CN101705184B - Full-automatic cell physiological parameter analyzer based on multi-parameter detection integrated chip - Google Patents

Abstract

Translated fromChinese

本发明公开了一种基于多参数检测集成芯片的全自动细胞生理参数分析仪，包括封闭的测量腔，测量腔内置有芯片接口装置和多参数集成芯片器件；芯片接口装置包括自下而上依次固定联接在一起的底座、芯片接口电路板和封盖，封盖的中心开有与多参数集成芯片器件相适配的通孔，封盖的通孔的上方联接有压力垫片，芯片接口电路板上焊接有弹簧顶针，多参数集成芯片器件置于封盖通孔中，且压力垫片、多参数集成芯片器件和弹簧顶针之间依次形成紧密接触；计算机、测量及控制电路的光寻址电位传感器检测电路、阻抗检测电路、微电极阵列检测电路分别与中央控制电路连接；光寻址电位传感器检测电路、阻抗检测电路、微电极阵列检测电路分别与芯片接口电路板连接。

The invention discloses a fully automatic cell physiological parameter analyzer based on a multi-parameter detection integrated chip, which comprises a closed measurement cavity, and a chip interface device and a multi-parameter integrated chip device are built in the measurement cavity; the chip interface device includes sequentially from bottom to top The base, the chip interface circuit board and the cover are fixedly connected together. The center of the cover is opened with a through hole suitable for the multi-parameter integrated chip device. The top of the through hole of the cover is connected with a pressure gasket. The chip interface circuit The spring thimble is welded on the board, and the multi-parameter integrated chip device is placed in the through hole of the cover, and the pressure pad, the multi-parameter integrated chip device and the spring thimble are in close contact in sequence; the optical addressing of the computer, measurement and control circuits The potential sensor detection circuit, the impedance detection circuit, and the microelectrode array detection circuit are respectively connected to the central control circuit; the light addressing potential sensor detection circuit, the impedance detection circuit, and the microelectrode array detection circuit are respectively connected to the chip interface circuit board.

Description

Detect the full-automatic cell physiological parameter analysis appearance of integrated chip based on multiparameter

Technical field

The present invention relates to a kind of cell physiological parameters analyser.

Background technology

The extracellular signal detecting method is owing to can realize can't harm; Grow the measurement of time-histories and receive increasing concern, relevant technical development is also maked rapid progress, like microelectrode array (MEA); LAPS (LAPS), cell impedance bioelectrical measurement or the like are also to commercialized development.But; When carrying out the cell physiological experiment; Monitoring to single parameter often can only be from reflecting the cells physiological activity in a certain respect; And the result of the complicacy of test environment (corrosion of various noisemakers, long-term test back nutrient solution counter electrode etc.) meeting distrubed test, so the announcement of pair cell physiological activity mechanism needs carry out analysis-by-synthesis to multiple relevant physiological parameter simultaneously.At present, existing cell chip and analysis device function singleness can only detect single parameter, can not realize that multi-physiological-parameter detects simultaneously, become cell physiological and analyze fast-developing bottleneck.

The one Chinese patent application that on April 9th, 2008, disclosed publication number was CN101158677 discloses a kind of cell electric physiology integrated chip and making method; This chipset microelectrode array transmitter (microelectrode array; MEA), (light-addressable potentiometric sensor is LAPS) with interdigitation cell-impedance transducer (interdigitated sensor for LAPS; IDA) three kinds of transmitters are in one; Can detect the adherent growth state of cell on device simultaneously, the concentration of multiple ion in action potential and the cell metabolism product, parallel detection when being used to realize multiparameter.

Along with the demand of cell multi-physiological-parameter analysis constantly rises, and the research of multiparameter integrated chip needs, and also do not have analyzer to can be used for the synchronous detection and the analysis-by-synthesis of multiparameter integrated chip and the multiple physiological parameter of cell at present.

Summary of the invention

Technical problem to be solved by this invention provides a kind of full-automatic cell physiological parameter analysis appearance based on multiparameter detection integrated chip, but the multiple physiological parameter of pair cell detects simultaneously and analyzes.

The present invention solves the technical scheme that its technical problem takes:

Should comprise the measurement chamber of sealing based on the full-automatic cell physiological parameter analysis appearance of multiparameter detection integrated chip, this measurement chamber is built-in with chip interface device and multiparameter integrated chip device; Said multiparameter integrated chip device is made up of culture chamber, multiparameter integrated chip and device substrate, and said multiparameter integrated chip is connected through spot welding with device substrate, culture chamber through nontoxic glue fixing on the multiparameter integrated chip; Said chip interface device comprises base, chip interface circuit plate and the capping that is fixedly connected sequentially together from bottom to top; The center of said capping has and the suitable through hole of multiparameter integrated chip device; The top of the through hole of said capping is connected with the pressure pad; Be welded with spring thimble on the chip interface circuit plate, multiparameter integrated chip device places the through hole of said capping, and forms closely contact successively between pressure pad, multiparameter integrated chip device and the spring thimble; Said analyser also comprises measurement and pilot circuit and computingmachine; Said measurement and pilot circuit comprise LAPS testing circuit, impedance detection circuit, microelectrode array testing circuit and central control circuit, and said LAPS testing circuit, impedance detection circuit, microelectrode array testing circuit and computingmachine are connected with central control circuit respectively; LAPS testing circuit, impedance detection circuit, microelectrode array testing circuit are connected with the chip interface circuit plate respectively.

Further, analyser of the present invention also comprises intake valve, and this intake valve links to each other with the measurement chamber through inlet pipe; Said measurement also is equipped with TP, humidity sensor, CO in the chamber₂Concentration sensor, well heater and humidifier; Said measurement and pilot circuit also comprise the environment pilot circuit, and this environment pilot circuit is connected with central control circuit; Said TP, humidity sensor, CO₂Concentration sensor, well heater, humidifier and intake valve are connected with the environment pilot circuit respectively.

Further; Analyser of the present invention also comprises sampling pump, T-valve and goes out a kind pump; Connect through the sample introduction conduit successively between said T-valve, sampling pump and the measurement chamber; Said go out kind pump and measure between the chamber connect through going out kind conduit, the outlet of said sample introduction conduit and the import that goes out kind conduit all place in the culture chamber of multiparameter integrated chip device; Said measurement and pilot circuit also comprise the flux analysis pilot circuit; Said sampling pump, T-valve and go out kind pump and be connected with the flux analysis pilot circuit respectively.

Further, the center of base according to the invention and chip interface circuit plate all has through hole, the central axes of the through hole of said base and chip interface circuit plate and the through hole of capping.

Further, impedance detection circuit according to the invention comprises active crystal oscillator, frequency divider, microcontroller, impedance transducer, bias adjustment circuit, programmable amplifying circuit, decay and bias adjustment circuit, impedance transducer and selective circuit of measuring range; Said active crystal oscillator, microcontroller, impedance transducer are connected with frequency divider respectively; Said microcontroller, decay and bias adjustment circuit, impedance transducer, selective circuit of measuring range, programmable amplifying circuit, bias adjustment circuit, impedance transducer link to each other successively; Said microcontroller links to each other with impedance transducer.

Further, LAPS testing circuit according to the invention comprises microcontroller, D/A converting circuit, Direct Digital Frequency Synthesizers, amplifying circuit, bias adjustment circuit, analog to digital conversion circuit, sin demodulator circuit, power amplification circuit, LAPS, programmable amplifying circuit, BPF., pre-amplification circuit, current-to-voltage converting circuit, cos demodulator circuit; Said microcontroller links to each other with D/A converting circuit, Direct Digital Frequency Synthesizers, analog to digital conversion circuit, programmable amplifying circuit, current-to-voltage converting circuit respectively; Said D/A converting circuit links to each other with bias adjustment circuit, LAPS respectively; Said Direct Digital Frequency Synthesizers, amplifying circuit, bias adjustment circuit, power amplification circuit, LAPS, current-to-voltage converting circuit, pre-amplification circuit, BPF., programmable amplifying circuit, sin demodulator circuit, analog to digital conversion circuit connect successively; Said amplifying circuit links to each other with sin demodulator circuit, cos demodulator circuit respectively; Said cos demodulator circuit links to each other with programmable amplifying circuit, analog to digital conversion circuit respectively.

Further; Microelectrode array testing circuit according to the invention comprises independently amplification and the data acquisition circuit more than ten six tunnel, and said amplification in every road and data acquisition circuit comprise prime amplifier, post-amplifier, low-pass filter, output buffer, data acquisition circuit; Said prime amplifier, post-amplifier, low-pass filter, output buffer, data acquisition circuit connect successively.

The principle of work of analyser of the present invention is: the multiparameter integrated chip device that cultivation is had cell; Be connected with analyser through the chip interface device; Control T-valve, sampling pump and go out the break-make and the rotating speed of kind pump by the flux analysis pilot circuit; Thereby accomplish the automatic sampling of nutrient solution and medicine and clean waste liquid, realize automatical analysis.By temperature, humidity and the CO of environment pilot circuit to the measurement chamber₂Concentration is carried out strict control makes it maintain the environment of suitable growth of cell.The variation that impedance transducer on the multiparameter integrated chip detects the cell impedance is detected and is sent to central control circuit by impedance detection circuit; The outer action potential signal of born of the same parents that the microelectrode array sensor detecting goes out cell obtains and sends to central control circuit by the microelectrode array testing circuit, and the ion variable signal that LAPS detects cellular metabolism obtains and be sent to central control circuit by the LAPS testing circuit.Central control circuit is controlled the process of whole physiology acquired signal, and after the signal data that obtains analyzed automatically, is sent on the computingmachine by the usb interface.

The multiparameter integrated chip is integrated microelectrode array; Three kinds of transmitters of LAPS and impedance transducer are in one; This chip utilizes between microelectrode array record action potentials of cells frequency, amplitude, waveform and cellular network parameters such as signal velocity; Utilize the growth of impedance transducer test cell and stick state and carry out the outer microenvironment of cell born of the same parents with LAPS and detect, like the metabolism composition detection of ion and molecule etc.

The multiparameter integrated chip is connected with analyser through specially designed chip interface device.Chip and analyser Interface design are mainly considered following 2 points: the one, and be convenient for changing multiparameter integrated chip device: because general cultivation the in the surface of multiparameter integrated chip device has cell, once multi-plate chip is changed in test possibly.Therefore to guarantee that chip changes quick and conveniently, and too strong vibrations can not be arranged when changing chip, in order to avoid influence the growth conditions of cell.The 2nd, the chip interface device will guarantee that electricity contacts well between multiparameter integrated chip device and the analyser, and the detection signal particularly signal amplitude of the outer action potential of born of the same parents is very faint, and bad contact is prone to bring noise and signal inaccurate.The present invention has adopted spring thimble, and the method for pressure pad realizes.The very convenient replacement multiparameter of this plan integrated chip device, vibrations that in the process of replacement, the measured chip device caused and damage are also minimum.The chip interface device also can detect the cell physiological chip of simple function, like microelectrode array chip, impedance transducer and the LAPS of standalone feature except detecting the multiparameter integrated chip simultaneously.

The main measuring object of analyser is an active somatic cell, in long-time the detection, needs to guarantee that cells physiological is active, and the growing environment of cell directly influences the signal detecting result of cell simultaneously.Therefore designed environmental control system in analyser can be to measuring temperature, humidity and the CO in the chamber₂Concentration is controlled and is regulated, and keeps normal physiological state and special experiment demand with the cell that guarantees to be incubated on the multiparameter integrated chip device.

In the cell physiological parameters test experience, needing often will change the nutrient solution of cell and apply medicine stimulates.Analyser is realized the automatical analysis of whole measuring process through flow analysis system.Nutrient solution and medicine are input to the surface of multiparameter integrated chip device through sampling pump; Automatic injection and the waste liquid of controlling sampling pump and the break-make that goes out kind pump and rotating speed completion nutrient solution and medicine through the flux analysis pilot circuit clean, and control the switching between nutrient solution and the medicine through flux analysis pilot circuit control T-valve.

The Core Feature of analyser is the various kinds of cell physiological signal that impedance transducer, microelectrode array, the LAPS on the parallel detection multiparameter integrated chip exported.Therefore analyser comprises the testing circuit of corresponding function part, and these circuit can carry out flexible combination according to the demand of chip, and carries out the complicacy sequential logic and measure.The plate card type design is adopted in the design of entire circuit, and independent functional units is an independently integrated circuit board.Mainboard is mainly by the environment pilot circuit, environment pilot circuit, impedance detection circuit, LAPS testing circuit, microelectrode array testing circuit, flux analysis pilot circuit, central control circuit, computer interface, compositions such as power module.Relatively independent between these circuit and the module, but can carry out communication and control each other through central control circuit.The design also has a powerful and influential advantage, is exactly that analyser can carry out flexible customization according to user's demand.Design according to different multiparameter integrated chips; Assemble flexibly; Do not need environment control section, some demand just to be used for detecting impedance transducer and two parts of light addressing circuit transmitter like some experiment, then do not need the circuit-board card of multi channel signals amplification and collecting unit.

This analysis appearance can be effective to the multiparameter integrated chip, can detect the action potential of cell, the concentration change and the cell impedance parameter of extracellular different kinds of ions simultaneously, have short, sample consumption analysis time less with the efficient advantages of higher.In measuring process, can regulate the temperature, humidity and the CO that measure in the chamber automatically₂Concentration is to satisfy the growing environment requirement of cell.Can realize the automatical analysis function through flow analysis system simultaneously.Can be widely used in the screening and the detection range of cell physiological, medicine.In addition, this analyser can be realized can quantizing detected result the real-time detection of cell physiological state behind the conduction of unicellular, cell neural network and the drug effect.Owing to be used in the unique design of chip interface device and plate card type circuit layout, the detection demand of the applicable multiple different multiparameter integrated chips of this analysis appearance and the demand of kinds of experiments scheme.

Description of drawings

Fig. 1 is the one-piece construction synoptic diagram of analyser of the present invention;

Fig. 2 is a multiparameter integrated chip structure iron;

Fig. 3 is the encapsulation synoptic diagram of multiparameter integrated chip device;

Fig. 4 is the perspective view of chip interface device;

Fig. 5 is the synoptic diagram of a kind of embodiment of chip interface circuit;

Fig. 6 measures chamber internal structure synoptic diagram;

Fig. 7 is a flux analysis pilot circuit schematic diagram;

Fig. 8 is the temperature-control circuit schematic diagram;

Fig. 9 is the humidity control circuit schematic diagram;

Figure 10 is CO₂Concentration pilot circuit schematic diagram;

Figure 11 is an impedance transducer testing circuit schematic diagram;

Figure 12 is a LAPS testing circuit schematic diagram;

Figure 13 is a microelectrode array testing circuit schematic diagram;

Figure 14 is the structural representation of measurement and pilot circuit assembled embodiment;

Figure 15 is the structural representation of analyser of the present invention;

Figure 16 is LAPS measuring result figure on the multiparameter integrated chip of the present invention;

Figure 17 is impedance transducer measuring result figure on the multiparameter integrated chip of the present invention;

Figure 18 is the measuring result figure of microelectrode array on the multiparameter integrated chip of the present invention.

Among the figure: 1, sampling pump, 2, T-valve, 3, the nutrient solution container, 4, medicament reservoir; 5, chip interface device pedestal, 6, chip interface device connecting struts, 7, multiparameter integrated chip device, 8, chip interface device thickness adjustment sheet; 9, chip interface device pressure pad, 10, spring thimble, 11, the capping of chip interface device, 12, the chip interface circuit plate; 13, the measurement chamber of sealing, 14, go out a kind pump, 15, go out a kind conduit, 16, waste fluid container; 17, TP, 18, humidity sensor, 19, CO₂Concentration sensor, 20, humidifier, 21, well heater, 22, intake valve, 23, CO₂Gas cylinder, 24, measurement and pilot circuit, 25, the chip interface device, 26, CO₂Gas passage, 27, the flux analysis passage, 28, power interface, 29, USB interface; 7.1, the multiparameter integrated chip, 7.11, impedance transducer, 7.12, reference electrode, 7.13, the microelectrode array transmitter; 7.14, LAPS, 7.21, culture chamber, 7.22, device substrate, 12.1, the spring thimble fixed orifices; 12.2, lead, 12.3, measurement and pilot circuit interface, 24.1, environment pilot circuit integrated circuit board, 24.2, environment pilot circuit interface; 24.3, the impedance detection circuit integrated circuit board, 24.4, LAPS testing circuit integrated circuit board, 24.5, microelectrode array testing circuit integrated circuit board, 24.6, flux analysis pilot circuit integrated circuit board; 24.7, the central control circuit integrated circuit board, 24.8, computer interface, 24.9, power module.

Embodiment

Fig. 1 has provided the one-piece construction figure of whole analyser.It comprises themeasurement chamber 13 of sealing, and thismeasurement chamber 13 is built-in with chip interface device, multiparameter integratedchip device 7,TP 17,humidity sensor 18, CO₂Concentration sensor 19,well heater 21 andhumidifier 20.

Analyser comprises that nutrient solution and medicine are automatically to an appearance system.Liquid innutrient solution container 3 and themedicament reservoir 4 flow into the surface of multiparameter integratedchip device 7 throughsampling pump 1, is used for the cell on the chip is stimulated.Break-make and rotating speed through the full flux analysis pilot circuitcontrol sampling pump 1 in measurement and thepilot circuit 24; Completion is injected automatically and is cleanednutrient solution container 3 and the liquid in themedicament reservoir 4; Waste liquid flows in thewaste fluid container 16 through going outkind pump 16, the switching between T-valve 2 control nutrient solutions and the medicine.Connect through the sample introduction conduit successively between T-valve 2,sampling pump 1 and themeasurement chamber 13; Go outkind pump 14 and measure between thechamber 13 and connect through going out kind conduit, the outlet of sample introduction conduit and the import that goes out kind conduit all place in the culture chamber 7.21 of multiparameter integrated chip device 7.Sampling pump 1, T-valve 2 and go outkind pump 14 and be connected with the flux analysis pilot circuit respectively.

TP 17,humidity sensor 18, CO₂Concentration sensor 19,well heater 21,humidifier 20,intake valve 22 is connected with the environment pilot circuit respectively, and the composing environment system realizes measuring temperature, humidity, the CO in the sky₂The control of concentration.

The output signal of multiparameter integrated chip device links to each other with measurement andpilot circuit 24 through the chip interface device.Measurement andpilot circuit 24 comprise LAPS testing circuit, impedance detection circuitry, microelectrode testing circuit, central control circuit and environment pilot circuit, and said LAPS testing circuit, impedance detection circuitry, microelectrode testing circuit, environment pilot circuit and computingmachine are connected with central control circuit respectively; LAPS testing circuit, impedance detection circuitry, microelectrode testing circuit are connected with chipinterface circuit plate 12 respectively.

Fig. 2 has provided the structure of the disclosed a kind of multiparameter integrated chip of one Chinese patent application that disclosed publication number is CN101158677 on April 9th, 2008.Integrated microelectrode array 7.13 in the middle of the Si substrate front surface, respectively integrated impedance transducer 7.11 and LAPS 7.14 are symmetrical set reference electrode 7.12 respectively in microelectrode array 7.13 other both sides in microelectrode array 7.13 both sides.The type integrated chip comprises a LAPS, an impedance transducer, and the microelectrode array that contains 35 microelectrodes.

Fig. 3 has provided the encapsulation synoptic diagram of multiparameter integrated chip device.Multiparameter integrated chip device is made up of culture chamber 7.21, multiparameter integrated chip 7.1, device substrate 7.22.Multiparameter integrated chip 7.1 and device substrate 7.22 are connected through spot welding, culture chamber 7.21 through nontoxic glue fixing above multiparameter integrated chip 7.1 and device substrate 7.22.

Fig. 4 is analyser and chip interface schematic representation ofapparatus.Base 5 in the chip interface device, chipinterface circuit plate 12 are fixedly connected sequentially from bottom to top withcapping 11 and are in the same place, and fix through connecting struts 6.The center ofcapping 11 has the through hole suitable with multiparameter integrated chip device 7.Base 5 all has through hole with the center of chipinterface circuit plate 12, the central axes of the through hole ofbase 5 and chipinterface circuit plate 12 and the through hole of capping 11.The top of the through hole ofcapping 11 is connected withpressure pad 9, is welded withspring thimble 10 on the chip interface circuit plate 12.Multiparameter integratedchip device 7 places the through hole ofcapping 11; And form closely contact betweenpressure pad 9, multiparameter integratedchip device 7 and thespring thimble 10 successively, wherein the solder joint on the multiparameter integratedchip device 7 respectively with chipinterface circuit plate 12 onspring thimble 10 form and contact one by one.When instrument is implemented, can add thickness adjustedsheet 8, place betweenpressure pad 9, the multiparameter integratedchip device 7, to strengthen pressure between multiparameter integratedchip device 7 and thespring thimble 10, to form betterelectricity contact.Base 5 processes with capping 11 usefulness synthetic glass or otherisolator.Pressure pad 9 can adopt Rotation Design,pressure pad 9 can connectingstruts 6 for the axle center is rotated, make things convenient for multiparameter integratedchip device 7 from the through hole ofcapping 11, to put into and take out.Chipinterface circuit plate 12 is connected with LAPS testing circuit, impedance detection circuitry, microelectrode testing circuit on measurement and the pilot circuit 24.The very convenient replacement measured chip of this plan, vibrations that in the process of replacement, measured chip caused and damage are also minimum.

Fig. 5 is the synoptic diagram of one of them embodiment of chip interface circuit plate 12.Wherein spring thimble fixed orifices 12.1 welding that is used forspring thimble 10 is fixed, and realizes contacting with the good of chip.Chip is through the 12.2 output signals of the lead on the circuit card, and the entire circuit plate is linked to each other with measurement andpilot circuit 24 by measurement and pilot circuit interface 12.3.

Fig. 6 ismeasurement chamber 13 structural representations of sealing.At the middle portion ofmeasuring chamber 13 is theinterface arrangement 25 of multi-functional integratedchip device 7 and instrument.Aroundmeasurement chamber 13, be furnished withTP 17,humidity sensor 18 and CO₂Concentration sensor 19, and correspondingwell heater 21,humidifier 20, and CO₂Gas passage 26.Flux analysis circulation 27 is used to place thefluid pipeline 15 of automatical analysis process.

Fig. 7 is a flux analysis pilot circuit schematicdiagram.Sampling pump 1, go outkind pump 14, T-valve 2 links to each other with MCU respectively,sampling pump 1, go out betweenkind pump 14 and the microcontroller to also have D/A convertingcircuit.Sampling pump 1 all use the Transistor-Transistor Logic level trip switch and is turned to going outkind pump 14, and the voltage swing of exporting with D/A converting circuit comes the rotating speed of control pump.The voltage of D/A converting circuit output is controlled by the numerary signal of microcontroller output.

Fig. 8 is the temperature-control circuit schematic diagram.TP 17 adopts RTD (Pt100), constitutes the three-wire system temperature measurement circuit with constant current supply, resistance and differential amplifier circuit.TP 17 RTDs (Pt100) convert temperature variation into resistance change, and temperature measurement circuit converts the resistance variation ofTP 17 into voltage output, is exported by differential amplifier circuit.The voltage of differential amplifier circuit output becomes numerary signal through analog and is input to microcontroller.Microcontroller adopts increment type PID algorithm relay break-make according to the temperature of setting, and then the heat-up time of control heater, realizes temperature control rapidly, continuously.RTD exists non-linear, adopts method of least squares to solve the nonlinearity erron of RTD.In the temperature control system of reality,, carry out actual experiment again parameter is carried out adjustment progressively through the initial value of the curve calculation pid parameter of ascending to heaven.

Fig. 9 is the humidity control circuit schematic diagram.Humidity control circuit is similar with the temperature-control circuitprinciple.Humidity sensor 18 adopts commercial environment-friendly type Humirel humidity module HTG3515CH, and its output form is a linear voltage output relative humidity.Significant parameter 0～100%RH relative humidity scope, precision ± 3%RH, operating temperature range-40～110 ℃, the 5s time of response, 0 ± 1%RH is sluggish.The analog voltage signal of humidity sensor output becomes numerary signal through analog and is input to microcontroller.Microcontroller is according to the humidity of setting, and the relay break-make is controlled humidification time of humidifier then.

Figure 10 is CO₂Concentration pilot circuit schematic diagram.CO₂Concentration sensor 19 has adopted the infrared CO of the C20 of Britain GSS company₂Concentration sensor is gas sensor and the system that adopts indium antimonide aluminium (NDIR LED technology), is output as TTL level RS232 signal.CO₂Concentration sensor is with CO₂Concentration information is input to MCU after being converted into numerary signal.MCU is according to the parameter of setting, relay, and then the closing of control intake valve 22.CO₂External gas cylinder 23 through specialty realizes that to gas external gas cylinder is not the standard component of this analysis appearance.

Shown in Figure 11 is the impedance detection circuit schematic diagram.Impedance detection circuit is core with the digital lock-in amplifier, and the core of lock-in amplifier adopts impedance transducer ASIC-AD5933.Impedance transducer AD5933 is a impedance measurement integrated chip that ADI company releases, and is a high-precision impedance transformation system scheme, the analog of inner integrated frequency generator and 12 1MSPS.Because the stimulus signal that need in impedance measurement, apply must could guarantee enough for a short time that the linear condition of system makes stimulus signal can not change the characteristic of system itself; The 2V peak-to-peak value of AD5933 output is not suitable for the actuation signal as transmitter, so need it is done the adjustment of decay and biasing.Attenuation circuit constitutes by 2 grades, decays 20 times and 10 times respectively.Make output at the 10mV peak-to-peak value, reach the requirement of transmitter input.Because the AD5933 input range can only be 0-3.3V voltage, so need to use the direct current biasing of bias adjustment circuit to a 1.5V of prime output signal stack.Realize the stack of biasing through an in-phase adder.

Figure 12 is a LAPS testing circuit schematic diagram.This partial circuit is a core with the simulation lock-in amplifier, adopts Direct Digital Frequency Synthesizers DDS as signal generator, through amplifying circuit, imports sensor unit into, and the output signal carries out demodulation with reference signal after treatment.The unitary main control unit of entire circuit has adopted the high-performance MSP430 micro-chip of TI company.Signal generating circuit has adopted direct digital frequency synthesis technology, and the AD9854 chip that utilizes ADI company to produce produces 2 tunnel frequencies and the adjustable orthogonal signals of amplitude, and frequency is 100HZ-100KHZ.The signal amplitude that Direct Digital Frequency Synthesizers DDS directly exports is less, needs could supply late-class circuit to use through amplifying.For the linear input signal that guarantees transmitter will be again could driving sensor improves accuracy of detection in order to reach to switch through range through overdamping and direct current biasing adjustment,, current/voltage switches feedback resistance in changing through analog switch.The OPA627 high performance amplifier that amplifier adopts TI company to produce can provide higher bandwidth (16MHZ), very little bias current (1PA) and and low-noise performance.The preposition amplification of voltage uses circuit to be the standard in-phase amplifying circuit.Magnification is set at 11 times, and that the corresponding levels are used is the broadband operational amplifier LMH6624 of ultra-low noise, and its wide spectrum noise is low to moderate 0.92nV/sqrtHz.Because the analog channel of lock-in amplifier is ac coupling, the 1/f noise of magnifying glass can not exert an influence to system performance.Programmable amplifier provides the fine setting of magnification, to reach the optimal input range of MXR and AD converter.The reference signal of detuner is provided by the DDS signal generator.Output after the demodulation comprise input signal and reference signal with frequently and difference frequency.Obtain difference frequency signal through low-pass filter, accomplish synchronous demodulation.Adopt 2 AD630 detuners can demodulate real part of impedance and imaginary part simultaneously.Because AD converter can only be imported positive voltage, but low pass output signal possibly be negative value.Signal is done and is input to AD converter through anti-aliasing circuit after a nearly step is managed and raised.The ADS1256 high-precision difference input AD converter that AD converter is produced for TI company.Its sampling rate can reach 30KSPS, and is enough fully for gathering direct current signal.Adopt DAC8811 current mode DA and cooperate external high-operational amplifier OPA2277 to accomplish the voltage of program-controlled output-2.5V-+2.5V.LTC1821 high-accuracy voltage type DA transmodulator be used to provide LAPS needed-the 10V-+10V direct current biasing.

Figure 13 is a microelectrode array testing circuit schematic diagram.The microelectrode array testing circuit comprises multichannel magnifying glass, and high-Speed Data-Acquisition Module.Signal after low-pass filter filtering, arrives data acquisition circuit after the output buffering after prime amplifier, post-amplifier two-stage are amplified.

Figure 14 has provided the detection and the pilot circuit embodiment synoptic diagram of analyser.The design of The whole design plate card type, independent functional units is an independently integrated circuit board.Mainboard mainly is made up of power module 24.9, central control unit 24.7 and computer USB interface unit 24.8, measurement chamber interface 24.2 and bus etc.The main circuit functional block that instrument comprises has environment pilot circuit 24.1, automatical analysis pilot circuit 24.6, impedance detection circuit 24.3, LAPS testing circuit 24.4 and microelectrode array testing circuit 24.5 etc.LAPS testing circuit 24.4, impedance detection circuitry 24.3, microelectrode array testing circuit 24.5, environment pilot circuit 24.1 and computingmachine are connected with central control circuit respectively.These functional modules are relatively independent each other, but can carry out communication and control each other through central control unit 24.7.The design also has a powerful and influential advantage, is exactly that instrument can carry out flexible customization according to user's demand.According to the design of different integrated chips, carry out instrument and listen flexible assembling, do not need the environment control section like some experiment.

Shown in Figure 15 is the structural representation of multi-functional cell physiological parameters analyser whole implementation example.Mainly, measure andpilot circuit 24,sampling pump 1, go outkind pump 14,nutrient solution container 3,medicament reservoir 4,waste fluid container 16, power interface 28, computer USB interface 29 integral parts such as grade by measuring chamber 13.Measure the top thatchamber 13 is positioned at analyser, containchip interface device 25 etc.Sampling pump 1, go outkind pump 14,nutrient solution container 3,medicament reservoir 4,waste fluid container 16 and correspondent control circuits form full-automatic flow analysis system through pipeline.Measurement andpilot circuit 24 are positioned at the rear portion of analyser, link to each other with computingmachine through USB interface 29.Whole instrument is supplied power by the 220V alternating-current.

Provide application implementation example of the present invention below.

LAPS on the multiparameter integrated chip is mainly used in the microenvironment that detects cell and detects, and promptly the outer ionic concn of born of the same parents detects.In the experiment, directly be Si on the LAPS surface₃N₄, be a kind of material responsive to hydrogen ion.Adopt pH to be respectively 6,7, four kinds of solution of 8,9 are measured, and the result is shown in figure 16.By finding out that along with reducing of pH, curve moves to left along bias voltage among the figure, side-play amount is directly proportional with pH value change amount substantially.Along with reducing of pH, Sorensen value increases in the solution, makes that the potential difference between solution and sensor surface two phases increases.This potential difference is added to behind the bias voltage, causes whole the moving to left of curve of measuring.According to Nernst equation, the logarithmic value (pH of ie in solution) of the hydrogen ion activity in two alternate potential differences and the solution is directly proportional.The skew of the i-v curve through detecting LAPS just can obtain the pH value of solution.This instrument of experimental result proof can correctly detect the output of the LAPS on the multiparameter integrated chip.

Impedance transducer measurement on the employing multiparameter integrated chip is incubated at the cell mass on the broad-area electrode or is incubated at the impedance characteristic of the individual cells on the microelectrode.These impedance measurements have shown propagation, migration and the cell adhesion information of electroactive excitable cell or non-excitable cell type.Select the ECV-304 endotheliocyte, adopt gold electrode at 25KHz (it is the most obvious that a large amount of experiments show that this frequency impedance changes), 10mV grows the experiment of the adherent property of time-histories under stimulating.Measuring result is shown in figure 17, has reflected the whole process of cell attachment and apoptosis more really.This instrument of experimental result proof can correctly detect the output of the impedance transducer on the multiparameter integrated chip.

Experiment utilize on the multiparameter integrated chip microelectrode array to former generation cardiac muscle cells carry out the detection of born of the same parents' volta potential.The myocardial cell is former to be commissioned to train to support and to be attached to the MEA device surface after 2 days, and cell is the transparent shape of heaving, and most of cell grows on the required electrode site.Shown in Figure 180 is the multi channel signals output of microelectrode array, can see the outer action potential signal of tangible born of the same parents.This instrument of experimental result proof can correctly detect the output of the microelectrode array on the multiparameter integrated chip.

Claims (7)

1. one kind is detected the full-automatic cell physiological parameter analysis appearance of integrated chip based on multiparameter, and it is characterized in that: it comprises the measurement chamber (13) of sealing, and this measurement chamber (13) is built-in with chip interface device and multiparameter integrated chip device (7); Said multiparameter integrated chip device is made up of culture chamber (7.21), multiparameter integrated chip (7.1) and device substrate (7.22); Said multiparameter integrated chip (7.1) is connected through spot welding with device substrate (7.22), culture chamber (7.21) through nontoxic glue fixing on multiparameter integrated chip (7.1); Said chip interface device comprises base (5), chip interface circuit plate (12) and the capping (11) that is fixedly connected sequentially together from bottom to top; The center of said capping (11) has and the suitable through hole of multiparameter integrated chip device (7); The top of the through hole of said capping (11) is connected with pressure pad (9); Be welded with spring thimble (10) on the chip interface circuit plate (12); Multiparameter integrated chip device (7) places the through hole of said capping (11), and forms closely contact successively between pressure pad (9), multiparameter integrated chip device (7) and the spring thimble (10); Said analyser also comprises measurement and pilot circuit and computingmachine; Said measurement and pilot circuit comprise LAPS testing circuit, impedance detection circuit, microelectrode array testing circuit and central control circuit, and said LAPS testing circuit, impedance detection circuit, microelectrode array testing circuit and computingmachine are connected with central control circuit respectively; LAPS testing circuit, impedance detection circuit, microelectrode array testing circuit are connected with chip interface circuit plate (12) respectively.

2. the full-automatic cell physiological parameter analysis appearance based on multiparameter detection integrated chip according to claim 1, it is characterized in that: this analyser also comprises intake valve (22), this intake valve (22) links to each other with measurement chamber (13) through inlet pipe; Also be equipped with TP (17), humidity sensor (18), CO in the said measurement chamber (13)₂Concentration sensor (19), well heater (21) and humidifier (20); Said measurement and pilot circuit also comprise the environment pilot circuit, and this environment pilot circuit is connected with central control circuit; Said TP (17), humidity sensor (18), CO₂Concentration sensor (19), well heater (21), humidifier (20) and intake valve (22) are connected with the environment pilot circuit respectively.

3. the full-automatic cell physiological parameter analysis appearance that detects integrated chip based on multiparameter according to claim 1 and 2; It is characterized in that: this analyser also comprises sampling pump (1), T-valve (2) and goes out a kind pump (14); Connect through the sample introduction conduit successively between said T-valve (2), sampling pump (1) and the measurement chamber (13); Said go out kind pump (14) and measure between the chamber (13) connect through going out kind conduit, the outlet of said sample introduction conduit and the import that goes out kind conduit all place in the culture chamber (7.21) of multiparameter integrated chip device (7); Said measurement and pilot circuit also comprise the flux analysis pilot circuit; Said sampling pump (1), T-valve (2) and go out a kind pump (14) and be connected with the flux analysis pilot circuit respectively.

4. the full-automatic cell physiological parameter analysis appearance that detects integrated chip based on multiparameter according to claim 1 and 2; It is characterized in that: the center of said base (5) and chip interface circuit plate (12) all has through hole, the central axes of the through hole of said base (5) and chip interface circuit plate (12) and the through hole of capping (11).

5. the full-automatic cell physiological parameter analysis appearance based on multiparameter detection integrated chip according to claim 1, it is characterized in that: said impedance detection circuit comprises active crystal oscillator, frequency divider, microcontroller, impedance transducer, bias adjustment circuit, programmable amplifying circuit, decay and bias adjustment circuit, impedance transducer and selective circuit of measuring range; Said active crystal oscillator, microcontroller, impedance transducer are connected with frequency divider respectively; Said microcontroller, decay and bias adjustment circuit, impedance transducer, selective circuit of measuring range, programmable amplifying circuit, bias adjustment circuit, impedance transducer link to each other successively; Said microcontroller links to each other with impedance transducer.

6. the full-automatic cell physiological parameter analysis appearance based on multiparameter detection integrated chip according to claim 1, it is characterized in that: said LAPS testing circuit comprises microcontroller, D/A converting circuit, Direct Digital Frequency Synthesizers, amplifying circuit, bias adjustment circuit, analog to digital conversion circuit, sin demodulator circuit, power amplification circuit, LAPS, programmable amplifying circuit, BPF., pre-amplification circuit, current-to-voltage converting circuit, cos demodulator circuit; Said microcontroller links to each other with D/A converting circuit, Direct Digital Frequency Synthesizers, analog to digital conversion circuit, programmable amplifying circuit, current-to-voltage converting circuit respectively; Said D/A converting circuit links to each other with bias adjustment circuit, LAPS respectively; Said Direct Digital Frequency Synthesizers, amplifying circuit, bias adjustment circuit, power amplification circuit, LAPS, current-to-voltage converting circuit, pre-amplification circuit, BPF., programmable amplifying circuit, sin demodulator circuit, analog to digital conversion circuit connect successively; Said amplifying circuit links to each other with sin demodulator circuit, cos demodulator circuit respectively; Said cos demodulator circuit links to each other with programmable amplifying circuit, analog to digital conversion circuit respectively.

7. the full-automatic cell physiological parameter analysis appearance that detects integrated chip based on multiparameter according to claim 1; It is characterized in that: said microelectrode array testing circuit comprises independently amplification and the data acquisition circuit more than ten six tunnel, and said amplification in every road and data acquisition circuit comprise prime amplifier, post-amplifier, low-pass filter, output buffer, data acquisition circuit; Said prime amplifier, post-amplifier, low-pass filter, output buffer, data acquisition circuit connect successively.

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