.Then it weighs
Reinspection is surveyed, and finally obtains in the second detection hole 3 cell culture fluid in each frequency f1,f2,……,fn-1,fnUnder it is corresponding
Impedance magnitude β1,β2,……,βn-1,βn。
Step 5: after the completion of the impedance measurement of cell culture fluid in the first detection hole 2 and the second detection hole 3, single-chip microcontroller will
The impedance data of first detection hole 2 and the second detection hole 3 measured at each frequency uploads to host computer 17.Host computer 17 is pressed
According to following formula computing impedance difference in magnitude Δ Zi:
ΔZi=Zi-βi(i=1,2 ..., n-1, n),
Each frequency f is just obtained in this way1,f2,……,fn-1,fnUnder corresponding impedance magnitude difference occurrence Δ Z1,
ΔZ2,……ΔZn-1,ΔZn。
Host computer 17 is according to preset impedance contrast threshold value Δ Z, from initial frequency f1Start to judge one by one, it is relatively more each one by one
Impedance magnitude difference Δ Z under frequencyiIt is poor when occurring impedance magnitude for the first time with the size between preset impedance contrast threshold value Δ Z
ΔZiWhen > Δ Z (1≤i < n/2), corresponding frequency is impedance spectrum lower limit of integral frequency f at this timelow.Host computer 17 later
Search impedance spectrum upper limit of integral frequency fhigh, i.e., from end frequency fnStart, backward more each frequency impedance difference in magnitude Δ one by one
ZiWith the size between impedance contrast threshold value Δ Z, when occurring impedance magnitude difference Δ Z for the first timeiWhen > Δ Z (n >=i > n/2), at this time
Corresponding frequency is impedance spectrum upper limit of integral frequency fhigh.Finally, according to impedance spectrum lower limit of integral frequency flowAnd impedance spectrum
Upper limit of integral frequency fhighDetermine integral frequency range [flow,fhigh]。
Step 6: after integral frequency range has been determined, right in the frequency range section, host computer 17 is using frequency as abscissa, impedance
Amplitude is ordinate, carries out multinomial to the impedance data of the first detection hole 2 and the second detection hole 3 respectively using least square method
Curve matching respectively obtains the cell culture fluid containing cell to be detected and the cell culture fluid without containing cell to be detected
Impedance spectrum function expression Z (f) and β (f), this is also to the cell in the relatively high frequency range of cell impedance measurement sensitivity
Impedance spectrum is fitted.
Then integral operation is carried out to frequency spectrum function in the frequency range section according to following formula, be calculated containing to be checked
The impedance spectrum integrated value for surveying cell and the cell culture fluid without containing cell to be detected is I respectivelyXAnd I0:
The I that will finally obtainX, I0It makes the difference, has just obtained the integrated value Δ I=I for indicating cell activityx-I0。
Step 7: according to the preset cell activity upper limit value I of host computer 17max, calculated through cell activity normalization formula
Cell activity index Vindex: Vindex=Δ I/Imax, cell activity index VindexIt is living to characterize current cell with integrated value
Property grade.
Claims (3)
1. a kind of detection method of the cytoactive detection device based on impedance spectrum integral feature, using based on impedance spectrum product
The cytoactive detection device of dtex sign, the cytoactive detection device include the cell with the first, second two detection holes
An interdigital electrode (4) is respectively arranged in impedance transducer (1), each detection hole bottom, and interdigital electrode (4) is through MCU control circuit
(14) host computer (17) are connected, MCU control circuit (14) includes high-speed microprocessor, DDS signal generator, sense channel gating electricity
Road, superimposed signal circuit, high-pass filtering circuit, the output end of high-speed microprocessor are separately connected DDS signal generator, sense channel
Gating circuit and superimposed signal circuit, for the output end of DDS signal generator through attenuator circuit connection signal supercircuit, signal is folded
Road is powered on sequentially through sense channel gating circuit, sensor connector port (15) connection interdigital electrode (4), sensor connector port
(15) output end sequentially connects the input terminal of single-chip microcontroller through current/voltage converter circuit, amplifying circuit, high-pass filtering circuit；
The cell culture fluid containing cell to be detected is injected in the first detection hole, injection is free of the phase of cell to be detected in the second detection hole
The cell culture fluid of same volume, it is characterized in that the following steps are included:
Step A: single-chip microcontroller first controls detection hole gating circuit and gates the first detection hole, sequentially controls DDS signal generator and generates
Increasing frequency f1,f2,……,fn-1,fnSignal, and respectively acquisition interdigital electrode (4) output to induction signal, calculate the first inspection
The corresponding impedance magnitude Z of cell culture fluid in gaging hole1,Z2,……,Zn-1,Zn；Then control detection hole gating circuit gating the
Two detection holes obtain in the second detection hole cell culture fluid in each frequency f1,f2,……,fn-1,fnUnder corresponding impedance
Amplitude β1,β2,……,βn-1,βn；Each frequency and impedance magnitude are finally uploaded into host computer (17)；
Step B: host computer (17) first presses formula Δ Zi=Zi-βiCalculate impedance magnitude difference Δ Zi, i=1,2 ..., n-1, n,
By impedance magnitude difference Δ ZiCompared with preset impedance contrast threshold value Δ Z, integral frequency range [f is determinedlow,fhigh], flowIt is impedance spectrum
Lower limit of integral frequency, fhighIt is impedance spectrum upper limit of integral frequency；The cell containing cell to be detected is fitted in the frequency range again
The impedance spectral function Z (f) and β (f) of culture solution and the cell culture fluid without containing cell to be detected, obtain impedance spectrum functional integration
To impedance spectrum integrated valueWithFinally calculate integrated value Δ I=
Ix-I0With cell activity index Vindex=Δ I/Imax, ImaxIt is cell activity upper limit value.
2. detection method according to claim 1, it is characterized in that: in step B, host computer (17) is from initial frequency f1Start
Impedance magnitude difference Δ Z under more each frequency one by oneiWith impedance contrast threshold value Δ Z, when occurring impedance magnitude difference Δ Z for the first timei>
When Δ Z, corresponding frequency is impedance spectrum lower limit of integral frequency flow；Later from end frequency fnIt is more each to start backward
Frequency impedance difference in magnitude Δ ZiWith impedance contrast threshold value Δ Z, when occurring impedance magnitude difference Δ Z for the first timeiWhen > Δ Z, corresponding frequency
Rate is impedance spectrum upper limit of integral frequency fhigh。
3. detection method according to claim 1, it is characterized in that: the every secondary control DDS signal of single-chip microcontroller occurs in step A
The frequency of device output is to increase identical step-length on the basis of previous output frequency.
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| CN111239197A (en) * | 2020-02-21 | 2020-06-05 | 中山大学 | Device and method for integrating cell full-spectrum impedance and rapid impedance measurement |
| CN112461751B (en) * | 2020-10-16 | 2022-04-26 | 江苏大学 | Cancer cell activity detection and evaluation device and method based on multi-adhesion strength fusion |
| CN113406153B (en) * | 2021-06-29 | 2022-06-07 | 毕玉琦 | Food flora short-term test device |
| CN114292749B (en) * | 2022-01-19 | 2023-10-24 | 中山大学 | Device and method for synchronously detecting high-flux intracellular and extracellular electrophysiological and mechanical pulsation activity of electrically excited cells |
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