CN101713757B - Photoelectric compound integral sensor for detecting cell physiological parameters and preparation method thereof - Google Patents
Photoelectric compound integral sensor for detecting cell physiological parameters and preparation method thereof Download PDFInfo
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- CN101713757B CN101713757B CN200910154615A CN200910154615A CN101713757B CN 101713757 B CN101713757 B CN 101713757B CN 200910154615 A CN200910154615 A CN 200910154615A CN 200910154615 A CN200910154615 A CN 200910154615A CN 101713757 B CN101713757 B CN 101713757B
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Abstract
The invention discloses a photoelectric compound integral sensor for detecting cell physiological parameters and a preparation method thereof. The sensor comprises a cavity body fixed on a printed circuit board (PCB) and a cavity cover matched with the cavity body; the bottom of the cavity body is fixedly provided with an integrated silicon chip, and the center of the integrated silicon chip is provided with a reference electrode; two ends of the reference electrode are respectively provided with a group of multisite electrode arrays; two sides of the middle part of the reference electrode are respectively provided with a pair of impedance electrodes; two sides of each of both end parts of the reference electrode are respectively provided with a first optical addressing potential electrode, and the back of the first addressing potential electrode is provided with a groove; the cavity cover is provided with at least two fluid through holes and at least four LED through holes, and the end face of a main body part of the cavity cover is fixedly provided with a second optical addressing potential electrode array; and the main body part of the cavity cover is arranged in the cavity body, and a gap is formed between the end face of the main body of the cavity cover and the integrated silicon chip. The invention can make stable, reliable nd synchronous detection of the cell physiological parameters for a long time, and can improve the signal-to-noise ratio of the detected electrode.
Description
Technical field
The present invention relates to fields such as cell electrophysiology, micro electronmechanical process technology, cell sensor.In monitoring, can be used for cultured in vitro physiological status of cells and physiological function are analyzed; Microenvironment through changing the cell growth, the variation of each physiological parameter of monitoring cell respond microenvironment are analyzed.
Background technology
Stechiology is a subject of the character and the function of bio signal in cellular level and molecular level studying physiological process, and functions such as ability cell membrane electrical characteristics etc. are analyzed, and are the bases of life science.Ion channel on the cell membrane is the important carrier of cell normal physiological activity.Patch-clamp and fluorescent dye are the transportation of ion channel intermediate ion and more cyto-architectural important method on the research cell membrane.Though these methods relatively directly and can more accurately detect, pair cell has damage and toxicity, so the long-term active procedure of pair cell is difficult to detect; Devices needed is relatively more expensive and not easy to operate in testing process simultaneously.
Along with the development of modern microelectric technique and micro-processing technology, the research platform of cell develops to no invasive, long-term detection, high flux, low-cost direction.Sensing range is also from the structure function expansion of traditional ion channel research to whole growth process and whole cell.
The cell impedance is a kind of means that states such as the attaching of cultured in vitro cell when on the double dish wall, growing, migration, apoptosis are studied.When cell is grown on the electrode of impedance chip, because the insulativity of cell membrane makes that the attaching space between electrode and the solution reduces, thereby makes impedance variations.The area of attachment of cell and electrode and dynamics can be used for the growth conditions of characterize cells on electrode.In the impedance electrodes commonly used, the electrode of interdigitation can increase the girth of electrode edge, increases the sensitivity of cell impedance detection.
For the electrical excitation sexual cell, action potential detects and can the each several part ion motion of ion channel be characterized in the born of the same parents of cell, is also embodied in simultaneously on the outer Electric Field Distribution of cell born of the same parents.In the detection method in modern times, FET and multidigit point electrode array are two kinds of methods commonly used.The processing of multidigit point electrode array is fairly simple by contrast, uses more.Usually, the site size and the cell size of multidigit point electrode are complementary, and are that multi-channel parallel detects, and exchange between pair cell simultaneously as the information transmission of myocardium plasomidum, neural network can synchronization map; In addition, also can describe the function of ion channel through the outer signal waveform of born of the same parents.
Because not every ion can both obtain mapping in born of the same parents' external electric field signal, for the signal of non-electrical excitation cell, the cellular metabolism ion can not form concentrated current potential granting on a large scale especially.Therefore the invention of little physiology meter is exactly the long-time metabolism that is used for detecting the various ions of cell, and is not a large amount of ion motions of short duration in the action potential.Light-addressable potentiometric electrode (LAPS) is a photoelectric effect of utilizing silicon type device, under illumination, is activated at generation electron-hole pair formation photogenerated current in the silicon through pulse laser.In the time of LAPS surface ion change in concentration, thereby the electric field in the whole electron-hole pair changes and makes photogenerated current also change.This method can characterize the ion concentration of electrode surface quickly.Because therefore the effects of ion complicated component when detecting, carry out ion selective membrane to the LAPS surface and handle, so that object ion is selected.
With above-mentioned three partial functions be integrated in same chip more fully the outer physiology parameter of pair cell born of the same parents detect.And existing method has deficiency in the following areas:
1) in processing, multidigit point electrode array and impedance electrodes have certain similarity, but and the difference between the LAPS bigger, the structure of device is different, the processing thickness of each layer is distinguished bigger.Three part job operations can not reach compatible in the existing method, and the LAPS back side is not thinned, and signal to noise ratio (S/N ratio) is not high;
2) adopting LAPS to detect Na outside the born of the same parents
+, K
+During Deng metabolism; The LAPS surface needs the corresponding ion selective membrane of coating; With multidigit point electrode array, the long-term co-operation of impedance electrodes the time, because cellular incubation needs in nutrient solution, to soak for a long time, cell culture fluid is polluted easily; Ion selective membrane is corroded easily, can cause the instability that detects;
3) in common integrated chip design, because chip area layout and area of chip restriction make that the electrode channel number of each several part is limited, the quantity of information that therefore provides is also abundant inadequately.
Therefore guaranteeing that the omnibearing physiological activity of cell can detect simultaneously, the compatibility processing between this three partial functions chip, detection etc. become the critical problem in the detection of cell multiparameter.
Summary of the invention
A technical matters to be solved by this invention provides a kind of integrated silicon chip that improves the detecting electrode signal to noise ratio (S/N ratio).
Another technical matters to be solved by this invention provides a kind of photoelectric compound integral sensor of stable, reliable detection cell physiological parameters.
The present invention solves the technical scheme that its technical matters takes: the center arrangement at integrated silicon chip has reference electrode; The two ends of said reference electrode respectively are furnished with one group of multidigit point electrode array; Both sides at the middle part of reference electrode respectively are furnished with a pair of impedance electrodes; Both sides in two ends of reference electrode respectively are furnished with one first light-addressable potentiometric electrode, and the back side of the said first light-addressable potentiometric electrode is provided with groove.
Further, the end of reference electrode according to the invention is provided with mask frame, and the site of said multidigit point electrode array is positioned at mask frame.
The photoelectric compound integral sensor that the present invention has the detection cell physiological parameters of above-mentioned integrated silicon chip mainly comprises cavity that is fixed on the pcb board and the chamber lid that is complementary with this cavity; Bottom at cavity is fixed with integrated silicon chip; The center arrangement of said integrated silicon chip has reference electrode; Respectively be furnished with one group of multidigit point electrode array at the two ends of reference electrode; The both sides at the middle part of reference electrode respectively are furnished with a pair of impedance electrodes, respectively are furnished with one first light-addressable potentiometric electrode in the both sides of two ends of reference electrode, and the back side of the said first light-addressable potentiometric electrode is provided with groove; The chamber covers and is provided with at least two fluid through-holes and at least four LED through holes, and the end face towards said integrated silicon chip of the main part of chamber lid is fixed with the second light-addressable potentiometric electrod-array; The main part of chamber lid places in the cavity, between the said end face of the main part of chamber lid and the integrated silicon chip space is arranged.
Further, the end of reference electrode according to the invention is provided with mask frame, and the site of said multidigit point electrode array is positioned at mask frame.
Integrated silicon chip preparation method of the present invention mainly comprises the steps:
1) be substrate with the silicon chip, after the cleaning respectively the adjacent domain at four of the etching silicon chip back side angles to form four grooves;
2) at the positive heat growth of silicon chip one deck the one SiO
2Layer is then at a SiO
2Deposition the one Si on the layer
3N
4Layer; Adopt the method for photoetching again, the SiO outside will the zone corresponding with groove
2A layer and a Si
3N
4Layer etching;
3) deposition one deck the 2nd SiO on the front of silicon chip
2Layer; At the 2nd SiO
2Sputter one deck Ti layer on the layer, the layer of sputter layer of Au again forms metal composite layer Ti/Au; Adopt the method for photoetching, metal composite layer Ti/Au is etched electrode and the lead pattern that presets; Center arrangement at said electrode and lead pattern has reference electrode; The two ends of said reference electrode respectively are furnished with one group of multidigit point electrode array; Both sides at the middle part of reference electrode respectively are furnished with a pair of impedance electrodes; Both sides in two ends of reference electrode respectively are furnished with one first light-addressable potentiometric electrode, and the position at said first light-addressable potentiometric electrode place is corresponding with the groove at the silicon chip back side;
4) continue deposition one deck the 2nd Si on the front of silicon chip
3N
4Layer, and with the 2nd Si on the electrode site of said multidigit point electrode array, pad and the zone corresponding with groove
3N
4Etching;
5) the 2nd SiO on will the zone corresponding on the front of silicon chip with groove
2Layer etching;
6) sputter Al film on the zone outside the groove at the silicon chip back side obtains integrated silicon chip.
Further, the end of reference electrode according to the invention is provided with mask frame, and the site of said multidigit point electrode array is positioned at mask frame.
Compared with prior art; The invention has the beneficial effects as follows: at the bottom of the chamber of the present invention's design-the more fully detection of physiological functions such as cell growth state, film potential, the outer metabolic ions of born of the same parents of chamber lid integral sensor, solved the problem that occurs mutual corrosion contamination between cellular incubation and the LAPS surface ion selective membrane; The chip that integrated silicon chip at the bottom of the chamber will have three kinds of functions adopts compatible method for processing, is machined on the same silicon base chip, satisfying with in the multiparameter synchronous detection of a cellular incubation, has also improved the performances such as signal to noise ratio (S/N ratio) of chip.This cavity integral sensor can provide more cell physiological movable effective information; Provide that an ability cell growth state and function carry out that real non-destructive detects stable, the cell multiparameter light is replied unification body sensor platform by cable reliably, can apply to fields such as basic cell physiological detection, drug screening assessment, environmental toxicity detection.
Description of drawings
Fig. 1 is the structural representation of photoelectric compound integral sensor of the present invention.
Fig. 2 (a) is the structural representation of the chamber lid of photoelectric compound integral sensor of the present invention.
Fig. 2 (b) is the upward view of Fig. 2 (a).
Fig. 3 (a) is the front layout of the integrated silicon chip in the photoelectric compound integral sensor of the present invention.
Fig. 3 (b) is the back side layout of the integrated silicon chip in the photoelectric compound integral sensor of the present invention.
Fig. 4 (a) is the structural representation of the multidigit point electrode array in the integrated silicon chip among the present invention.
Fig. 4 (b) is the structural representation of the LAPS electrode in the integrated silicon chip among the present invention.
Fig. 5 (a) is the synoptic diagram of the work flow step 1-4 of the integrated silicon chip among the present invention.
Fig. 5 (b) is the synoptic diagram of the work flow step 5-7 of the integrated silicon chip among the present invention.
Fig. 5 (c) is the synoptic diagram of the work flow step 8-9 of the integrated silicon chip among the present invention.
Fig. 6 is the impedance spectrogram of the impedance electrodes of the integrated silicon chip among the present invention.
Fig. 7 is the impedance index curve diagram of growing on the impedance electrodes in the Human umbilical vein endothelial cells ECV304 integrated silicon chip in the present invention.
Fig. 8 is the impedance amplitude performance diagram of multidigit point electrode array electrode before and after platinum plating is black in the integrated silicon chip of the present invention.
Fig. 9 is cardiac muscle cell's extracellular signal figure of the multidigit point electrode array detection among the present invention.
Figure 10 is LAPS photogenerated current-bias voltage (I-V) curve map among the present invention.
Figure 11 is that the LAPS in the integrated silicon chip among the present invention detects cellular metabolism acidification rate figure.
Figure 12 is the detected Na of the 2nd LAPS array chip in the lid of chamber of the present invention
+Concentration curve.
Among the figure, 1 integrated silicon chip, 1.1 multidigit point electrode arrays, 1.1.1 silicon chip, 1.1.2 the 2nd SiO
2Layer, 1.1.3 the 2nd Si
3N
4Layer, 1.1.4 lead-in wire, 1.1.5 electrode sensing site, 1.1.6Ti layer, 1.1.7Au layer, 1.1.8 photoresist, 1.2 impedance electrodes, 1.3 the one LAPS, 1.3.2 the one SiO
2Layer, 1.3.3 the one Si
3N
4Layer, 1.3.4Al film, 1.3.5 groove, 1.4 reference electrodes, 1.4.1 mask frame, 2. the 2nd LAPS; 3.PCB plate, 4. the chamber is covered, 4.1 chambeies lid main body, 4.2 fluid through-holes, 4.3LED through hole; 4.4 end face, 5. cavity, 6. cell, 7. single pin, 8.LED light source power supply line; 9. chamber lid LAPS output line, 10. fluid input tube, 11. fluid output tubes, 12.LED, 13. gold threads.
Embodiment
Below in conjunction with accompanying drawing and embodiment the present invention is described further.
1. sensor production
(1) design of cavity bottom integrated silicon chip and processing
Fig. 3 (a) is the front layout of integrated silicon chip 1 of the bottom of cavity 5, and it is integrated in multidigit point electrode array 1.1, impedance electrodes 1.2 and the first light-addressable potentiometric electrode (LAPS) 1.3 on the same silicon base.
Shown in Fig. 3 (a), the center arrangement of integrated silicon chip 1 has reference electrode 1.4, and the shape of reference electrode 1.4 is isolated multidigit point electrode array 1.1, impedance electrodes 1.2 and a LAPS 1.3 3 parts on spatial vision.Multidigit point electrode array 1.1 is 4 * 4 distribution, and electrode site is rounded, and diameter is 25 μ m, and spacing is 200 μ m.Fig. 4 (a) is the processing structure synoptic diagram of multidigit point electrode array 1.1, and on the silicon chip 1.1.1 as substrate, at first having deposited a layer thickness is the 2nd SiO of 500nm
2Layer 1.1.2 is Ti/Au complex metal layer (30nm/300nm) above that, and uses two Si of thickness as 800nm
3N
4Layer 1.1.3 covers the lead-in wire 1.1.4 in the metal level, but electrode sensing site 1.1.5 exposes.
In the integrated silicon chip 1, the both sides at the middle part of reference electrode 1.4 respectively are furnished with a pair of impedance electrodes 1.2.In design, impedance electrodes 1.2 is interdigitated, and finger beam is 40 μ m, and adjacent finger spacing is 60 μ m, and interdigital logarithm has 18 to (in the synoptic diagram of Fig. 3 (a), for clear expression, having reduced interdigital number), effectively refers to the long 2.65mm that is.The similar of the processing structure of impedance electrodes 1.2 and multidigit point electrode array 1.1.On silicon chip 1.1.1, having deposited a layer thickness is the 2nd SiO of 500nm
2Layer 1.1.2 is Ti/Au complex metal layer (30nm/300nm) above that, and uses two Si of thickness as 800nm
3N
4Layer 1.1.3 covers the lead-in wire in the metal level, but effective interdigital part exposes.
In the integrated silicon chip 1, the effective coverage area that the both sides of two end 1.4.1 of reference electrode 1.4 respectively are furnished with one the one LAPS, 1.3, the one LAPS 1.3 is 3.4mm * 3.4mm.Fig. 4 (b) is the processing structure figure of a LAPS 1.3.Shown in Fig. 5 (a)-2, with the back side of silicon chip 1.1.1 be thinned to 100 μ m with a LAPS 1.3 region facing, form four groove 1.3.5, these four groove 1.3.5 are positioned at the adjacent domain at four angles of silicon chip 1.1.1.Silicon chip 1.1.1 goes up by down from the last SiO that thickness 50nm is arranged successively
2Layer 1.3.2 and thickness are the Si of 100nm
3N
4Layer 1.3.3.Sputter Al film 1.3.4 is used for LAPS1.3 signal output as Ohmic contact on the silicon chip outside the LAPS 1.3 at the back side of silicon chip 1.1.1.
Middle large-area reference electrode 1.4 is used for the each several part zone is isolated on spatial vision.Wherein 1.4 pairs of outer rings, site at multidigit point electrode array 1.1 of reference electrode have formed a mask frame 1.4.1, and the electric field that can help isolating from other partial function electrodes disturbs, and improve the signal to noise ratio (S/N ratio) of multidigit point electrode array 1.1.The processing structure and the impedance electrodes of reference electrode 1.4 are similar.
In the process of silicon chip 1.1.1, adopt the order of LAPS-multidigit point electrode array & impedance electrodes-LAPS to carry out.Fig. 5 is the process flow diagram of process.
1) selection thickness is that the silicon chip 1.1.1 of 430 μ m is substrate (Fig. 5 (a)-1); Cleaning the back protects with photoetching method; It is 100 μ m that four angle adjacent domains in the silicon chip 1.1.1 back side (being the zone of a LAPS 1.3 correspondences among Fig. 3 (a)) are thinned to thickness; Form four groove 1.3.5 (, one of them groove only being shown in the work flow synoptic diagram) for convenient signal.
2) then hot growth one layer thickness of the silicon chip 1.1.1 behind the thinning back side is the SiO of 50nm
2Layer 1.3.2, and use chemical vapor deposition (PECVD) technology to deposit the Si of a layer thickness in the above as 100nm
3N
4Layer 1.3.3.On the front of silicon chip 1.1.1, adopt the photoresist protection, will be at the SiO outside the corresponding zone of groove 1.3.5
2A layer and a Si
3N
4Layer etching (Fig. 5 (a)-3), silicon chip a---SiO of groove area at this moment
2Layer 1.3.2 a---Si
3N
4Layer 1.3.3 formed the preliminary structure of a LAPS 1.3.Because the etching of groove 1.3.5 has certain gradient, therefore at the positive SiO of silicon chip
2Layer 1.3.3 and the 2nd Si
3N
4Layer 1.3.3 area can be bigger than the area of groove.
3) be to avoid the influence of the processing of multidigit point electrode array 1.1 and impedance electrodes 1.2 parts to a LAPS 1.3, the zone at a LAPS 1.3 structures place needs protected.On the basis of former step processing, utilize PECVD to deposit two SiO of a layer thickness on the silicon chip 1.1.1 for 500nm
2Layer 1.1.2, on this basis, sputter one layer thickness is the Ti layer 1.1.6 of 30nm and the Au layer 1.1.7 that a layer thickness is 300nm successively again.Wherein, the 2nd SiO
2Layer 1.1.2 both had been used for the insulation between silicon chip 1.1.1 and the complex metal layer (Ti/Au), also was used for the protection to a LAPS 1.3 body structure surfaces; In the metal composite layer, Ti layer 1.1.6 increased Au layer 1.1.7 and deposited the 2nd SiO
2Degree of adhesion (Fig. 5 (a)-4) between the substrate of layer 1.1.2; Au layer 1.1.7 is because therefore its stability and good bio-compatibility are often adopted the collection that is used for bio signal.Utilize the photoetching method protection again, according to the layout shown in Fig. 3 (a), etching sheet metal, and manifest the electrode 1.1.5 and lead-in wire 1.1.4 pattern (Fig. 5 (b)-5) in multidigit point electrode array 1.1 and the impedance electrodes 1.2.
4) because lead-in wire 1.1.4 partly needs and external insulation is isolated, so thickness is the 2nd Si of 800nm
3N
4Layer 1.1.3 deposits on it (Fig. 5 (b)-6).Then, photoresist 1.1.8 is applied to the 2nd Si
3N
4On the layer 1.1.3, and expose the zone that electrode pair is answered through photoetching method, and with the 2nd Si of exposed region
3N
4Layer 1.1.3 etching, this moment, electrode exposed, but photoresist 1.1.8 keeps (Fig. 5 (b)-7); On above-mentioned processing basis, continue the 2nd SiO in the substrate
2Layer 1.1.2 remains in the partial etching (Fig. 5 (c)-8) in the corresponding zone of groove, photoresist 1.1.8 washed again.
5) with the method for photoetching, the regional sputter Al film 1.3.4 outside the groove 1.3.5 at the silicon chip 1.1.1 back side as Ohmic contact conduction (Fig. 5 (c)-9), is used to export the signal of a LAPS 1.3.
Adopt this process, multidigit point electrode array 1.1, impedance electrodes 1.2 and a LAPS1.3 need not because SiO separately
2Layer and Si
3N
4Layer thickness inconsistent and mutual compromise, and attenuate has been carried out at a LAPS 1.3 back sides form groove have reduced the noise of LAPS when not influencing multidigit point electrode array 1.1 and impedance electrodes 1.2, improved the signal to noise ratio (S/N ratio) of LAPS.
(2) encapsulation of cavity bottom integrated silicon chip
Be the signal of each several part electrode in the derivation integrated silicon chip 1, and integrated silicon chip 1 is supported, need integrated silicon chip 1 is fixed on the pcb board 3 of customization.
The central authorities of pcb board 3 are the gold-plated disk of a diameter 4mm, have the integrated silicon chip 1 of Al film 1.3.4 to be fixed on the pcb board 3 back spatter with conductive silver glue, and guarantee the Al film 1.3.4 and the contact of the good ohmic between the pcb board 3 at the back side of integrated silicon chip 1.On pcb board 3, opened four holes (diameter is 3mm) and be used for printing opacity with a LAPS 1.3 corresponding places of integrated silicon chip 1.LED lamp 12 is fixed in pcb board 3 back sides, and through four holes the one LAPS 1.3 being carried out light stimulus.Stamp gold thread 13 between the pad of the multidigit point electrode array 1.1 on the integrated silicon chip 1 and the pad of impedance electrodes 1.2 and pcb board 3, use epoxy sealing, do signal output.
On the integrated silicon chip that has encapsulated 1, settling an internal diameter is the organic glass cavity 5 of 25mm, and with epoxy resin itself and pcb board 3 bases that have integrated silicon chip 1 is fixed.This moment, the bottom of cavity 5 completed.
(3) chip design, processing and the making of chamber lid
Chip on the chamber lid 4 is 2 * 2 the 2nd LAPS 2 electrod-arrays, and the area of each unit is 4mm * 4mm, the LAPS 1.3 of its similar in integrated silicon chip 1.It is processed as: on silicon chip, adopt the SiO of PECVD growth one deck 50nm
2The Si of layer and 100nm
3N
4, and the Al film that detects one deck 500nm on the silicon chip overleaf is as Ohmic contact, as signal output.Become independent LAPS electrode unit after the chip scribing of making, being combined to form is 2 * 2 arrays.
(4) encapsulation of chamber lid
Fig. 2 (a) is the organigram of chamber lid 4, and Fig. 2 (b) is the upward view of Fig. 2 (a).The diameter of the main part 4.1 of chamber lid 4 and the internal diameter of cavity 5 match, and make chamber lid 4 can seal with cavity 5 and screw on.Visible from Fig. 2 (a), in chamber lid 4, can have the bigger LED through hole 4.3 of four diameters (fluid through-hole 4.2 less of Φ=3mm) (Φ=1mm) at least with at least two diameters.The 2nd LAPS 2 is inverted; There are the one side of Al film and cover end face 4.4 usefulness conductive silver glues to fix sputter; And in LED through hole 4.3, each places a LED 12, for LAPS provides light source excitation; Two fluid through-holes 4.2 connect fluid input tube 10 and fluid output tube 11 respectively, are used for cell culture chamber and add medicine or change liquid.The Na that chamber lid main body 4.1 detects in the cell culture fluid at the 2nd LAPS 2
+, K
+When reaching the ion of similar need film selection, screw in the cavity.
(5) the total encapsulation of sensor
Fig. 1 is the encapsulation and the structural representation of whole sensor.Cavity 5 and chamber lid 4 fix through screw thread, and chamber lid 4 and cavity 5 are twisted and closed the part height is 2cm, stay that vacant height is 1mm at the bottom of the chamber, can be full of the nutrient solution of fixed amount, detect the each several part functional parameter of cell simultaneously.For the 2nd LAPS 2 to integrated silicon chip 1 at the bottom of the chamber and chamber lid end face 4.4 provides light stimulus, all be placed with LED 12 up and down at sensor.Can be in detection for extracellular environment change liquid, liquid is substituted between the fluid input tube 10 on the chamber lid 4 and fluid output tube 11 are used for the 2nd LAPS 2 of integrated silicon chip 1 and chamber lid 4.
2. the sign of sensor and characteristic
(1) impedance electrodes characteristic and cell growth status detection
In cell growth, migration in the processes such as apoptosis, has adherent variation between at the bottom of cell and the double dish, and the available impedance electrodes 1.2 of this variation characterizes quantitatively.Do not cultivate in 1 last time of integrated silicon chip when cell, the impedance spectrum of impedance electrodes 1.2 (Fig. 6) is used for characterizing its basic impedance.Visible in the drawings, be 10 in frequency
3-10
4.5During Hz, phase place is constant-60 °, and impedance raises along with frequency and reduces; In frequency is 10
4.5-10
6During Hz, phase place is similar linear the reduction, and impedance raises along with frequency and reduces, but 10
5.5-10
6During Hz, impedance amplitude stops downtrending, is shown as constant.Therefore, 10
4.5-10
5.5Hz is the higher Frequency Band Selection of impedance sensitivity.
Human umbilical vein endothelial cells ECV304 is the clone a kind of commonly used of research angiogenic growth etc.In this experiment, cell is with 10
6The density of individual/mL is cultivated on impedance electrodes 1.2.Impedance when Fig. 7 is cultivation detects in real time.Whole process comprises adherent-stable-three processes of apoptosis of growing, and uses the impedance variations index, and the impedance after promptly cell attaches and the ratio of difference between the basic impedance and basic impedance characterize the variation of impedance.In 0-4h, the impedance index is in rising trend, and this moment, cell was adherent gradually to integrated silicon chip 1, and the impedance variations index is risen; Reaching remain unchanged after stable (4-8h); When 9-12h, add the HgCl that 0.1 μ M has severe toxicity
2After, the impedance index occurs reducing and fluctuation, and cell begins to take off wall and apoptosis occurs.These phenomenons can be observed at the CCD microscope synchronously synchronously.
(2) multidigit point electrode array characteristic reaches the detection to born of the same parents' external electric field
In the multidigit point electrode array 1.1, when cellular incubation on electrode site, cell and electrode form electric coupling and detected electrical excitation cell corresponding born of the same parents' external electric field when action potential afterwards and distribute.Usually come electrode-liquid surface is characterized with impedance spectrum, also can the thermonoise of electrode be characterized simultaneously.
Fig. 8 is the map of magnitudes in the impedance spectrum of 1 μ M electrode.It is thus clear that when the frequency of 1KHz, electrode-solution interface impedance is 500K Ω.In this device, also electrode surface has been done the processing of electroplating platinum black,, enlarged the contact area of electrode and solution, reduced impedance, reduced noise to increase electrode surface roughness.
When extracellular signal is detected, at first will be on integrated silicon chip 1 with cellular incubation.Usually multidigit point electrode array 1.1 is mainly used in and detects cardiac muscle cell and two big types of electrical excitation sexual cells of neural network.In the embodiment of the invention, mainly be example with cardiac muscle cell.The heart of newborn SD mouse (0-2 days) is taken out, smash to pieces and enzymolysis, cultivate on the chip that scribbles gelatin in advance, after 2-3 days, carry out the multi-channel detection electric potential signal and detect.Fig. 9 is detected ten passage cardiac muscle cell extracellular signals.Visible baseline noise is about 14-18 μ V from signal, and measured cardiac muscle cell's extracellular signal mainly divides unimodal and bimodal two kinds; Its electric potential signal duration is 7-9ms, and amplitude is 30-150 μ V, and the frequency of beating is about 36 times/min, and the signal forming time through passage, can infer the direction of transfer of signal in cardiac muscle cell's plasomidum.When cell in drug effect following time, these parameter values can change, thereby learn the effect of medicine to the cardiac muscle cell.
(3) the LAPS characteristic reaches extracellular acidification rate and Na
+The detection of metabolism
Commonly used, the I-V curve is represented the performance of LAPS, and it has been expressed photogenerated current and has been added in the relation between the bias voltage on the sensor.Figure 10 is a LAPS 1.3 typical S type I-V curves, and the I-V curve of the 2nd LAPS 2 that the chamber covers and a LAPS's 1.3 is similar.At cut-off region, there is not photogenerated current to produce; In the perform region, photogenerated current increase and voltage reduce to be linear approximate relationship; In the saturation region, the variation of bias voltage can not cause the variation of photogenerated current again.Therefore detect in the application of the outer ion metabolism of born of the same parents at LAPS, usually bias voltage is arranged on the bending point of curve and remains unchanged, so this point voltage is to electrode surface voltage sensitivity.
In acidification rate detects, according to surperficial basic mode type at (Si
3N
4/ SiO
2)-solution interface can be because of H in the solution
+And the formation voltage difference.And this voltage difference and bias voltage acting in conjunction are on a LAPS 1.3.H in solution
+When concentration changes, can make change in voltage, cause that photogenerated current changes, thereby cause of the negative direction of principal axis skew of I-V curve to bias voltage.Bias voltage is constant when keeping, and the variation of photogenerated current has just characterized H in the solution
+The variation of concentration.According to this principle, the cardiac muscle cell is cultivated on seal chamber integrated silicon chip 1, can detect its time dependent acidification rate figure (Figure 11).At first, in stopping to change five minutes of liquid, the magnitude of voltage that is converted to according to photogenerated current reduces, and explains that cell has carried out H within these five minutes
+Metabolism, the extracellular acidification rate improves; After changing liquid, after having got back to original level, visible voltage begun second metabolic process; Similar the 3rd metabolic process taken place; In the end when the acetylcholine with 100mg/ml feeds cell culture fluid, find that voltage no longer descends, this has characterized the H of cell
+Metabolism slows to and stops.
Na outside born of the same parents
+In the detection, utilize the 2nd LAPS 2 array detection of chamber lid in the sensor.On the surface of the 2nd LAPS 2, adopting the PVC film is carrier, and NPOE adopts liquid ions exchange membrane (sensitive materials are Bis (12-crown-4)) as Na as plastifier
+Sensitive membrane.Figure 12 is that the 2nd LAPS 2 is detecting different N a
+I-V scanning curve in the solution of concentration.Can see, along with the reduction of concentration, the curve skew of turning right, and sensitivity is 45.2mV/pNa.
Claims (6)
1. integrated silicon chip; It is characterized in that: the center arrangement of said integrated silicon chip has reference electrode (1.4); The two ends of said reference electrode respectively are furnished with one group of multidigit point electrode array (1.1); Both sides at the middle part of reference electrode respectively are furnished with a pair of impedance electrodes (1.2), respectively are furnished with one first light-addressable potentiometric electrode (1.3) in the both sides of two ends of reference electrode, and the back side of the said first light-addressable potentiometric electrode is provided with groove.
2. integrated silicon chip according to claim 1 is characterized in that: the end of said reference electrode is provided with mask frame, and the site of said multidigit point electrode array is positioned at mask frame.
3. the photoelectric compound integral sensor of the detection cell physiological parameters of the integrated silicon chip with claim 1, it is characterized in that: it comprises cavity (5) that is fixed on the pcb board (3) and the chamber lid (4) that is complementary with this cavity; Be fixed with said integrated silicon chip (1) in the bottom of cavity; The chamber covers and is provided with at least two fluid through-holes (4.2) and at least four LED through holes (4.3), and the end face towards said integrated silicon chip (1) (4.4) of the main part of chamber lid is fixed with the second light-addressable potentiometric electrod-array (2); The main part of chamber lid places in the cavity, between the said end face of the main part of chamber lid and the integrated silicon chip (1) space is arranged.
4. photoelectric compound integral sensor according to claim 3 is characterized in that: the end of said reference electrode is provided with mask frame, and the site of said multidigit point electrode array is positioned at mask frame.
5. the described integrated silicon chip preparation method of claim 1 is characterized in that comprising the steps:
1) be substrate with silicon chip (1.1.1), after the cleaning respectively the adjacent domain at four of the etching silicon chip back side angles to form four grooves;
2) at the positive heat growth of silicon chip one deck the one SiO
2Layer is then at a SiO
2Deposition the one Si on the layer
3N
4Layer; Adopt the method for photoetching again, the SiO outside will the zone corresponding with groove
2A layer and a Si
3N
4Layer etching;
3) deposition one deck the 2nd SiO on the front of silicon chip
2Layer; At the 2nd SiO
2Sputter one deck Ti layer on the layer, the layer of sputter layer of Au again forms metal composite layer Ti/Au; Adopt the method for photoetching, metal composite layer Ti/Au is etched electrode and the lead pattern that presets; Center arrangement at said electrode and lead pattern has reference electrode (1.4); The two ends of said reference electrode respectively are furnished with one group of multidigit point electrode array (1.1); Both sides at the middle part of reference electrode respectively are furnished with a pair of impedance electrodes (1.2); Both sides in two ends of reference electrode respectively are furnished with one first light-addressable potentiometric electrode (1.3), and the position at the said first light-addressable potentiometric electrode (1.3) place is corresponding with the groove at the silicon chip back side;
4) continue deposition one deck the 2nd Si on the front of silicon chip
3N
4Layer, and with the 2nd Si on the electrode site of said multidigit point electrode array, pad and the zone corresponding with groove
3N
4Etching;
5) the 2nd SiO on will the zone corresponding on the front of silicon chip with groove
2Layer etching;
6) sputter Al film on the zone outside the groove at the silicon chip back side obtains integrated silicon chip.
6. integrated silicon chip preparation method according to claim 5 is characterized in that: the end of said reference electrode is provided with mask frame, and the site of said multidigit point electrode array is positioned at mask frame.
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| CN102559483B (en) * | 2012-01-11 | 2013-11-20 | 浙江大学 | Device and method for real-time monitoring of behaviors and states of cells |
| CN102680526B (en) * | 2012-05-16 | 2014-07-02 | 清华大学 | Single cell array microchip and manufacturing, electrical measurement and electroporation methods thereof |
| CN109374714B (en) * | 2018-10-25 | 2023-11-14 | 深圳刷新生物传感科技有限公司 | Assembled biosensor chip |
| CN109628291B (en) * | 2018-12-04 | 2021-02-12 | 浙江大学 | Micro-cavity impedance sensor for real-time monitoring of 3D cell activity and proliferation capacity and preparation method thereof |
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| CN1284166A (en) * | 1997-12-25 | 2001-02-14 | 松下电器产业株式会社 | Cell potential measuring electrode and measuring appts using same electrode |
| CN101158677A (en) * | 2007-10-29 | 2008-04-09 | 浙江大学 | Cell electric physiology integrated chip and preparation method |
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| CN1284166A (en) * | 1997-12-25 | 2001-02-14 | 松下电器产业株式会社 | Cell potential measuring electrode and measuring appts using same electrode |
| CN101158677A (en) * | 2007-10-29 | 2008-04-09 | 浙江大学 | Cell electric physiology integrated chip and preparation method |
| CN101358941A (en) * | 2008-08-22 | 2009-02-04 | 浙江大学 | Double-face nanometer band electrode array integration sensor capable of being cut and method for manufacturing same |
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