CN1902305A

CN1902305A - Real time electronic cell sensing systems and applications for cell-based assays

Info

Publication number: CN1902305A
Application number: CN 200480040230
Authority: CN
Inventors: 徐晓; 亚马·阿巴斯; 王小波; 甘江波
Original assignee: Acea Biosciences Inc
Current assignee: Agilent Technologies Inc
Priority date: 2003-11-12
Filing date: 2004-11-12
Publication date: 2007-01-24
Anticipated expiration: 2024-11-12
Also published as: CN100478436C

Abstract

The present invention includes devices, systems, and methods for assaying cells using cell-substrate impedance monitoring. In one aspect, the invention provides cell-substrate impedance monitoring devices that comprise electrode arrays on a nonconducting substrate, in which each of the arrays has an approximately uniform electrode resistance across the entire array. In another aspect, the invention provides cell-substrate monitoring systems comprising one or more cell-substrate monitoring devices comprising multiple wells each having an electrode array, an impedance analyzer, a device station that connects arrays of individual wells to the impedance analyzer, and software for controlling the device station and impedance analyzer. In another aspect, the invention provides cellular assays that use impedance monitoring to detect changes in cell behavior or state.

Description

Real time electronic cell sensing systems and based on the application in the mensuration of cell

The application requires the right of priority of following patent application: U.S. Provisional Application number 60/519,567 (submitting on November 12nd, 2003); U.S. Provisional Application number 60/542,927 (submitting to) on February 9th, 2004; And U.S. Provisional Application number 60/548,713 (submitting to) on February 27th, 2004.All mentioned in this section applications all are hereby incorporated by.

The application also introduces following patent application as a reference: Application No. 10/705,447 (submitting to) on November 10th, 2003, its name is called " Impedance BasedDevices and Methods For Use in Assays " (patent agent is numbered ACE-101.P.1.1-US); U.S. Patent application No.10/705,615 (submitting to) on November 10th, 2003, its name is called " Impedance Based Apparatuses and Methods forAnalyzing Cells and Particles " (patent agent is numbered ACE-00101.P.1.2-US); U.S. Provisional Application 60/397,749 (submitting to) on July 20th, 2002; U.S. Provisional Application 60/435,400 (submitting to) on December 20th, 2002; U.S. Provisional Application 60/469,572 (submitting to) on May 9th, 2003; PCT application PCT/US03/22557 (submitting to) and PCT application PCT/US03/22537 (submitting to) on July 18th, 2003 on July 18th, 2003.All mentioned in this section applications all are hereby incorporated by.

Background of invention

Technical field

The present invention relates to field based on the mensuration of cell.Specifically, the invention provides device, equipment and system to carry out cell analysis and to carry out mensuration based on cell based on impedance.

Background technology

A. the electronic analysis of cell

Biological electronics is the integrated research field interdisciplinary that relates to biomaterial and electronic installation that is developing.The biological electronics method has applied to analysis of cells and has measured biomolecules and cell.In one type application, on microelectrode, and measurement and definite cell-electrode impedance are with the change of monitoring cell with cell cultures.

(name is called " IMPEDANCE BASEDDEVICES AND METHODS FOR USE IN ASSAYS " at PCT application No.PCT/US03/22557, submit on July 18th, 2003) in, a kind of be used to the detect cell that is positioned at electrode surface and/or the device of molecule are disclosed.This device is by measuring adhering to or combining the impedance variations that causes and detect cell and/or molecule by cell and/or molecule and electrode surface.This patent application also discloses the embodiment of multiple this device and has used described device to carry out equipment, system based on the mensuration of cell.

B. allergic disease and IgE the mediation cell-stimulating

Allergic disease also often is called the immediate hypersensitivity illness, is modal function of immune system obstacle, and it influences 20% of all U.S.'s individualities.Immediate hypersensitivity shows as from simple rash or scratches where it itches and epiphora any to the most extreme anaphylaxis situation, and respiratory tract is limited to and suffocates and dead point in described extreme case.Because the seriousness of allergy, the high per-cent that lacks suitable therapeutic method and suffer from the crowd of various forms of described situations, pharmacy corporation have been attached great importance to developing new drug and have been used for treating effectively and resisting the symptom that these damage the illness of physical abilities and potential threat life.

Participating in allergic main cell in the immunity system is mastocyte, basophilic granulocyte and eosinophilic granulocyte.Basophilic granulocyte and eosinophilic granulocyte break up in marrow, are circulated in the blood, and raise in the inflammation tissue site in the late period of inflammatory reaction.On the contrary, the mastocyte proper distribution and participates in allergic immediate phase (immediate phase) (people such as Sharma, the Clin RevAllergy Immunol.2002 Apr of immunoglobulin E (IgE)-mediation in whole reticular tissue; 22 (2): 119-48).The individual first allergen that meets with causes producing IgE, and this IgE will be attached to the IgE acceptor (Fc (ε) RI) of the high-affinity that is positioned at the mastocyte surface, thereby cause the sensitization of mastocyte.Meet with this allergen subsequently and can cause and be positioned at the crosslinked of the lip-deep Fc of mastocyte (ε) RI-IgE mixture, and stimulate mastocyte to discharge the medium of immediate hypersensitivity.Incident in a series of cells of crosslinked triggering of the IgE of the receptors bind on mastocyte surface is referred to as mast cells activation, and it discharges strong inflammatory mediator with the extracellular is terminal point, and many such media are stored in the secretory granules, comprise histamine.Mast cells activation can be divided into complementary early stage and late period.The fusion of the generation of the activation that comprises phosphorylation and protein tyrosine kinase and substrate thereof in early days, second messenger's inositoltriphosphoric acid and the diacylglycerol of mast cells activation, the rising of intracellular Ca2+ level and secretory granules and film (people such as Stassen, Crit Rev Immunol.2002; 22 (2): 115-40).Comprise that the obvious form that the reconstruct owing to actin cytoskeleton causes changes the late period of mast cells activation, cause the synthetic and excretory genetic expression of strong inflammatory cytokine, and the lipid medium of the various effects of generation in blood vessel, bronchial smooth muscle and white corpuscle is synthetic.

Based on a plurality of steps that immediate hypersensitivity initial sum implementation relates to, a plurality of potential targets that are used for drug intervention are arranged.Mostly existing for the symptom of seeking to alleviate described situation such as the methods of treatment of the immediate hypersensitivity of asthma, rather than directly at the cause of disease.Yet, carrying out effort full of hope, with by use humanized mono-clonal anti-IgE antibodies come in and IgE antibody, and obtain clinical symptom long-term mitigation (D ' people such as Amato, MonaldiArch Chest Dis.2003 Jan-Mar; 59 (1): 25-9).Equally, to the inherent signal transduction pathway of the mast cells activation of IgE mediation illustrate and the appearance of combinatorial chemistry provides and uses micromolecular inhibitor and come target to participate in the key protein of mast cells activation and the chance of enzyme.These compounds can provide novel immunomodulator with treatment immediate hypersensitivity illness potentially.The micromolecular inhibitor of several kinases (comprising PKC and Tyrosylprotein kinase Syk) in the rodent research of some immediate hypersensitivitys of blocking-up, provide inspirer clinical before result (people such as Seow, Eur J Pharmacol.2002 May 17; 443 (1-3): 189-96).

Increasing company utilizes big chemical libraries to screen the potential inhibitor of the signal transducting system that may participate in various disease states.Therefore be badly in need of a kind of high-throughout molecule and raji cell assay Raji, to screen the potential modulator of these signal transduction pathway.About the signal conduction of IgE mediation, used mensuration is the medium that is discharged into behind the measurement flailing action in the substratum.This is by measuring enzymic activity (the Rac and phosphatidylinositol 3-kinaseregulate the protein kinase B in Fc epsilon RI signaling in RBL 2H3mast cells.J Immunol.2001 Feb 1 of these media; 166 (3): 1627-34), use radioactivity precursor (people such as Guillermot, J Cell Sci.1997 Sep; 110 (Pt 18): 2215-25) or by ELISA realize, thereby the amount of the medium that discharged is carried out quantitatively (people such as Berger, Allergy.2002 Jul; 57 (7): 592-9).These mensuration are single point assay or endpoint determination, and it measures the cumulative release of these media, and also relate to use reagent and manipulating cells, for example fixing or cracking.The adaptability that exists the fact of single point assay can not guarantee to screen the required high throughput analysis of big chemical libraries, described single point assay uses expensive reagent (such as antibody) and cell manipulation.

C. screening anticancer medicine and discovery

In cancer therapy drug exploitation, be an important factor that obtains to be used to develop the information of clinical administration strategy to the research of the time-dependent manner of the cytotoxicity of medicine and cell inhibitory effect effect.Specifically, the different time-dependent sexual norm of the IC50 of derivation time-dependent manner and observation IC50 (for example, referring to Hassan SB, Jonsson E, Larsson R and KarlssonMO in J.Pharmacology and Experimental Therapeutics, 2001, Vol.299, No.3, pp 1140-1147; Levasseur LM, Slocum HK, Rustum YM and Greco WR, in Cancer Research, 1998, vol.58, pp 5749-5761.).In general, these researchs use the terminal point list to measure mensuration.For the dose concentration of medicine that is applied to cultured cells or compound, each time point all needs different experiments.These have all limited the temporal resolution in this time-dependent manner Study of cytotoxicity and the number of time point.Therefore, need new technology or method high time resolution to be provided and to allow at many point in time measurement.

The present invention has further expanded the invention of following patent application: PCT application number PCT/US03/22557 (submitting on July 18th, 2003), its name is called " IMPEDANCEBASED DEVICES AND METHODS FOR USE IN ASSAYS ", and Application No. 10/705,447 (submitting on November 10th, 2003), its name is called " IMPEDANCE BASED DEVICES AND METHODS FOR USE INASSAYS " (patent agent is numbered ACE-00101.P.1.1-US).The invention provides based on measuring cell-substrate impedance and carry out real-time electronic cell sensor-based system, and provide and use this system to carry out method for measuring based on cell based on the mensuration of cell.In addition, the present invention also is intended to solve signal conduction and the method for cell-stimulating and the limitation of technology of existing mensuration IgE mediation.

Summary of the invention

On the one hand, the present invention relates to be used to monitor the device of cell-substrate impedance, described device comprises: a) insulating substrate; B) two or more are installed on suprabasil electrod-array, and wherein each of two or more electrod-arrays all comprises two electrode structures; And c) at least two connection gaskets (connection pad), each connection gasket all is positioned at the edge of substrate.Each of two or more electrod-arrays for device, first electrode structure of two electrode structures is connected in two or more connection gaskets, and second electrode structure of two electrode structures is connected in two or more connection gaskets another.Each of two electrode structures of two or more electrod-arrays comprises a plurality of electrode members, and the electrode resistance distribution of each electrod-array of device on whole array is uniformly approximate.The substrate of device has the surface that is suitable for cell attachment or growth, cell described suprabasil adhere to or grow can cause between the electrode structure in each electrod-array can be detected impedance variations.In a kind of preferred embodiment, be connected with fluid-tight container at suprabasil each electrod-array of apparatus of the present invention.

Again on the one hand, the present invention relates to cell-substrate impedance measurement system, it comprises: a) at least one Multiple-Aperture Device, and it can monitor cell-substrate impedance, comprises electrod-array at the bottom of wherein having each comfortable hole, two holes in the porous at least; B) electric impedance analyzer; C) the device operator's console that is used for engaging one or more Multiple-Aperture Devices and can selects and the electrod-array of any porous is electrically connected on electric impedance analyzer; And d) is used for control device operator's console and electric impedance analyzer measured impedance value is carried out data obtain software program with data analysis.In a preferred embodiment of the invention, each electrod-array of Multiple-Aperture Device is independent addressing (addressed).

On the other hand, the invention provides a kind of method of utilizing apparatus of the present invention monitoring cell-substrate impedance.This method comprises: many array apparatus of the present invention are provided; These many array apparatus are connected in electric impedance analyzer; With cell deposition in the device two or more arrays at least one array on; And the cell on one or more electrod-arrays of monitoring device-substrate impedance.

On the other hand, the invention provides the method for calculation of cell index (Cell Index), with quantitative and relatively cell-substrate impedance.

On the other hand, the invention provides the method for a kind of calculating with the resistance of the array of cell-substrate monitoring device and the electric trace that connection gasket couples together (electrical trace).The resistance of this electric trace is used to calculate cell index.

On the other hand, the invention provides the method for using cell of the present invention-substrate impedance measurement system to monitor cell-substrate impedance.This method comprises: cell of the present invention-substrate impedance measurement system is provided, cell is made an addition at least one hole of the Multiple-Aperture Device that comprises electrod-array, and monitor one or more cell-substrate impedances that comprise the hole of cell.Impedance can be monitored in rule or irregular time intervals.In a kind of preferred embodiment, carry out cell-substrate impedance monitoring at least in two holes on Multiple-Aperture Device.

On the other hand, the invention provides a kind of method of carrying out based on the The real time measure of cell, it can study the effect of one or more compound pair cells, and this method comprises: above-mentioned cell-substrate impedance measurement system is provided; Cell is imported at least one hole of the system that comprises electrod-array; In being arranged, one or more holes of cell add one or more compounds; Before adding one or more compounds and monitor cell-substrate impedance on the electrod-array in one or more holes afterwards.Preferably, after in one or more holes that comprise cell, adding one or more compounds, on the regular or irregular timed interval, monitor cell-substrate impedance.The variation of the impedance of time-dependent manner can provide the information of the cell state that adds the time-dependent manner before the compound, and the information of the cell state of the time-dependent manner when reacting with compound.These information can be in order to determine the effect of compound pair cell.

On the other hand, the invention provides and a kind of at least a compound is carried out the method for real-time cytotoxic assay, it comprises: above-mentioned cell-substrate impedance measurement system is provided; Cell is imported in one or more holes of the system that comprises electrod-array; In comprising one or more holes of cell, add one or more compounds; Before adding one or more compounds and monitor the cell-substrate impedance in one or more holes afterwards, wherein the impedance variations of time-dependent manner provides the Cytotoxic information of the time-dependent manner of one or more compounds.Preferably, after in one or more holes that comprise cell, adding one or more compounds, in regular or irregular timed interval monitoring cell-substrate impedance.The variation of the impedance of time-dependent manner can provide any Cytotoxic information that may exist of compound.

In an embodiment of aforesaid method, use a plurality of holes with same cell type, wherein in the different holes that comprise cell, add the compound of different concns.This method can provide time-dependent manner and cell-cytotoxic reaction concentration dependent.

Another aspect, the invention provides a kind of the analysis and the method for the cytotoxic effect of first kind of compound and second kind of compound time-dependent manner that a certain cell type is produced relatively, it comprises: a) use first kind of compound to carry out the real-time cytotoxic assay of pair cell type with aforesaid method; B) use second kind of compound to carry out real-time cytotoxic assay with aforesaid method to described cell type; And c) cell-cytotoxic reaction of the time-dependent manner of first kind of compound of comparison and second kind of compound.

In an embodiment of this method, the cell-cytotoxic reaction of the time-dependent manner of first kind of compound is measured under a plurality of administration concentration.In another embodiment, the cell-cytotoxic reaction of the time-dependent manner of second kind of compound is measured under a plurality of administration concentration.In another embodiment, the cell-cytotoxic reaction of the time-dependent manner of first kind of compound and second kind of compound is measured under a plurality of administration concentration.

On the other hand, the invention provides a kind of Cytotoxic method of summarizing compound to the various kinds of cell type, it comprises: a) carry out the real-time cytotoxic assay of compound to different cell types with aforesaid method, with b) analyze the real-time cell-cytotoxic reaction of different cell types to compound, so that the cytotoxicity overview of compound to be provided.

In another embodiment, use aforesaid method to carry out of the cytotoxicity general introduction of multiple compound to the various kinds of cell type.

In yet another aspect, the present invention relates to use the electronics resistance technique to estimate and the method for the morphological change that quantitatively takes place in the cell, described morphological change is stimulated by IgE and IgE acceptor and antigen cross-linking cause.This method comprises: cell of the present invention-substrate impedance measurement system is provided; Transfered cell in one or more holes of system, and in the one or more holes that comprise cell, add IgE; In the one or more holes that comprise cell and IgE, add antigen; Cell-substrate impedance with one or more holes of supervisory system.Can and monitor impedance afterwards before adding IgE, and preferably before adding IgE, add after the IgE and add before the antigen and add antigen and measure afterwards.Cell-substrate impedance can be used as a kind of indication of reaction that cell stimulates IgE, and this indication changes realization by morphocytology.

In yet another aspect, the present invention relates to by utilizing electronics measurement and cell sensing to screen the method that is attached to the inhibitor of the signal transduction pathway that IgE-Fc (ε) RI mixture starts by antigen.This method comprises: cell of the present invention-substrate impedance measurement system is provided; Transfered cell in one or more holes of system, and in one or more holes that comprise cell, add at least a test compounds; The control wells that comprises cell of at least one no test compounds is provided; IgE is added in one or more holes that comprise cell and test compounds and in one or more control wells; In one or more holes that comprise cell and test compounds and in one or more control wells, add antigen; And the cell in one or more holes of supervisory system-substrate impedance.Can and detect impedance afterwards before adding IgE, and preferably before adding IgE, add after the IgE and add before the antigen and add antigen and measure afterwards.This cell-substrate impedance can be used as a kind of indication of cell to the reaction of IgE stimulation, and this indication realizes by the morphocytology variation.Cell-substrate impedance in more one or more holes that comprise test compounds and the cell-substrate impedance in one or more control wells can provide the effect of the reaction that the test compounds pair cell stimulates at IgE.Particularly, the IgE reaction suppressor can reduce the cell handled with test compounds by it and identified with respect to the character of the cell-impedance response of the reaction of control cells.

In an embodiment aspect this, the present invention relates to use the electronics resistance technique to screen the method for potential inhibitor that IgE is attached to high-affinity Fc (ε) the RI acceptor of mastocyte or basophil cellular surface.

In another embodiment in this respect, the present invention relates to use the electronics resistance technique to screen the method for key enzyme and proteic micromolecular inhibitor, described key enzyme and albumen participate in combining caused signal transduction pathway by antigen and cell high-affinity Fc (ε) RI acceptor when crosslinked being with or without IgE.

On the other hand, the present invention relates to use the electronics resistance technique to come the method for target key enzyme and proteic affirmation purpose, described key enzyme and albumen participate in combining caused signal transduction pathway by antigen and cell high-affinity Fc ε RI acceptor when crosslinked being with or without IgE-.This method comprises: cell of the present invention-substrate impedance measurement system is provided; The cell that quiding gene is transformed in one or more holes of system; Provide at least one to comprise control wells without the cell of genetic modification; In the hole of the cell that comprises genetic modification and in one or more control wells, add IgE, and in the hole of the cell that comprises genetic modification and in one or more control wells, add antigen; In the hole of the cell that comprises genetic modification and in one or more control wells, add antigen; Monitor the hole of the cell that comprises genetic modification and the cell-substrate impedance of one or more control wells; And analyze impedance data to determine of the effect of gene alteration pair cell at the reaction of IgE stimulation.

On the other hand, the present invention relates to use the electronics resistance technique to come the method for reaction of the different genetic background cells of comparison IgE-mediation.This method can be used for screening and confirms genetic marker, for example gene expression profile, gene splicing variant, protein expression profiles, crucial single nucleotide polymorphism (SNP) or sudden change and other hereditary variants, these genetic markers can determine or influence that the IgE acceptor that IgE stimulations, IgE acceptor interaction, antigen mediate is crosslinked, intracellular signal transduction path and threshing.

On the other hand, the present invention relates to use the electronics resistance technique to screen, find and confirm specificity or non-specific the method that causes crosslinked antigen of IgE (allergen) and haptenic chemical structure.

The accompanying drawing summary

Fig. 1 is the synoptic diagram of a kind of design of cell of the present invention-substrate impedance measurement device.A) substrate that has 16 electrod-arrays (perhaps 16 electrode structure unit) of arranging with 2 row x8 row configurations in substrate has been described.B) the single electrod-array that installs has been described.C) shown the synoptic diagram of electrod-array, shown that it is uniformly approximate that the electrode resistance of requirement on whole array distributes.

Fig. 2 shows the image of 16x device of the present invention.

Fig. 3 shows the image of 96x device of the present invention.

Fig. 4 shows a kind of design of 16x device operator's console of the present invention, is connected with 6 16x devices on operator's console.

Fig. 5 shows a kind of design of 96x device operator's console of the present invention, is placed with 96 orifice plates on operator's console.

Fig. 6 shows the different pages of real-time electronic cell sensing software.A) lab note page or leaf (experimental note page) allows the experimenter to write down the key message of experiment, for example experiment purpose and experiment flow.B) experimental design page or leaf (experimental layout page) allows to write down cell, cell number, compound and the compound concentration that is added in each hole.C) test duration is provided with page or leaf (Test time setting page), allow record and control to carry out cell-employed timed interval of substrate impedance measurement, and a plurality of experimental procedures can be set, and each step all has the time of different time interval value and different lengths.D) cell index page or leaf (Cellindex page) is a result page, wherein after a predetermined time interval that page or leaf is provided with was set by the test duration finishes each and measure, software system upgrade automatically that institute's foraminous measures with the cell index value of deriving.E) experimental data drawing page or leaf (Experimental data plot page) permission is drawn flexibly to experimental data and is put in order.

Fig. 7 is presented at the bottom of the cell based of the present invention on the supervisory system the real-time monitoring with the H460 cell proliferation of different initial cells inoculation numbers inoculation.Every continuous recording cell proliferation in 15 minutes, continue above 125 hours.The cell growth curve of drawing with logarithmic scale has shown growth of index cell and the cell of stationary phase.

Fig. 8 shows cell attachment and the diffusion of using impedance monitoring system at the bottom of the cell based of the present invention to monitor the NIH3T3 cell in real time.Inoculating cell on the device that is coated with poly-L-Lysine or fibronectin.Different bags is measured more than 3 hours every monitoring in 3 minutes in real time altogether by lip-deep cell attachment and cellular invasion process.

Fig. 9 shows that use cell of the present invention-substrate impedance monitoring system monitors the morphological change of Cos-7 cell in real time.Made the cell serum starvation 8 hours, and stimulate with 50ng/mL EGF.With the morphological change of 3 minute timed interval monitoring cell, monitor 2 hours altogether, monitored 14 hours altogether with 1 hour timed interval then.The initial saltus step (jump) that occurs in the signal of the cell that EGF handles is crumple and the Actin muscle dynamic response owing to the film that produces in response to EGF.Arrow mark indication place is the EGF stimulation point.

Figure 10 shows the drawing of the time-dependent manner cell index of the H460 cell of handling with anti-cancer medicine paclitaxel.The H460 cell of the cultivation in different holes is the taxol treatment with different concns in its exponential phase of growth.After processing, cell was used cell of the present invention-substrate impedance monitoring system to monitor in real time the dynamic response of the taxol of various dose every 15 minutes, continued 50 hours.

Figure 11 shows the drawing of the time-dependent manner cell index of the H460 cell of handling with cancer therapy drug AC101103.AC101103 with different concns handles the H460 cell of the cultivation in different holes in its exponential phase of growth.After processing, cell was used cell of the present invention-substrate impedance monitoring system to monitor in real time the dynamic response of the AC101103 of various dose every 30 minutes, continued 20 hours.

Figure 12 shows the dynamic drug response curve of the A549 cell of handling with Zorubicin.In each hole of 16X cell-substrate impedance monitoring device, inoculate 10,000 A549 cells.Before processing, grow with cell-substrate impedance monitoring system monitoring cell attachment and cell.When cell is in exponential phase of growth, in cell, add the Zorubicin of different concns.Cell is got off by real time record as shown in the figure to the time-dependent manner and the drug dose dependency of Zorubicin.

Figure 13 shows mastocyte cell index recording drawing to the reaction of IgE when antigen being arranged or do not have antigen.In each hole of 16X device, inoculate 20,000 RBL-2H3 mastocyte.Make cell attachment and growth 22 hours, simultaneously record data.In the time of 22 hours, cell is resisted-DNP mouse IgE processing with non-specific mouse IgG of 1 μ g/mL or 1 μ g/mL.Continue other 20 hours of the electronics impedance response of record cell, the substratum in the sucking-off hole subsequently, and change fresh serum free medium.Made cellular-restoring 30 minutes, and used the antigen of 1 μ g/mL then, DNP-BSA handles.Measure the electronics impedance response 3 hours of cell again.

Figure 14 shows another result of experiment, and wherein monitoring is reacted with cell-electrode impedance Anti-DNA IgE sensitization and that pass through application DNP-BSA activated RBL-2H3 mastocyte.On the 16X device with the density of 20,000 cells/well inoculation RBL-2H3 mastocyte, continuously measured impedance, and used cell of the present invention-substrate impedance monitoring system log (SYSLOG) cell index every 30 minutes.100ng/mL DNP-BSA with cell and the anti-DNPIgE incubation of 100ng/mL, is used then in about 14 hours of inoculation back after 24 hours.After using DNP-BSA, carry out impedance measurement (showing) by cell index with 5 minutes the timed interval.

Figure 15 shows the cell-electrode impedance reaction of RBL-2H3 mastocyte and the dependency between morphocytology kinetics and the medium release.(A) with 100ng/mL IgE sensitization RBL-2H3 mastocyte or with contrast IgG antibody treatment RBL-2H3 mastocyte, activate with DNP-BSA then.Cell is fixed, is changed thoroughly with Paraformaldehyde 96 and dye with rhodamine-Phalloidine at indicated time point.Manifest cell and take the cell photo with the Nikon E-400 immunofluorescence microscopy that the CCD photographic camera is installed.(B) by the IgE sensitization, by using DNP-BSA activated RBL-2H3 mastocyte.

Figure 16 show IgE separately the mastocyte of mediation-electrode impedance reaction raise and to the effect of the activation step of antigen mediation.(A) be inoculated in RBL-2H3 cell on the microelectronics sensor array not treated or with shown in anti--DNP IgE of concentration handle.The resistance value that uses cell-substrate impedance monitoring system monitoring continuously and record to show by cell index.(B) with 15ng/mL or 1 μ g/mL anti--the RBL-2H3 cell of DNP-IgE sensitization activated with 100ng/mLDNP-BSA.The resistance value that the using system monitoring continuously is shown by cell index.

Figure 17 shows with the activated restraining effect of cell-electrode impedance measuring system monitoring inhibitors of protein kinase C bisindolylmaleimidesfor (Bisindolymaleimide) to the RBL-2H3 mastocyte of IgE-mediation.With the RBL-2H3 mastocyte with 100ng/mL anti--the DNP-IgE sensitization, then with shown in the bisindolylmaleimidesfor incubation 1 hour of concentration, add 100ng/mL DNP-BSA afterwards again.The resistance value that uses cell of the present invention-substrate impedance monitoring system monitoring to show by cell index.

The Hex activity of the RBL-2H3 mastocyte of the IgE-mediation of measuring when Figure 18 is presented at pharmacological inhibitor.The RBL-2H3 mastocyte is by sensitization, and respectively with DMSO, 16.6 micromole SU, 6656,5 micromole U73122,16.6 micromole's bisindolylmaleimidesfors, 16.6 micromole Piceatannol and 16.6 micromole PD, 98059 incubations 1 hour.Come activating cells by adding 100ng/mL DNP-BSA.

Detailed Description Of The Invention

A. definition

Clear for disclosure, rather than for affected certain restriction, the following detailed description of the present invention will be divided into a plurality of chapters and sections.

Unless other definition is arranged, as used herein all technology and scientific terminology all with the technical field of the invention in the term of technician institute common sense have identical meaning.All patents mentioned herein, application, disclosed application and other publications all intactly are attached among the application with the form of reference.If the definition that proposes in the definition that proposes in this chapter and patent, application, disclosed application and other publications is opposite or inconsistent, then the definition that proposes among this chapter is better than the definition that proposes in those documents that are incorporated herein by reference.

" one (a) " or " one (an) " refers to " at least one " or " one or more " as used herein.

" film " is meant material piece as used herein.

" biocompatible membrane " refers to pair cell as used herein, the film that comprise viability, adhere to, diffusion, motility, growth or cell fission does not have deleterious effect.

When suspension survival, unimpaired epithelial cell or endotheliocyte was added in the container, when the cell adhesion that quite big per-cent was arranged in 12 hours during in vessel surface, the surface that is called container " was suitable for cell attachment ".Preferably, in 12 hours at least 50% cell adhesion in vessel surface.More preferably, be suitable for surface properties that the surface of cell attachment has and make and pave in the plate 12 hours (that is, after cell adds container) that at least 70% cell adhesion is in the surface.Even more preferably, the surface properties that is suitable for the surface of cell attachment makes paves in the plate 12 hours at least 90% cell adhesion in the surface.Most preferably, Biao Mian surface properties makes and to pave in the plate 8,6,4,2 hours at least 90% cell adhesion in the surface.In order to have the required surface properties that is used for cell attachment, the surface need be through chemical treatment (as: with acid and/or alkaline purification), and/or physical treatment (as: using plasma treatment), and/or biological chemistry is handled (as: with the molecule or the biomolecules bag quilt of one or more promotion cell attachment.In the present invention, biocompatible surface (for example film) is preferably suitable for the cell attachment of employed type in mensuration, described mensuration is used biocompatible surface (for example, film), and most preferably makes in mensuration the adhering to of at least 90% cell of the biocompatible film of contact.

" biomolecules bag by " is meant the bag quilt on the surface, and it comprises the molecule as naturally occurring biomolecules or biochemical reagents, or derived from or based on the biochemical reagents of one or more naturally occurring biomolecules or biochemical reagents.For example, the biomolecules bag can be comprised extracellular matrix components (as fibronectin, collagen), and or derivatives thereof maybe can comprise biochemical reagents, as polylysine or poly ornithine, they are based on the polymerizable molecular of naturally occurring biochemical reagents Methionin and ornithine.Based on naturally occurring biochemical reagents such as amino acid whose polymerizable molecular, can utilize the isomer or the enantiomer of naturally occurring biochemical reagents.

" extracellular matrix components " is meant the molecule in the extracellular matrix that is present in the animal.It can be the composition that derives from the extracellular matrix of any species and any types of organization.The non-limiting instance of the composition of extracellular matrix comprises ln, collagen fibronectin, other glycoprotein, peptide, glycosaminoglycan, proteoglycan etc.Extracellular matrix components also comprises somatomedin.

" electrode " is meant a kind of structure with high conductivity, that is, and and the projecting far away conductivity of electrolyte materials of specific conductivity.

" electrode structure " is meant single electrode as used herein, particularly electrode (as the electrode structure of a spiral type) with complex construction, or the combination of at least two electrode members that are electrically connected.All electrode members in " electrode structure " all are electric connections.

" electrode member " is meant the single structure feature in the electrode structure as used herein, for example, and the digitation structure in the interdigitated electrodes structure.

" electrod-array " or " electrode structure unit " is meant two or more electrode structures as used herein, it is configured to have certain size and interval, thereby when being connected in signal source, can be used as a unit operation, to produce electric field in electrode structure surrounding space zone.The impedance variations that produces at electrode surface owing to cell attachment can be measured in preferred electrode structure unit among the present invention.The unitary limiting examples of electrode structure comprises interdigitated electrodes structural unit and concentric electrode structure unit.

" electrode bus (electrode bus) " is a part of electrode, and it connects single electrode member or substructure.Electrode bus provides from single electrode member or single electrode substructure and has led to the common conducting wire of another electric connection point.In device of the present invention, electrode bus is each electrode member of contact electrode structure all, and the electrical interconnection that is connected to electric trace is provided, and described electric trace can be linked to connection gasket.

" electrode trace " or " conductive trace " or " electric trace " are meant from electrode or electrode member or electrode structure leads to device or equipment is terminal or peripheral conducting wire, so that electrode or electrode member or electrode structure and electric impedance analyzer are coupled together.Terminal or the periphery of device can be corresponding to the connection gasket on device or the equipment.

" connection gasket " is a zone on equipment of the present invention or the device, it be electrically connected to described equipment or the device at least one electrode or at least one electrode structure in all electrode members on, and operationally be connected to (for example, impedance measuring circuit or signal source) on the circuit external.Electrical connection between connection gasket and impedance measuring circuit or the signal source can be direct or indirect, can be by any suitable conductive tool, and for example lead or metal wire are realized.This conductive tool also can realize through being positioned at last other regional electrodes of equipment or device or conductivity circuit.

" interdigitated " be meant the raised structures that comes from a direction with from the raised structures of another direction mode interlaced (should point out that the interdigitated electrodes element preferably can not be in contact with one another) with the finger of the both hands that close.

" high probability of contacts electrode element " is meant if cell is positioned at the sensor region of device of the present invention or equipment randomly as used herein, so the probability of cell (or particle) contacts electrode element by on device of the present invention or the equipment or the size of the mean diameter of use therein cell, electrode member, gap length between the electrode member calculate, greater than about 50%, more preferably greater than about 60%, more preferably greater than about 70%, even more preferably greater than about 80%, greater than about 90%, or greater than about 95%.

" at least two electrodes processing in described substrate " refer to that at least two electrodes are processed or make or produce in substrate as used herein.These at least two electrodes can be in the same one side of substrate or the not coplanar of substrate.Substrate can have multilayer, and these at least two electrodes can be on the different layers of the same one deck of substrate or substrate.

As used herein " described suprabasil with one side at least two electrodes of processing " be meant that at least two electrodes are formed on same of substrate.

" at least two electrodes processing on described suprabasil same plane " are meant as used herein, if dielectric base is a multiwalled, have at least two electrodes to be machined on same one deck of substrate so.

" described ... that electrode (or electrode structure) has essentially identical surface-area " is meant that the surface-area of mentioned electrode does not have very big difference mutually as used herein, thereby, adhering on mentioned any one electrode (or electrode structure) or the impedance variations that causes of growing will be to adhere on mentioned any other electrodes (or electrode structure) with cell or the same or analogous degree of impedance variations that causes of growing contributes to total detectable variation of impedance by cell.In other words, when electrode (or electrode structure) when having essentially identical surface-area, after adhering on arbitrary electrode or growing, any one electrode all can contribute to total impedance variations at cell.In the most applications, have the electrode of maximum of " essentially identical surface-area " and minimum interelectrode surface-area ratio less than 10.Preferably, the interelectrode surface-area ratio of the electrode of the maximum of electrod-array and minimum is less than 5,4,3,2,1.5,1.2 or 1.1.More preferably, at least two of described electrode structure electrodes all have and are bordering on identical or identical surface-area.

" described device has the surface that is suitable for cell attachment or growth " is meant that electrode and/or non-electrode region on the equipment have suitable physics, chemistry or biological nature as used herein, thereby target cell can be attached on this surface with surviving, and when cell cultures was grown, newly-generated cell can continue on the surface attached to equipment.Yet, the device or its surface comprises cells survival or the necessary material of growing is optional.The material that these are necessary, for example nutrition or somatomedin can be provided by substratum.More preferably, when the suspension of epithelial cell that will survive, intac or endotheliocyte joins " surface that is suitable for cell attachment " when going up, has 50% cell adhesion in 12 hours at least to the surface.More preferably, the surface that is suitable for cell attachment has such surface properties, thus pave plate have at least in 12 hours 70% cell adhesion to the surface (that is, adding cell) to the chamber or the hole that constitute described device.Even more preferably, the surface properties that is suitable for the surface of cell attachment will cause paving plate and have 90% cell adhesion in 12 hours at least to the surface.Most preferably, the surface properties that is suitable for the surface of cell attachment will make and have at least 90% cell to adhere on the surface in 8,6,4,2 hours paving plate.

As used herein " described interelectrode detectable impedance variations " (or " the detectable impedance variations between described electrode structure ") be meant when the molecule association reaction when electrode surface takes place, to cause the impedance between described electrode (or electrode structure) to produce sizable variation, this variation can be recorded by electric impedance analyzer or impedance measuring circuit.The difference between resistance value when impedance variations refers on the electrode surface of equipment the molecule association reaction takes place and when on electrode surface, the molecule association reaction not taking place.Perhaps, impedance variations refers to as cell when electrode surface adheres to and the cell difference between the resistance value when electrode surface adheres to not, or the variation of the cell number, kind, activity, tackiness or the morphology that contain the surface of electrode on being attached to equipment resistance value when changing.In most applications, it promptly is detectable that impedance changes above 0.1%.Preferably, detectable impedance variations surpasses 1%, 2%, 5% or 8%.More preferably, detectable impedance variations surpasses 10%.The function of the electric field frequency that is used to measure that interelectrode impedance is normally applied." detectable impedance variations between electrode " also do not require that in all frequencies detectable impedance variations is all arranged." detectable impedance variations between electrode " only requires that the variation of impedance can be recorded at any single frequency (or a plurality of frequency).In addition, impedance has two compositions, is called resistance and reactance (reactance can be divided into two classes, capacitive reactance and induction reactance)." in detectable impedance variations between the described electrode " only requires resistance and reactance on any one or more frequencies, and a detectable variation is arranged.In this application, impedance is electric impedance or electronic impedance.The method of measuring this impedance is, (1) voltage (or a plurality of frequency of the given frequency of application between described electrode, or has a concrete voltage waveform), and monitoring is in described frequency (or a plurality of frequency, or have a concrete waveform) electric current by described electrode down, with the voltage amplitude amplitude divided by the current amplitude value resistance value of deriving; (2) use monofrequency composition (or a plurality of frequency, or have a concrete current waveform) electric current come by described electrode, and monitoring is in described frequency (or a plurality of frequency, or have a concrete waveform) voltage between the described electrode down, with the voltage amplitude amplitude divided by the current amplitude value resistance value of deriving; (3) can measure or measure other method of electric impedance.Note, in above-mentioned " resistance value that the voltage amplitude amplitude is derived divided by the current amplitude value ", " divided by " be that the value to current amplitude and voltage amplitude is carried out under identical frequency.Measuring this electric impedance is a kind of electronic processes or electric process, and it does not relate to uses any reagent.

" described at least two electrodes have visibly different surface-area " is meant that the surface-area of any electrode all is inequality each other as used herein, thereby the impedance variations that causes in cell attachment on the larger electrode or growth will be not can not contribute to total detectable impedance with the same or analogous degree of impedance variations that causes with cell attachment on smaller electrode or growth.Preferably, any impedance variations that causes in cell attachment on the larger electrode or growth can be significantly less than the impedance variations that on smaller electrode cell attachment or growth cause.In general, the ratio of the surface-area of extreme electrode and minimum electrode is greater than 10.Preferably, the ratio of the surface-area of extreme electrode and minimum electrode surpasses 20,30,40,50 or 100.

The many counter electrode or the electrode structure that " carry out steric many counter electrode or electrode structure according to the hole on the porous microtest plate " as used herein and be on finger device or the equipment are steric, with with the porous microtest plate on the sterie configuration coupling in hole, thereby, when needs, described device (for example can be inserted into, is attached to or is attached to porous plate, the porous plate at the no end), thus the porous in the porous microtest plate will comprise electrode or electrode structure.

" arrange " as used herein and be meant by row-Lie configuration, just electrical connection, the position of electrode, electrod-array or switch circuit is identified by line position number and column position number.

As used herein " each hole all comprises essentially identical number ... cell " be that the minimum value of cell number is at least cell number maximum in the hole in the finger-hole 50%.Preferably, in the hole the minimum value of cell number be cell number in the hole maximum at least 60%, 70%, 80%, 90%, 95% or 99%.More preferably, every hole all comprises identical cell number.

As used herein " each hole all comprises ... the cell of same kind " be meant that with regard to its intended purposes, every hole comprises the cell of same kind, but unnecessary every hole comprises identical kind cell.For example, if being every hole, its intended purposes comprises mammalian cell, so, if each hole comprises the mammalian cell of identical type, as the human cell, or different types of mammalian cell, as human cell and other inhuman mammalian cell, for example mouse, goat or MC etc. allow so.

As used herein " every hole comprises ... the test compounds of a series of different concns " be meant that every hole comprises the test compounds of serial dilution concentration, be 1M, 0.1M, 0.01M etc. as concentration with 1/10th serial dilutions.

" dose response curve " is meant the dependence of cell response to the test compounds dose concentration as used herein.The reaction of cell can be measured by many different parameters.For example, test compounds is under a cloud to have cytotoxicity, and can cause necrocytosis.So, the percentage ratio of the cell that the cell response after handling cell with test compounds can be by do not survive (or survival) records.

" electrode has than the much smaller length of the maximum one dimension size of the particulate that will analyze along the length of microchannel " is meant that electrode has at least 90% length less than the maximum one dimension size of the particulate that will analyze in the length along the microchannel as used herein.Preferably, electrode has at least 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% length less than the maximum one dimension size of the particulate that will analyze in the length along the microchannel.

As used herein " microelectrode strides across the whole height of microchannel " be meant microelectrode stride across the microchannel whole height at least 70%.Preferably, microelectrode stride across the microchannel whole height at least 80%, 90%, 95%.More preferably, microelectrode stride across the microchannel whole height at least 100%.

" hole with the aperture that is equal to or slightly greater than described granular size " is meant that the aperture in this hole should equal the particulate size at least as used herein, but less than 300% of granular size.Here aperture and particulate size are measured according to the one dimension value.

" microelectrode bar or electrode strip " is meant a kind of dielectric base bar as used herein, is processed with or is integrated with electrode or electrode structure unit on it.The limiting examples of dielectric base bar comprises isolator epitaxial wafer (insulator-on-semiconductor), the glass fibre (resembling the sort of material that is used to make printed circuit board (PCB)) of growing on polymeric film, glass, plastic sheet, pottery, the semi-conductor.Has little manufacturing (microfabricated), little processing (micromaching) or additive method manufacturing that the electrode structure unit of different geometries can be by any appropriate or processing on base strip.The limiting examples of electrode geometry comprises interdigitated electrode, " circle is (circle-on-line) on line " electrode, " rhombus is (diamond-on-line) on line " electrode, castle electrode or sinusoidal electrode.The characteristic dimension of the geometrical shape of these electrodes may diminish to and is less than 5 microns, or is less than 10 microns, arrives greater than 200 microns, greater than 500 microns, greater than 1 millimeter to big.The characteristic dimension of electrode geometry is meant the minimum width of electrode member, or the interelement minimum clearance of adjacent electrode, or recurrent part dimension on the electrode geometry.In the present invention, the microelectrode bar can be any geometrical shape.An exemplary geometry of microelectrode bar is a rectangle, and the width of its discal patch is between arriving greater than 10 millimeters less than 50 microns, and the length of bar is between arriving greater than 15 millimeters less than 60 microns.The geometrical shape of exemplary microelectrode bar can have the geometrical shape of 200 microns wide and 20 millimeters long.Single microelectrode bar can have two electrodes as measuring unit, or has a plurality of this two electrodess as a plurality of measuring units, or has single electrode structure unit as measuring unit, or has a plurality of electrode structures unit as a plurality of electrode structures unit.In a kind of exemplary, when a plurality of electrode structures unit was formed on the single microelectrode bar, these electrode structure unit were localized along the length direction of bar.The electrode structure unit can be square or rectangle or circle.The size that each electrode structure unit may have arrives greater than 2mm * 2mm from less than 50 μ m * 50 μ m.

As used herein " sample " be meant the equipment, microtest plate or the method that comprise available the application separate, operate, measure, quantitatively, any material of the part that detects or analyze.Sample can be biological sample, as biological fluid or biological organization.The example of biological fluid comprises the suspension of cell in substratum such as cell culture medium, urine, blood, blood plasma, serum, saliva, seminal fluid, stool, phlegm, cerebrospinal fluid, tear, mucus, amniotic fluid etc.Biological organization is generally the cell of particular type and the aggregate of their intercellular substance, the class formation material that it forms in people, animal, plant, bacterium, fungi or the virus structure comprises reticular tissue, epithelium, muscle tissue and nervous tissue.The example of biological organization also comprises organ, tumour, lymphoglandula, artery and individual cells.Biological sample also can further comprise cell suspending liquid, comprises the solution of biomolecules (as albumen, enzyme, nucleic acid, carbohydrate, be attached to the chemical molecular with biomolecules).

" liquid (fluid) sample " is the natural sample that exists with liquid or flow morphology as used herein, as biological fluid." liquid sample " also can refer to natural in the on-liquid state, the sample that exists of solid or gas for example, but be made into to contain liquid, fluid, solution or the suspension of solid or gaseous sample material.As, liquid sample can comprise liquid, fluid, solution or the suspension that contains biological organization.

102. the device and the system of monitoring cell-substrate impedance

Measure the device of cell-substrate impedance

The present invention includes the device that is used to measure cell-substrate impedance, it comprises an insulating substrate; Be installed on suprabasil two or more electrod-arrays, each of wherein said two or more electrod-arrays comprises two electrode structures; And at least two connection gaskets, each connection gasket is positioned at the edge of substrate.Each electrod-array of device has approximate uniform electrode resistance on whole array.The substrate of device has the surface that is suitable for cell attachment or growth; Cell described suprabasil adhere to or grow can cause that the detectable impedance between electrode structure in each electrod-array changes.

Electrod-array is two or more electrode structures, and it is configured to have certain size and interval, thereby when being connected in signal source, can be used as a unit operation, to produce electric field in electrode structure surrounding space zone.Electrode structure refers to single electrode, specifically is the single electrode with complex construction.(for example, electrode structure can comprise the electrode member that two or more are electrically connected.) in device of the present invention, electrod-array comprises two electrode structures, each electrode structure comprises a plurality of electrode members, or substructure.In a preferred embodiment of the invention, the electrode structure of each of two or more electrod-arrays of device has essentially identical surface-area.In the preferred embodiment of apparatus of the present invention, each of two or more electrod-arrays of device all comprises two electrode structures, and each electrode structure comprises a plurality of electrode members.Each of two electrode structures of electrod-array all is connected in the independent connection gasket that is positioned at basal edge.

Like this, in device of the present invention, for in two or more electrod-arrays of device each, first of two electrode structures is connected in two or more connection gaskets, and in two electrode structures second is connected in two or more connection gaskets another.Preferably, each array of device is separately addressable, mean the electric trace of array and the layout of connection gasket array is connected with electric impedance analyzer by this way, that is, can measuring voltage be applied to single electrod-array a preset time by using switch (as electronic switch).

Each electrod-array of device has approximate uniform electrode resistance and distributes on the entire electrode array." homogeneous resistance cloth in the whole array " be meant when measuring voltage is added on the electrode structure of array, and being positioned on the electrode resistance of arbitrary given position of array and the array arbitrarily, the electrode resistance of other positions is approximately uniform.Preferably, differ at the electrode resistance of first position of the array of device and electrode resistance and be no more than 30% in second position of identical array.Preferred, differ at the electrode resistance of first position of the array of device and electrode resistance and to be no more than 15% in second position of identical array.Even preferred, differ at the electrode resistance of first position of the array of device and electrode resistance and to be no more than 5% in second position of identical array.Preferred, differ at the electrode resistance of first position of the array of device and electrode resistance and to be no more than 2% in second position of identical array.

For device of the present invention, be used to make the measured resulting impedance of all cells counter electrode array to change the similar contribution of performance the preferred arrangement of the electrode member in the given electrod-array, interelectrode gap and electrode bus, and no matter where cell is positioned at and attached to which position of electrode surface.Like this, need when measuring voltage is added on electrod-array, the strength of electric field of any two positions in the given array of any on the device is similar.At any given position of array, the potential difference between the closest approach on closest approach on first electrode structure of strength of electric field and array and second electrode structure of array is relevant.Therefore need be concerning a given array, the potential drop that strides across electrode member and electrode bus is similar.Based on this requirement, it is uniformly approximate preferably striding across whole array top electrode distribution of resistance, wherein the electrode resistance a target location equals (to refer to be positioned at a bit on first electrode structure at the closest approach on first electrode structure, it and target location are nearest) and be connected to electrode resistance between the connection gasket of first electrode structure add closest approach on second electrode structure (refer to be positioned on first electrode structure a bit, it is nearest with the target location) and be connected to electrode resistance between the connection gasket of second electrode structure with.

The array that device of the present invention is designed to device strides across whole array and has approximate uniform distribution.This can realize by the following method, for example, make electrode structure and electrode bus that specific interval and size (length, width, thickness and geometrical shape) be arranged, thereby the resistance of single other positions is approximate identical arbitrarily on the resistance of arbitrary single position of array and array.In most of embodiments, electrode member in the given array (or electrode structure) has equal interval and similar thickness and width, electrode bus in the given array has similar thickness and width, and by the electric trace that given array exports to connection gasket closely similar thickness and width is arranged.Thereby, in these preferred embodiments, the design of array makes the length of the length of the length of electrode member or structure and geometrical shape, electrode trace and geometrical shape and bus and geometrical shape allow to stride across that electrode resistance in the array distributes is uniformly approximate.

In the preferred embodiment of some cells-substrate impedance measurement device, electrode structure comprises a plurality of electrode members, and each electrode member is directly connected in electrode bus.The electrode member of first electrode structure is connected in first electrode bus, and the electrode member of second electrode structure is connected in second electrode bus.In these embodiments, each of two electrode bus is connected in connection gasket separately through electric trace.Although the resistance of trace is poised for battle the resistance list a position contribution is arranged, for any two positions of array, it is identical being connected with trace from second bus to the second connection gasket from first bus to first connection gasket.Thereby, in these preferred embodiments, so that being provided, the even resistance that strides across array need not to consider the resistance of trace in the geometry proterties of array of designs.

With electrod-array shown in Fig. 1 C is example, and the cell that hope is attached to the B position is similar with the cell that is attached to the A position to the contribution that resulting impedance changes.Thereby in the time of just need being added with measuring voltage on electrod-array, position A has similar strength of electric field with position B.Thereby need stride across electrode member and similar potential drop is arranged with electrode bus.Based on these requirements, preferably striding across whole array has approximate uniform electrode resistance to distribute, wherein the electrode resistance of target location equal the electrode points of this position with two connection gaskets that are connected in electrod-array between electrode resistance with.The electrode resistance of position A and position B is among Fig. 1 C

R _{Position _ A}=R _{Trace 1}+ R _1-arrives-A1+ R _A1-arrives-A+ R _A-arrives-A2+ R _{A2-is to-2}+ R _{Trace 2}(1)

R _{Position _ B}=R _{Trace 1}+ R _1-arrives-B1+ R _B1-arrives-B+ R _B-arrives-B2+ R _{B2-is to-2}+ R _{Trace 2}(2)

Wherein, R _{Trace 1}For by the impedance (be resistance, because reactive component very very little) of connection gasket to the electrical connection trace of the position 1 that is positioned at the top electrodes bus; R _{Trace 2}For by the impedance (be resistance, because reactive component very very little) of connection gasket to the electrical connection trace of the position 2 that is positioned at the bottom electrode bus; R _1-arrives-A1Be 1 impedance (being resistance) from the position because reactive component is very very little along the top electrodes bus to position A1; R _2-arrives-A2Be 2 impedances (being resistance) from the position because reactive component is very very little along the bottom electrode bus to position A2; R _A1- _To-ABe the impedance (be resistance, because reactive component very very little) of position A1 along the electrode member that links to each other to position A from the top electrodes bus; R _A2-arrives-ABe the impedance (be resistance, because reactive component very very little) of position A2 along another electrode member that links to each other to position A from the bottom electrode bus.The requirement of the uniform electrode resistance of pairing approximation refers to R _{Position _ A}Be similar to R _{Position _ B}

The design of electrode structure can be satisfied the requirement of approximate even electrode resistance shown in Fig. 1 C.Among Fig. 1 C, electrode or electrode member are round configuration on line, about 90 microns of its circular diameter, and about 30 microns of line width, the gap between line is about 80 microns.The internal diameter of electrode bus is about 5.75mm, and the external diameter of electrode bus is about 6.070mm.

In a preferred embodiment of the invention, for the measurement of cell-substrate impedance, electric impedance analyzer is connected to suprabasil connection gasket indirectly through electronic switch.The impedance Z that electric impedance analyzer records _{Amount to}For:

Z _{Amount to}=Z _Switch+ Z _Trace+ Z _{Electrode _ array}(3)

Z wherein _SwitchBe electronic switch in the impedance in " opening " stage, Z _TraceBe the impedance of suprabasil electrical connection trace between connection gasket and the electrode bus, Z _{Electrode _ array}It is the impedance of the electrod-array when cell being arranged or not having cell.Z _SwitchAnd Z _TraceMain component be resistance.If Z _SwitchAnd Z _TraceValue for being determined, they are to resulting impedance Z so _{Amount to}Contribution just can from the impedance that records, be removed.By selecting superior in quality electronic switch, the Z of different switches _Open _CloseValue can be near constant.Z like this _SwitchValue is to total impedance Z that records _{Amount to}Contribution can from the impedance that records, be removed.Z _TraceDifficulty is removed because the resistance value of different electrod-arrays is different, and be used to make the thickness of the conductive film of electrode structure between the different device also may be different.For accurately measuring cell-substrate impedance, preferably use little electrical connection trace (or electric trace) impedance Z _TraceFor this reason, need the electric trace of big width, can cause the minimizing of electrod-array area like this.For this reason, in an embodiment of apparatus of the present invention, in order there to be suitable electric trace wide, make the diameter of electrod-array area, in the example shown in Figure 1B, promptly the internal diameter of arc-shaped electrode bus is less than the bottom diameter of standard microtiter plate.For electrode bus is not exposed in mensuration in the solution, plate with holes can be attached to substrate, enough little of the diameter of described plate bottom be not so that electrode bus can be in the inside in hole.

In a preferred embodiment of the invention, the device that is used to monitor cell-substrate impedance has two or more electrod-arrays, its shared connection gasket.Preferably, an electrode structure at least one electrod-array of device is connected in connection gasket, and described connection gasket is connected in the electrode structure on another electrod-array at least of device simultaneously.Preferably, at least two arrays for device, each of two or more arrays all has first electrode structure to be connected in connection gasket, described connection gasket is connected in the electrode structure of at least one other electrod-array, and each of two or more electrod-arrays all has second electrode structure to be connected in connection gasket, and described connection gasket is not attached to any other electroplax structure or arrays of device.Thereby, in the preferred design of device, there are at least two electrod-arrays, each electrod-array wherein has first electrode structure to be connected in shared connection gasket, and second electrode structure is connected in independently connection gasket.

In certain preferred embodiments of the present invention, each electrode structure of electrod-array all links to each other with electrode bus, and electrode bus is connected in two or more connection gaskets on the device one through conductive trace.In preferred embodiments, each in two electrode structures all is connected in single bus, thereby each array is connected in two buses, and each is connected in an electrode structure.In such arrangement, each of two buses all is connected in the connection gasket that separates in the substrate.

The conductive trace that is connected in bus can be made by any electro-conductive material.Trace can be in the surface of substrate, and can randomly be insulated layer covering.Selectively, trace also can be positioned over suprabasil second plane.The description of the arrangement of conductive trace and design is found in U.S. Patent application 10/705 on the impedance measurement device, 447, this with this patent application in the disclosure of the manufacturing of conductive trace and design in the explanation substrate be incorporated in the present patent application in the mode of reference.

Figure 1A shows an example of apparatus of the present invention.This device comprises substrate of glass (101), is processed with 16 electrod-arrays in substrate.Each electrod-array (102) comprises two electrode structures (seeing Figure 1B for details).Each electrod-array is connected in two electric traces (103), wherein in two electrode structures of each connection of two traces.All be connected in the connection gasket (104) at substrate (101) edge from these electrical connection traces (103) of electrod-array (102) beginning.Shown in Figure 1A, each of four electrod-arrays of each of four quarterns in the substrate (101) all has their electrical connection traces separately to be connected in shared connection gasket (104).Thereby whole device has four shared connection gaskets (104), the shared connection gasket of per quart device.And each electrod-array has an independent electrical connection trace (103), and it is connected in an independently connection gasket (104).The edge of therefore, substrate (101) has 20 connection gaskets (104).

An example that can be used for the electrod-array (as Figure 1A) of device of the present invention is seen Figure 1B.Show an electrod-array at this.This electrod-array has two electrode structures, and wherein each electrode structure comprises a plurality of at the round electrode member of geometrical shape (105) on line of having of this demonstration.In this electrode array configurations, the electrode member (105) of another electrode structure alternately occurs in the electrode member (105) in electrode structure and the array.Be directly connected in the mode of electrode bus (106) by electrode member (105), each electrode structure all separate connection in its electrode bus (106).Electrode bus (106) forms the arc around the array periphery, wherein two of array buses not in abutting connection with or crossover.Conduction connects trace (Figure 1A 103) each bus is connected to the connection gasket that is in substrate (Figure 1A 101) edge (Figure 1A 104).

Suitable electronics fastening means, as be bonded in the substrate with the printed circuit board (PCB) that is connected on connection gasket on metal clip, the connection gasket and the electronics between the external electronic circuits (for example electric impedance analyzer) that can be used for forming on the device connect.U.S. Patent application No.10/705 is seen in the cell-design of substrate impedance means and the description of its manufacture method, 447, will be wherein be incorporated among the application at this about the description of design, characteristic and the manufacturing of the impedance means that comprises electrod-array and disclosure mode with reference.

Preferred dielectric base is a planar, and is smooth or near flat.Exemplary substrate can comprise many materials, silicon epitaxial wafer (SOI) wafer of growing on the silicon-dioxide epitaxial wafer (silicon dioxide on silicon) that includes but not limited to grow on the silicon, the isolator, glass (as silica glass, lead glass or borosilicate glass), sapphire, pottery, polymkeric substance, glass fibre, plastics, as polyimide (as Kapton, the Kapton that DuPont provides), polystyrene, polycarbonate, polyvinyl chloride, polyester, polypropylene and urea resin.Preferably, the molecule association reaction that occurs in substrate surface will not be disturbed on the surface of substrate and substrate.For cell-substrate impedance monitoring, it is preferably biocompatible that dielectric base any is exposed to the surface of cell in the use of apparatus of the present invention.Biological inconsistent base material can be by covering other materials, for example polymkeric substance or biomolecules coating and become biocompatible.

The all surfaces of substrate or part surface can be included, but are not limited to by chemical treatment, for example by adding functional group or adding charged or hydrophobic grouping and modification of surfaces.

The description that can be used on the electrod-array in the impedance measurement of apparatus of the present invention has been described in U.S. Patent application No.10/705, in 447, at this with wherein all are incorporated among the application in the disclosure of the suprabasil making method mode with reference about electrod-array (or structural unit), electrode structure, electrode materials, electrode size and electrode.

Electrod-array in preferred apparatus of the present invention comprises the array that comprises two electrode structures, for example, and the electrod-array of spiral type and interdigitated electrod-array.In preferred embodiment more of the present invention, electrod-array processing is in substrate, wherein array comprises two electrode structures, and each electrode structure comprises a plurality of circles electrode member on line, and the electrode member of the electrode member of one of them structure and electrode of opposite structure alternately.For example, Figure 1B has shown a such array.

Preferably, the electrode member of the array in apparatus of the present invention (or electrode structure) has roughly the same width.Preferably, the width of the electrode member of the array of apparatus of the present invention (or electrode structure) is greater than 30 microns, and preferred width is between about 50 to about 300 microns, and preferred width is about 90 microns.

Preferably, the electrode member of the array in apparatus of the present invention (or electrode structure) roughly is evenly spaced apart.Preferably, the gap width between the electrode member of the array of apparatus of the present invention (or electrode structure) is less than 50 microns, and more preferably width is between about 5 to about 30 microns, and more preferably width is about 20 microns.

Apparatus of the present invention can comprise one or more fluid-tight containers, and it can be used as fluid container.This container can reversibly or irreversibly be attached to, or is formed in, on the part of substrate or substrate (hole that for example, forms in microtiter plate).In another example, the microelectrode bar that apparatus of the present invention comprise reversibly or irreversibly is attached on the plastic housing, and this plastic housing has corresponding to the unitary opening of electrode structure that is positioned on the microelectrode bar.Suitable fluid container material comprises the material (for example pottery, glass, metal etc.) of plastics, glass or plastic coat.The description and the disclosure that comprise the device of fluid container are seen female case U.S. Patent application No.10/705,447, to wherein comprise fluid container and the fluid container structure that electrode is used for the substrate of impedance measurement about engaging at this, all disclosures that comprise its size, design, composition and making method are incorporated among the application in the mode of reference.

In a preferred embodiment, being positioned at suprabasil each electrod-array of apparatus of the present invention all links to each other with a fluid-tight container.Preferably, device of the present invention be assembled into a no end, porous has the plastic plate or the bar of fluid sealing, as shown in Figures 2 and 3.Assemble described device so that single array is positioned at the bottom in container or hole in the substrate.Preferably, each array of device is connected on the hole of porous plate.In some preferred embodiments, the Multiple-Aperture Device that is used for cell-substrate impedance measurement has " non-array " hole, and they are attached to substrate but are not connected with array.Such hole can randomly be used to carry out non-mensuration based on impedance, or is used for the observation of microscopically cell.

See U.S. Patent application No.10/705 about the design of porous impedance measurement device and the description of assembling, 447, will be at this wherein about the porous impedance measurement device, the disclosure that comprises its design, forms and make is incorporated among the application in the mode of reference.Device of the present invention preferably has 2 holes to 1,536 holes, more preferably 4 holes are to 384 holes, in addition more preferably 16 holes to 96 holes, all holes or be less than on all holes electrod-array is arranged.

In some preferred embodiments, the tissue culturing plate of market sale is applicable to device of the present invention.The also no base plate of customizable preferred size.Preferably, the aperture of bottom, hole (being in the end of substrate) is about 1 millimeter to about 20 millimeters, more preferably about 2 millimeters to about 8 millimeters.Can there be the diameter of homogeneous in the hole, or can be towards the bottom and be tapered, thereby the diameter of the end that container contacts with substrate is less than the diameter of opposite end.

Preferred device

Following description about device is not used for the present invention is imposed any restrictions.

Fig. 2 is a kind of image of design with 16x device of substrate of glass (201), and it has the electrod-array of 16 little manufacturings to be installed on the printed circuit board (PCB) (208) through metal clip (209).Metal clip (209) is engaged in suprabasil connection gasket (204) and is welded on the wire (210) on the printed circuit board (PCB) (208).Wire (210) on the printed circuit board (PCB) (208) connects the connection pin (211) that is positioned at the device edge successively.Plastics of the no end 16 hole bars (207) are attached in the substrate (201) through double-sided pressure-sensitive, constitute 16 independent fluid containers.In this device, the diameter of the bottom of fluid container is about 5mm.

The image that has shown a kind of a kind of design of the 96x device with 6 substrate of glass among Fig. 3, the every electrod-array that substrate of glass has 15 or 16 little manufacturings to form.The bottom surface of Fig. 3 A display unit, the hole placed face down that it is open, and Fig. 3 B shows the device that has plastic cover, the placement that faces up of its open hole.96 orifice plates (307) at the no end are installed and are sealed in 6 substrates (301), thereby 95 holes in 96 holes comprise electrod-array (when open hole faces up) in the bottom surface, hole.Not belt electrode (shown in (312) among Fig. 3 A) of one hole is arranged in 96 holes.The hole base diameter is about 5mm.Metal clip is bonded on the connection gasket of substrate, and is welded on the wire of top side face of little printed circuit board (PCB) (308) of the end that is positioned at substrate (301) (as described in Fig. 3 B, when 96 orifice plates face up when placing with open hole).Printed circuit board (PCB) (308) is two-sided, and has the wire of identical numbering to be in the relative position of circuit card two sides respectively.Link the wire of bottom side at the wire of circuit card top side face by conductive path, be electrically connected with the device operator's console at this.

Using method

The present invention also provides the method that the device that is positioned at the fluid container on the electrod-array carries out cell-substrate impedance measurement that comprises of the present invention of using.This method comprises: the device that is positioned at the fluid container on the electrod-array that comprises of the present invention is provided, electric impedance analyzer is attached on the device of the present invention, in one or more fluid containers of device, add cell, and measure the impedance on the one or more arrays on the device.The method of using impedance measurement device to carry out raji cell assay Raji is seen female case U.S. Patent number application 10/705,447, will be wherein be incorporated herein by reference at this, also introduce described content as a reference at the application's D and E chapters and sections about the disclosure of the method for using impedance measurement device.

Cell-substrate impedance measurement system

On the other hand, the present invention relates to cell-substrate impedance measurement system, it comprises A) at least one porous cell-substrate impedance measurement device, at least two of wherein said porous comprise electrod-array at the bottom of the hole; B) be electrically connected to the electric impedance analyzer of porous cell-substrate impedance measurement device; C) device operator's console, it can engage with one or more Multiple-Aperture Devices, and comprises and can select and connect the electronic circuit of the electrod-array in arbitrary hole in the porous to electric impedance analyzer; And D) software system of coupling device operator's console and electric impedance analyzer are used for the control device operator's console and carry out obtaining data and analytical data from electric impedance analyzer.

In cell of the present invention-substrate impedance measurement system, electric impedance analyzer is engaged in the connection gasket of one or more Multiple-Aperture Devices, to measure impedance.Cell-substrate measurement system can by numerical switch, from the impedance that the array in a hole measured in record, be transformed into the impedance of the array of measuring another hole by the circuit of using appts operator's console, measures to be used for effectively and side by side carrying out porous.

Porous cell in the system of the present invention-substrate impedance measurement device can be arbitrary porous cell-substrate impedance measurement device, comprise electrod-array at the bottom of wherein having two Kong Zaikong in the porous at least, and wherein in the porous at least two holes comprise separately addressable electrod-array.The used device of system of the present invention can be measured the difference of the resistance value relevant with cell behavior when being connected in electric impedance analyzer.For example, this cell-substrate impedance measurement device that is used for system of the present invention can be measured when cell attachment during in electrod-array and the difference of the resistance value when cell is not attached to electrod-array, the difference of resistance value in the time of maybe can detecting cell number when the surface that comprises electrode of the equipment of being attached to, type, activity, tackiness or morphological change.

Can become the preferred device of a part of cell-substrate impedance monitoring system, can be at female case U.S. Patent application No.10/705,447 described those devices, will be at this comprising the disclosure of the cell-substrate impedance monitoring device of electrod-array, the disclosure that comprises its design, forms and make is incorporated among the application in the mode of reference.The preferred embodiment that can become the part of cell-substrate impedance monitoring system also can be those devices that the application describes.

Preferably, the Multiple-Aperture Device of system of the present invention comprises 4-1,536 holes, and wherein part or all of hole can comprise electrod-array.In some embodiments of the present invention, the device operator's console can comprise that one or more platforms or one or more groove are so that place one or more Multiple-Aperture Devices.These one or more platforms or one or more groove can comprise that socket, pin or other are used for the device that is electrically connected between device and device operator's console.Preferably, carrying out between cell impedance measurement test period, the device operator's console is placed in the incubator for tissue culture.It can be electrically connected on electric impedance analyzer and the computer that preferably places incubator for tissue culture outer.

The device operator's console comprises the electronic circuit that is connected to impedance monitoring device and electric impedance analyzer and electronic switch, described electronic switch can be switched on or switched off with system in each be connected of two or more electrod-arrays on the used Multiple-Aperture Device.The electronic switch of device operator's console is controlled by software program.Software program guiding device operator's console is connected the array of device with electric impedance analyzer, and monitors the impedance of one or more electrod-arrays.During impedance monitoring, electric impedance analyzer can be monitored impedance a frequency or under more than a frequency.Preferably, in given mensuration, monitor impedance more than a time point, and more preferably, the impedance of at least 3 time points of monitoring.The device operator's console can be connected to electric impedance analyzer with the single array of device, to monitor at a Measuring Time point one, some or all of array on the device.To each required time point of monitoring, the switch of device operator's console makes selecteed array can access quick monitoring continuously.In fact each time point of monitoring is the narrow time range of measuring in measuring (for example being less than 1 second to several minutes), carries out impedance monitoring during this period.In certain preferred embodiments of the present invention, device operator's console software is programmable, and impedance monitoring is carried out in the timed interval of selecting in any one hole that comprises the device of array with guidance.

The software of impedance monitoring system also can store and display data.The data that data can be presented on the screen, conduct is printed, or both have concurrently.Preferably, software can allow input and show test parameter, for example comprises the descriptive information such as the timed interval of cell category, compound concentration, monitoring.

Preferably, software also can be analyzed impedance data.In preferred embodiments, software can be at the cell index in one or more holes of one or more time points calculating Multiple-Aperture Devices.

Fig. 4 shows a kind of design of a 16x device operator's console, and it has 6 16 aperture apparatus to be connected in this operator's console.The device operator's console has be used for 6 devices 6 independently grooves, and wherein each groove comprises the Zero plug-in force socket (zero-insertionforce socket) (416) with connection pilot lamp (418).When device joined the device operator's console to correct way, because the circuitry lines of pilot lamp (418) on the PCB of device linked power supply, this lamp (418) can be bright.Operator's console comprises electronic switch, and it can be opened (connection) or close (disconnection) with digital form, to connect electrod-array in the single hole to electric impedance analyzer.

Fig. 5 shows the 96 aperture apparatus operator's console that can engage 96 aperture apparatus as shown in Figure 3.Operator's console uses POGO pin (520) to be connected to wire on the little printed circuit board (PCB), and (device has and uses metal clip to be connected in the little PCB of the connection gasket at device edge.POGO pin (520) is connected in the circuit of device operator's console inside.Circuit comprises electronic switch, and it can be opened (connection) or close (disconnection) with digital form, be connected with electric impedance analyzer with the electrod-array in the hole that those is comprised electrode.

Fig. 6 has shown the different pages of real-time electronic cell sensing software, has illustrated the input of the experiment parameter that can import and data analysis result's demonstration.A) lab note page or leaf allows the experimenter to write down the key message of experiment, for example experiment purpose and experiment flow.B) experimental design page or leaf allows to write down cell, cell number, compound and the compound concentration that is added in each hole.C) test duration is provided with page or leaf, allows record and control to carry out cell-employed timed interval of substrate impedance measurement, and a plurality of experimental procedures can be set, and each step all has the time of different time interval value and different lengths.D) the cell index page or leaf is a result page, wherein after a predetermined time interval that page or leaf is provided with was set by the test duration finishes each and measure, software system upgrade automatically that institute's foraminous measures with the cell index value of deriving.E) permission of experimental data drawing page or leaf is drawn flexibly to experimental data and is put in order.

C. calculate the method for cell index

Based on the impedance that records, cell number (more precisely, the viable cell number, or the cell number that adheres to) and the relation of interdepending between the cell attachment state, may derive so-called " cell number index " or " cell index " by the impedance frequency spectrum that records, it provides quantitatively and the useful index based on the cell behavior in the mensuration of impedance more of the present invention.During some are used in the present invention, " cell index " among the application is identical with " cell number index " in the following patent application: PCT application No.PCT/US03/22557 (submitting on July 18th, 2003), its name is called " IMPEDANCE BASED DEVICES ANDMETHODS FOR USE IN ASSAYS ", with U.S. Patent application No.10/705,447 (submitting on November 10th, 2003), its name is called " IMPEDANCE BASEDDEVICES AND METHODS FOR USE IN ASSAYS " (patent agent is numbered ACE-00101.P.1.1-US).Be incorporated among the application about cell index and the discussion of cell number exponential and disclosure mode at this U.S. Patent application 10/705,447 and PCT application No.PCT/US03/22557 with reference.

Can use this cell number exponential of various calculating method, disclose some new methods at this.

The invention provides the method for several calculating cell indexs, these methods are suitable for the cell attached to two or more essentially identical arrays of cell-substrate impedance means, wherein monitor the impedance variations of cell.In a preferred embodiment of the invention, these methods can be calculated cell index more accurately than the method for the cell index of the cell on two or more arrays of in the past calculating cell-substrate monitoring device.In some preferable methods of the present invention, the method for calculating cell index depends on the novel method of the resistance of the electric trace that calculates two or more essentially identical arrays of guiding.Therefore, the present invention has also comprised the method for the resistance of the electric trace that calculates two or more the basic identical arrays on the directed toward substrate.

" essentially identical electrod-array " or " essentially identical array " refers to: electrode size on the indication array and arrangement, electrode structure and electrode member all are identical.So two essentially identical electrod-arrays will have the electrode structure of same size (length and width, thickness), wherein electrode structure will have the electrode member of similar number, and the arrangement of each array upper electrode arrangement and electrode member also is identical.The arrangement here is meant structure or interelement distance (interval width), their mutual physical locations and their geometrical shape (angle, sinuousness, circle on line or the geometrical shape of castle etc.), comprises the same characteristic features of any electrode bus that is connected in electrode structure or electrode member.The electrode of basic identical array also comprises identical materials.With regard to calculating resistance of traces and cell index, substrate can have the essentially identical array of arbitrary number.

Following discussion provides the novel method of resistance of electrical connection trace that can calculate attached to the novel method of the cell index of the cell of the array of cell-substrate impedance monitoring device and be used to calculate two or more electrod-arrays of guiding cell-substrate impedance monitoring device.

Impedance (Z) is grouped into by two one-tenth, i.e. resistance R s and reactance Xs.On mathematics, impedance Z is expressed as follows,

Z＝Rs+j Xs

Wherein,

j = \sqrt{- 1},

Finger for (series connection) reactive component Xs, add thereon voltage with by its electric current phase quadrature is arranged.For (series connection) resistance, the voltage that adds thereon has identical phase place with electric current by it.Because it is well-known in electronics and electrical engineering, impedance also can as followsly be represented by shunting resistance Rp and parallel reactance Xp,

Z＝Rp*(jXp)/(Rp+jXp)，

Wherein,

j = \sqrt{- 1} .

Yet these expression formulas (series resistance and series reactance, or shunting resistance and parallel reactance) are of equal value.The technician of those electricity or electronic engineering can easily derive another kind of expression formula from a kind of parameter value of expression formula.For clear and consistence, use series resistance and series reactance expression formula in description in the present invention and the discussion.For simply, abbreviate series resistance and series reactance as resistance and reactance.

As U.S. Patent application No.10/705,447 (submitting to) on November 10th, 2003, its name is called " Impedance based devices and methods for use in assays " and PCT application number PCT/US03/22557, its name is called described in " Impedance based devicesand methods for use in assays " (submitting on July 18th, 2003), monitoring cell-substrate impedance can realize by measuring impedance in the range of frequency that is fit to arbitrarily with the variation that detects or measure impedance, at this this two patent application is incorporated in the present patent application about the disclosure of the cell-substrate impedance monitoring mode with reference.For example, can measure impedance between about 1Hz between about 100MHz in range of frequency.In another example, can measure impedance between about 100Hz between about 2MHz in range of frequency.Impedance generally is the function of frequency, and promptly resistance value changes with frequency change.Monitoring cell-substrate impedance both can also can be carried out under a plurality of frequencies at single frequency.If under multi-frequency, carry out impedance measurement, then can obtain the impedance spectrum of frequency dependence, promptly the frequency in each measurement obtains a resistance value.As mentioned above, impedance has two compositions, i.e. resistance and reactance.Resistance components or reactive component or both changes all can cause the variation of impedance.

As U.S. Patent application No.10/705,447 (submitting to) on November 10th, 2003, its name is called " Impedance based devices and methods for use in assays " and PCT application number PCT/US03/22557, it is described that its name is called " Impedance based devicesand methods for use in assays " (submitting on July 18th, 2003), at this these two patent applications are incorporated in the present patent application about the mode of the anti-disclosure of measuring resistance with reference, can measure the method for electricity (or electronics) impedance by following realization, (1) voltage (or a plurality of frequency of the given frequency of application between described electrode, or has a concrete voltage waveform), and monitoring is in described frequency (or a plurality of frequency, or have a concrete waveform) electric current by described electrode down, with the voltage amplitude amplitude divided by the current amplitude value resistance value of deriving; (2) use monofrequency composition (or a plurality of frequency, or have a concrete current waveform) electric current come by described electrode, and monitoring is in described frequency (or a plurality of frequency, or have a concrete waveform) voltage between the described electrode down, with the voltage amplitude amplitude divided by the current amplitude value resistance value of deriving; (3) can measure or measure other method of electric impedance.Note, in above-mentioned " resistance value that the voltage amplitude amplitude is derived divided by the current amplitude value ", " divided by " be that the value to current amplitude and voltage amplitude is carried out under identical frequency.Well-known as electricity and electronic engineering technician, in this calculating (be above-mentioned divided by), current amplitude and voltage amplitude all are expressed as plural form, and it has considered the size of electric current and voltage, and differing between electric current and voltage sinusoidal waveform.Similar, resistance value is represented by plural form that also it has resistance and reactive component as shown in above-mentioned equation.

As U.S. Patent application No.10/705,447 (submitting to) on November 10th, 2003, its name is called " Impedance based devices and methods for use in assays " and PCT application number PCT/US03/22557 (submitting on July 18th, 2003), it is described that its name is called " Impedance based devices and methods for use in assays ", cell-substrate the impedance of measuring can be used for calculating and is called cell index or cell number exponential parameter, relates to cell index or cell number exponential disclosure in two patent applications at this and is incorporated in the present patent application in the mode of reference.Can use the described cell number exponential of multiple calculating method, described method based on when cell attachment during in electrode structure with respect to the acellular resistance that causes when being attached to or the variation of reactance in electrode structure.Sometimes, adhere to but have the impedance (resistance and reactance) on the electrode structure of identical cell culture medium to be called baseline impedance acellular on the electrode structure.Baseline impedance can be obtained by following one or more methods: the impedance of the electrode structure that (1) is measured, wherein add acellular substratum in the hole of containing electrode structure, wherein the substratum of using during impedance measurement under this substratum and the condition of monitoring cell attachment is identical; (2) (adding contains behind the substratum of cell in the short time period, and cell does not have the enough time to be attached to electrode surface use short period of time (for example 10 minutes) measured impedance behind the substratum that contains cell to the hole that comprises electrode structure in hole bottom.The length of this short time period depends on the surface treatment or the modification of cell category and/or electrode surface); (3) when all cells in the hole all by certain processing (as pyroprocessing) and/or reagent (as stain remover) when killing, the impedance of the electrode structure of measuring (for this method that will use, processing and/or reagent should not influence the dielectric properties of the substratum on the electrode).

In an example (A), cell index or cell number index can followingly calculate:

(A1) in the frequency of each measurement, the resistance of using electrod-array when cell exists and/or is attached to electrode is divided by baseline electrical resistance calculated resistance ratio;

(A2) in frequency spectrum, find or the maximum value of definite resistance ratios,

(A3) maximum value from resistance ratios deducts 1.

The applied mathematics formula, cell index is derived as:

Wherein N is a number of measuring the Frequency point of impedance.For example, if the frequency that is used to measure is 10kHz, 25kHz and 50kHz, N=3 then, f ₁=10kHz, f ₂=25kHz, f ₃=50kHz.R _Cell(f _i) be in frequency f _iThe time resistance (cell-substrate resistance) of when cell exists in electrode electrod-array or electrode structure and R arranged _b(f _i) be in frequency f _iThe time electrod-array or structure baseline electrical resistance.

In this case, zero or do not have cell or only have seldom the cell of number exist or be attached to electrode surface near " cell index or cell number index " expression of zero.Higher " cell number index " value representation has more cell attachment in electrode surface for the cell of same type and cell under similar physiological condition.Higher " cell index " value is also represented the cell for same type and similar number, cell can adhere to better at electrode surface (such as, cell spreads more or cell is adhered to electrode surface more strongly).

In another example (B), the cell number index can calculate with the following method:

(B1) in the frequency of each measurement, calculate the reactance ratio divided by the baseline reactance with the reactance of electrod-array when cell exists or is attached to electrode.

(B2) in frequency spectrum, find or the maximum value of definite reactance ratio,

(B3) maximum value from the reactance ratio deducts 1.

In this case, zero or do not have cell or only have seldom the cell of number exist or be attached to electrode surface near " cell number index " expression of zero.Higher " cell number index " value representation has more cell attachment in electrode surface for the cell of same type and cell under similar physiological condition.

In another example (C), cell index can calculate with the following method:

(C1) in the frequency of each measurement, deduct baseline electrical resistance from the resistance of electrod-array when cell exists or is attached on the electrode, the resistance when existing to define cell changes with respect to the resistance of baseline electrical resistance;

(C2) find then or maximum value that definite resistance changes.

In this case, " cell number index " derives with respect to the maximum change value of the resistance of baseline electrical resistance based on the resistance in the range of frequency of whole measurement when cell exists.The dimension of cell index is Europe.

In another example (D), cell index can calculate with the following method:

(D1) in the frequency of each measurement, the size of computing impedance (equals

R wherein _sAnd X _sBe respectively series resistance and reactance).

(D2) size of electrod-array impedance deducts the size of baseline impedance when existing when cell or be attached to electrode, to determine that impedance phase when the cell is for the variation of the size of baseline impedance.

(D3) find or the maximum value of the size variation of definite impedance.

In this case, " cell number index " derives for the maximum change value of the size of baseline impedance based on the impedance phase in the range of frequency of whole measurement when cell exists.The dimension of this cell index is Europe.

In another example (E), index can calculate with the following method:

(E1) in the frequency of each measurement, with the resistance of electrod-array when the cell existence or when being attached to electrode divided by baseline electrical resistance, the calculated resistance ratio,

(E2) then by deduct the relative change of 1 resistance when obtaining the frequency of each measurement from resistance ratios.

(E3) then with all relative change value integrations (all relative change value additions when being about to different frequency).

In this case, " cell number index " based on a plurality of Frequency points, rather than as above-mentioned example in obtain in the single peak frequency.Equally, zero or approaching zero " cell index " expression does not have cell to be present in electrode.Higher " cell number index " refers to more cell attachment is arranged in electrode for the cell of same type and cell under similar physiological condition.

In another example (F), cell index can followingly calculate:

(F1) in the frequency of each measurement, deduct baseline electrical resistance with the resistance of electrod-array when cell attachment is on electrode, the resistance when defining cell is with respect to the resistance change of baseline impedance; (herein, in frequency f _iThe time resistance the available following formula of change: Δ R (f _i)=R _{The s-cell}(f _i)-R _{The s-baseline}(f _i) provide R _{The s-cell}And R _{The s-baseline}Be respectively series resistance and the baseline series resistance of cell when on electrod-array, existing.)；

(F3) frequency dependence of analysis resistance change can quantitatively described dependent some parameter to derive.In an example, can by Calculate described parameter.In another example, this parameter can by

Calculate.This parameter can be used as cell index or cell number index.

In this case, " cell number index " is based on the analytical derivation of the frequency spectrum of resistance change.Depend on described parameter and how to calculate, the dimension of cell index is Europe.

In another example (G), cell index can followingly calculate:

(G1) in the frequency of each measurement, the size of computing impedance (equals R wherein _sAnd X _sBe respectively series resistance and reactance).

(G2) deduct the size of baseline impedance from size, to determine that impedance phase when the cell is for the variation of the size of baseline impedance when cell attachment electrod-array impedance during in electrode.(be f in frequency herein, _iThe time, the change of impedance magnitude is by formula Δ Z (f _i)=| Z _Cell(f _i) |-| Z _Baseline(f _i) | obtain.

R _{The s-cell}And X _{The s-cell}Be respectively series resistance and the reactance of cell when being present on the electrod-array.| Z _Cell(f _i) | the size of the impedance of electrod-array when being present on the electrod-array for cell.| Z _Baseline(f _i) | be the size of the baseline impedance of electrod-array.

(G3) frequency dependence of the change of analysis impedance magnitude is with quantitatively this dependent some parameter of deriving.In an example, this parameter can by

Calculate.In another example, this parameter can by Calculate.This parameter can be used as cell index or cell number index.

In this case, " cell number index " is based on the analytical derivation of the frequency spectrum of impedance magnitude change.Depend on described parameter and how to calculate, the dimension of cell index is Europe.

As U.S. Patent application No.10/705,447 (submitting to) on November 10th, 2003, its name is called " Impedance based devices and methods for use in assays " and PCT application number PCT/US03/22557 (submitting on July 18th, 2003), its name is called described in " Impedance based devices and methods for use in assays ", can use diverse ways, calculate from the cell-substrate impedance (resistance or reactance) that records and to be called cell index or cell number exponential parameter, the description cell index of this two patent application or the disclosure of cell number index and calculating thereof will be incorporated into herein with the form of reference.Cell index or cell number index are the quantitative measurments of cell in the hole when cell-substrate impedance measurement.

It is worthy of note utilize impedance information come to derive in the cell state on the monitoring electrode this " cell number index " optional.In fact, people can directly use resistance value (for example, in single fixed frequency; Or maximal phase is to changing frequency, or on a plurality of frequencies) as indication at cell state.

Yet deriving " cell index " or " cell number index " and using this class index to monitor cell state has advantage.Use " cell number index " to monitor the cell growth and/or adhere to and/or the viability situation has several advantages.

At first, people can be with the performance of the more different electrode geometries of this cell number index.

Second, for given electrode geometry, may add the impedance of cell of the different numbers of electrode by measurement to, set up " working curve ", with the mutual relationship between showed cell number and the cell number index (in this experiment, will guarantee that importantly the cell of inoculating is attached to electrode surface well).With such working curve, when carrying out new impedance measurement, may be from the cell number index that newly records and estimate cell number.

The 3rd, the cell number index also can be used for more different surface appearances.For the identical electrode geometry and the cell of similar number, have to have at this electrode surface and better adhere to and/or this electrode surface is more suitable for cell attachment than maxicell number exponential surface treatment explanation cell.

As implied above, for calculating cell index or cell number exponential certain methods, importantly to know the impedance (resistance and/or reactance) of described electrode structure when being with or without cell is present on the electrode structure.Based on equation (1), the impedance of electrod-array (being with or without cell on the electrode) is provided by following formula:

Z _{Electrode _ array}=Z _{Amount to}-Z _Trace-Z _Switch(5)

Z wherein _SwitchBe that electronic switch " is opened " impedance in stage, Z at it _TraceBe the impedance that is electrically connected trace (or conductive trace) between connection gasket and the electrode bus in the substrate, Z _{Amount to}It is the resulting impedance that records at electric impedance analyzer.By selecting superior in quality switch, all electronic switch have the impedance (be mainly resistance) of consistent state for " opening ".For example, the state of electronic switch is about 3 Europe (+/-10%) for the resistance of " opening ", and state is the reactance of " opening " can ignore (for example, in range of target frequencies less than 0.2 Europe).Thereby, if determine or calculated trace impedance, the impedance of electrod-array when formula (5) can be used for calculating cell or acellular the existence so.

The present application a kind of method, with mutual relationship based on two or more the essentially identical arrays on cell-substrate impedance monitoring device, determine the impedance (mainly be resistance of traces, for thin conducting film trace, reactance is very little) of conductive trace (or being electrically connected trace).Below, Figure 1A has showed that four electrod-array A, B, C and D illustrate present method.The reactance (series reactance) of reactance of electronic switch (series reactance) and electrical connection trace is less than corresponding resistance (series resistance).Therefore, we the resistance that weight analysis is electrically connected trace.The impedance that is recorded by electric impedance analyzer comprises resistance (series resistance, R _{Amount to}) and reactance (series reactance).For electrod-array A-D, the total electrical resistance R that records _{Amount to}, conduction (electrical connection) resistance of traces (R _Trace), switch resistance (R _Switch) and the resistance (R of electrod-array _{The e-array}) meet following formula:

R _E-array-A=R _Total-A-R _Trace-A-R _Switch-A(6A)

R _E-array-B=R _Total-B-R _Trace-B-R _Switch-B(6B)

R _E-array-C=R _Total-C-R _Trace-C-R _Switch-C(6C)

R _E-array-D=R _Total-D-R _Trace-D-R _Switch-D(6D)

When the electronic switch of selecting has consistent state for the resistance of " opening ", R _Switch-A, R _Switch-B, R _Switch-CAnd R _Switch-DClosely similar value is arranged and can be assumed to identical value R _SwitchThereby in above-mentioned equation, known parameters is R _Total-A, R _Total-B, R _Total-CAnd R _Total-D, and R _Switch-A, R _Switch-B, R _Switch-CAnd R _Switch-D, and 8 unknown parameter R are arranged _E-array-A, R _E-array-B, R _E-array-CAnd R _E-array-DAnd R _Trace-A, R _Trace-B, R _Trace-CAnd R _Trace-DCan not directly solve 8 known variables of these equatioies from these four equatioies.Need between these variablees other to concern to come solve equation.Each resistance of traces (R _Trace-A, R _Trace-B, R _Trace-CAnd R _Trace-D) depend on the type of employed metallic membrane and the geometrical shape of trace, as trace how many square matrix sections, segmental film thickness, segmental width, segmental length etc. are arranged.For example

Wherein N is the segments of trace-A, t _A-i, d _A-iAnd L _A-iBe i segmental thickness, width and length of the trace of electrod-array A, p is the specific resistance of film.Equation provided herein is applicable to the film that comprises the monometallic type.Equation can be easy to be used to comprise the film (as be covered on the chromium bonding coat golden film) of two or more metal types after revising.

If the thickness of film is (for example, variation in thickness is less than 10%) quite uniformly in the whole substrate, the relation between the resistance of traces is only by predetermined geometrical shape (for example segmental length, width) decision so.The ratio α of the resistance of the resistance of conductive trace that for example, can direct calculating electrod-array A as described below and the conductive trace of electrod-array D _A-D, the thickness of film is identical everywhere to suppose these traces here, and these traces specific resistance everywhere also all is identical.

Similarly, based on predetermined electrod-array B, C and the trace geometric relationship of D, people can determine α _B-DAnd α _C-DIt should be noted that for the film in these traces and comprise situation more than a kind of metal types, the top equation of can deriving similarly.Thereby, based on equation

R _Switch-A=R _Switch-B=R _Switch-C=R _Switch-D=R _Switch, (9A)

R _Trace-A=α _A-DR _Trace-D, (9B)

R _Trace-B=α _B-DR _Trace-D, (9C)

And R _Trace-C=α _C-DR _Trace-D(9D)

Equation (6A)-(6D) can be changed into following form:

R _E-array-A=R _Total-A-α _A-DR _Trace-D-R _Switch(10A)

R _E-array-B=R _Total-B-α _B-DR _Trace-D-R _Switch(10B)

R _E-array-C=R _Total-C-α _C-DR _Trace-D-R _Switch(10C)

R _E-array-D=R _Total-D-R _Trace-D-R _Switch-D(10D)

To (10D), 5 known variables are wherein arranged, R for equation (10A) _E-array-A, R _E-array-B, R _E-array-CAnd R _E-array-DAnd R _Trace-DThese known variables can not be calculated by these equatioies on the mathematics.Need extraneous information to solve these variable R _E-array-A, R _E-array-B, R _E-array-CAnd R _E-array-DAnd R _Trace-D

Invent and described a kind of method in the present invention.In this method, identical biological or chemical solution or suspension are applied on the electrod-array A-D.Because electrod-array A has essentially identical electrode structure to D, thus the condition when all being exposed to identical biological or chemical solution or suspension when all electrod-arrays, the electrod-array resistance R _E-array-A, R _E-array-B, R _E-array-CAnd R _E-array-DShould be identical, or very similarly value, i.e. R are arranged _E-array-A≈ R _E- _Array-B≈ R _E-array-C≈ R _E-array-DIf we suppose that average electrode array resistors is R _{The e-array}, then following approximation relation is set up: R _E-array-A≈ R _E-array-B≈ R _E-array-C≈ R _E-array-D≈ R _{The e-array}Therefore, equation (10A)-(10D) deformable is:

R _{The e-array}≈ R _Total-A-α _A-DR _Trace-D-R _Switch(11A)

R _{The e-array}≈ R _Total-B-α _B-DR _Trace-D-R _Switch(11B)

R _{The e-array}≈ R _Total-C-α _C-DR _Trace-D-R _Switch(11C)

R _{The e-array}≈ R _Total-D-R _Trace-D-R _Switch-D(11D)

Thereby we need find the R of the above-listed equation of As soon as possible Promising Policy _Trace-DAnd R _{The e-array}A kind of mathematical method is for finding R _Trace-DAnd R _{The e-array}, described R _Trace-DAnd R _{The e-array}Make following expression formula obtain minimum value, this expression formula is used for the poor of quantitative description approximated equation (11A, 11B, 11C and 11D) both sides:

F (R _Trace-D, R _{The e-array})=[R _{The e-array}-(R _Total-A-α _A-DR _Trace-D-R _Switch)] ²+

[R _{The e-array}-(R _Total-B-α _B-DR _Trace-D-R _Switch)] ²+ [R _{The e-array}-(R _Total-C-α _C-DR _Trace-D-R _Switch)] ²+

[R _{The e-array}-(R _Total-D-R _Trace-D-R _Switch)] ²(12)

Expression formula F (R _Trace-D, R _{The e-array}) be approximated equation (11A, 11B, 11C and 11D) both sides differences square and.F (R _Trace-D, R _{The e-array}) more little, then approximated equation (11A, 11B, 11C and 11D) both sides are approaching more.Therefore, can cause F (R _Trace-D, R _{The e-array}) obtain the R of minimum value _Trace-DAnd R _{The e-array}Value should be determined.Mathematical method relates to calculates F (R _Trace-D, R _{The e-array}) to R _Trace-DAnd R _{The e-array}First order derivative, and to make described first order derivative be 0.Causing these first order derivatives is 0 R _Trace-DAnd R _{The e-array}Value be to cause F (R _Trace-D, R _{The e-array}) obtain the value of minimum value.First order derivative is as follows:

2 α _B-D[R _{The e-array}-(R _Total-B-α _B-DR _Trace-D-R _Switch)]+

2 α _C-D[R _{The e-array}-(R _Total-C-α _C-DR _Trace-D-R _Switch)]+

2[R _{The e-array}-(R _Total-D-R _Trace-D-R _Switch)]

＝0 (13A)

2[R _{The e-array}-(R _Total-B-α _B-DR _Trace-D-R _Switch)]+

2[R _{The e-array}-(R _Total-C-α _C-D-R _Trace-D-R _Switch)]+

2[R _{The e-array}-(R _Total-D-R _Trace-D-R _Switch)]

＝0。(13B)

Equation (13A) and (13B) deformable be:

R _{The e-array}[α _A-D+ α _B-D+ α _C-D+ 1]+R _Trace-D[α _A-D ²+ α _B-D ²+ α _C-D ²+ 1]=

α _A-D[R _Total-A-R _Switch]+α _B-D[R _Total-B-R _Switch]+

α _C-D[R _Total-C-R _Switch]+[R _Total-D-R _Switch]

(14A)

4R _{The e-array}+ R _Trace-D[α _A-D+ α _B-D+ α _C-D+ 1]=

[R _Total-A-R _Switch]+[R _Total-B-R _Switch]+[R _Total-C-R _Switch]+[R _Total-D-R _Switch]

(14B)

Can solve R like this _Trace-D:

A wherein ₁₁=[α _A-D+ α _B-D+ α _C-D+ 1];

S ₁=α _A-D[R _Total-A-R _Switch]+α _B-D[R _Total-B-R _Switch]+

α _C-D[R _Total-C-R _Switch]+[R _Total-D-R _Switch];

B ₁₂＝[α _A-D+α _B-D+α _C-D+1]；

S ₂=[R _Total-A-R _Switch]+[R _Total-B-R _Switch]+[R _Total-C-R _Switch]+[R _Total-D-R _Switch].

Thereby, determining R _Trace-DThe time, resistance of traces R _Trace-A, R _Trace-BAnd R _Trace-CCan be by equation (9B), (9C) and (9D) calculate.And electrod-array resistance R _E-array-A, R _E-array-B, R _E-array-CAnd R _E-array-DCan be respectively with equation (10A), (10B), (10C) with (10D) by the resistance R that records _Total-A, R _Total-B, R _Total-CAnd R _Total-DCalculate.

Thereby one aspect of the present invention relates to a kind of method, and described method is used for from the total electrical resistance that records, and (resistance of) electrical connection trace for example, the array A-D among Figure 1A, it comprises the following steps: to calculate two or more basic identical electrod-arrays

(1) electrod-array is exposed to solution with same or analogous solution or suspension;

(2) with electric impedance analyzer or impedance measuring circuit, measure the resistance (series resistance) of each electrod-array, this resistance is the resistance (for example for electrode structure shown in Figure 1A, between connection gasket and electrode bus) of the electrical connection trace between electronic switch resistance, connection gasket and the electrode structure when having solution or suspension to exist and the resistance sum of electrod-array;

(3) obtain the resistance that is electrically connected trace with equation (15) and equation (9B), (9C) with (9D), notice that with equation (15) when calculating, the geometric relationship between the electrod-array is in order to definite factor-alpha _A-D, α _B-DAnd α _C-D

Another aspect of the present invention relates to a kind of method, if described method is used for same or analogous solution or suspension being joined determination of electrode or contacting with described determination of electrode, from the total electrode resistance that records, (for example calculate two or more basic identical electrod-arrays, the resistance of the electrod-array array A-D among Figure 1A), it comprises the following steps:

(2) with electric impedance analyzer or impedance measuring circuit, measure the resistance (series resistance) of each electrod-array, this resistance is to be electrically connected the resistance (for example for electrode structure shown in Figure 1A, between connection gasket and electrode bus) of trace and the resistance sum of electrod-array between electronic switch resistance, connection gasket and the electrode structure when having solution or suspension to exist;

(4) use equation (10A, 10B, 10C and 10D) to calculate the resistance of electrod-array.

In many application, be used for the solution of every kind of electrod-array or suspension (for example cell suspending liquid) by different forming.For example, can use the cell suspending liquid of different cell numbers, thereby the suspension that is applied to each electrod-array is very different.In this case, the resistance of the electrod-array when defining cell and existing just requires to determine to be electrically connected resistance of trace by carrying out once " reference measure " or " calibration measurement ", when carrying out described " reference measure " or " calibration measurement ", electrod-array is exposed to identical reference solution.Can determine to be electrically connected the resistance of trace by " reference measure ".In another was tested separately, electrod-array was exposed to target solution or cell suspending liquid, and used electric impedance analyzer or impedance measuring circuit to measure the total electrical resistance of this condition lower electrode array.The resistance of the electrod-array when having cell suspending liquid to exist can deduct the electronic switch resistance of respective electrode array with electrical connection resistance of traces sum and from the resistance that records definite (or continuously determining) by the resistance that records.

Thereby, another aspect of the present invention relates to a kind of method, if described method is used for different target solution or suspension are applied to determination of electrode, (for example calculate essentially identical electrod-array from the total electrical resistance that electric impedance analyzer records, the resistance of the electrod-array array A-D among Figure 1A), it comprises the following steps:

(1) electrod-array is exposed to solution with same or analogous solution or suspension (reference solution);

(2) with electric impedance analyzer or impedance measuring circuit, measure the resistance (series resistance) of each electrod-array, this resistance is the electronic switch resistance when having reference solution to exist, be electrically connected the resistance sum of resistance of traces (for example for electrode structure shown in Figure 1A, between connection gasket and electrode bus) and electrod-array between connection gasket and the electrode structure;

(3) obtain the resistance that is electrically connected trace with equation (15) and equation (9B), (9C) with (9D), notice that with equation (15) when calculating, the geometric relationship between the electrod-array of Figure 1A is in order to definite factor-alpha _A-D, α _B-DAnd α _C-D

(4) to each electrod-array applying soln or suspension; With electric impedance analyzer or impedance measuring circuit, measure the resistance (series resistance) of each electrod-array, this resistance is the electronic switch resistance when having solution or suspension to exist, and is electrically connected the resistance of trace and the resistance sum of electrod-array between connection gasket and the electroplax structure;

(5) with equation (10A), (10B), (10C) with (10D) calculate the resistance of electrod-array, its resistance by deducting electronic switch the resistance that records from step (4) and the resistance realization of electrical connection trace.

Attention is exposed to reference solution with electrod-array and can carrying out before or after the step (step (4)) of electrod-array application target solution or suspension and measurement total electrical resistance with the step (step (1), (2) and (3)) of determining conductive trace resistance in aforesaid method.For example, can carry out step (4) earlier.Then, target solution or suspension are removed from electrod-array.Add reference solution (step (1)) to electrod-array again.Carry out step (2) and step (3) again to determine to be electrically connected the resistance of trace.At last, carry out step (5).

In another approach, step (1) and (2) can be carried out in that step (4) is preceding.

Another aspect of the present invention relates to a kind of method, and the total electrical resistance of the basic identical electrod-array that described method records based on electric impedance analyzer is used for determining the resistance of electrod-array when the mensuration cell based on cell exists.In this method, electrod-array is exposed to identical reference solution (for example, not containing the identical cell culture medium of any cell), and carries out electrical measurement to determine to be electrically connected the resistance of trace.After having determined to be electrically connected the resistance of trace, the resistance of the electrod-array when adding cell suspending liquid to electrod-array can be calculated by the total electrical resistance that records on electric impedance analyzer.The resistance of the electrod-array when this total electrical resistance includes cell and exists, the resistance of electronic switch and be electrically connected the resistance of trace.This method comprises the following steps:

(1) electrod-array is exposed to solution (reference solution) with same or analogous solution or suspension;

(2) with electric impedance analyzer or impedance measuring circuit, measure the resistance (series resistance) of each electrod-array, this resistance is to be electrically connected the resistance (for example for electrode structure shown in Figure 1A, between connection gasket and electrode bus) of trace and the resistance sum of electrod-array between electronic switch resistance, connection gasket and the electrode structure when having reference solution to exist;

(3) obtain the resistance that is electrically connected trace with equation (15) and equation (9B), (9C) with (9D), notice that with equation (15) when calculating, the geometric relationship among Figure 1A between the electrod-array is in order to definite factor-alpha _A-D, α _B-DAnd α _C-D

(4) to each electrod-array application target cell suspending liquid; And with electric impedance analyzer or impedance measuring circuit, measure the resistance (series resistance) of each electrod-array, this resistance is to be electrically connected the resistance of trace and the resistance sum of electrod-array between electronic switch resistance, connection gasket and the electrode structure when having target cell suspension to exist;

(5) with equation (10A), (10B), (10C) with (10D) calculate the resistance of electrod-array, it is realized with the resistance of electrical connection trace by the resistance that deducts electronic switch the resistance that records from step (4).

Attention is exposed to reference solution with electrod-array and can carrying out before or after the step (step (4)) of electrod-array application target solution or target cell suspension and measurement total electrical resistance with the step (step (1), (2) and (3)) of the resistance of definite conductive trace in aforesaid method.For example, step (4) be can carry out earlier, step (1) and (2) carried out then.In one approach, after step (4), target cell suspension is removed from electrod-array.Add reference solution to electrod-array then.In another approach, after step (4), with cell with the whole cracking of some cell pyrolysis liquids, thereby electrodes exposed is in identical reference solution, to carry out the measurements and calculations of step (2) and (3).Then, carry out the resistance of the electrod-array of step (5) when existing to define target cell suspension.

The resistance of conductive trace of determining the electrod-array of substantially the same electrod-array can be used as or not as a part that is used for based on the monitoring cell-substrate impedance of the mensuration of cell.This depends on the analytical procedure of the impedance data (in single or multiple frequency measurements, at a plurality of point in time measurement) of electrod-array.

In some were measured, the resistance or the impedance phase of the electrod-array that people may be when having cell to exist were interested for the relative change of baseline electrical resistance or impedance.In this case, preferably determine the resistance (or impedance) of electrod-array, it is by deducting the resistance of conductive trace from total resistance that records (or impedance) and the resistance of electronic switch is realized.So just need to determine the resistance or the impedance of conductive trace.

In other were measured, the resistance (or impedance) of the electrod-array when people exist cell is arranged was with respect to the absolute change of baseline electrical resistance (or impedance).In this case, directly deduct resistance or impedance under the base line condition that records the resistance that records under can the condition when electrod-array has cell to exist of people or the impedance.The resistance (or impedance) of resistance of electronic switch (or impedance) and conductive trace can be cancelled in such subtraction the contribution of the total resistance that records (impedance) value.So just need not determine the resistance of conductive trace.

In some were measured, people were interested in resistance value calculating cell index or cell number index based on monitoring.Depend on which method and calculate cell index, can need to determine, also can not need determine the resistance of conductive trace.For example, for above-mentioned cell index method of calculation (A), need conductive trace resistance to remove conductive trace resistance to the influence of resistance or impedance phase to mutation analysis.In another example, for above-mentioned cell index method of calculation (F), need not determine conductive trace resistance, in calculating because the influence of conductive trace resistance has been cancelled.

The monitoring of cell-substrate impedance can based on or not based on change with respect to baseline impedance (or resistance).For example, use the effect of estimating the test compounds pair cell based on the mensuration of cell.A kind of method is, by monitoring cell-substrate impedance and the variation of the cell-substrate impedance after determining to add test compounds before the cell and adding cell carry out described mensuration.The monitoring of cell-substrate impedance can be at the single frequency point after dosing or a plurality of Frequency point, and single time point or a plurality of time point carry out.For example, tightly before adding test compounds, measure the impedance of the electrod-array when having cell to exist under single frequency or a plurality of frequency.Add test compounds toward cell then.After adding test compounds, measure the impedance of the electrod-array when having cell to exist under identical single frequency or a plurality of frequency once more.Measurement behind such adding compound can be carried out many time points continuously in the regular or irregular timed interval.The change of cell-substrate impedance can deduct the impedance (resistance and/or reactance) that records before the adding test compounds and come definite or quantitative by the impedance (resistance and/or reactance) that records after the adding test compounds.If measure at a plurality of Frequency points, so for each time point that adds behind the compound, can be based on the change of the calculating of cell-substrate impedance derive single parameter or a plurality of parameter.These parameters are used to quantitatively to add the cellular change behind the compound.This method can further be used to analysis of cells to the reaction of the test compounds of multiple concentration with derivation dose dependent response curve.

D. carry out method based on the The real time measure of cell

The invention provides the mensuration based on cell, it can carry out assessing with on cell proliferation, cell growth, necrocytosis, morphocytology, cytolemma characteristic (as the composition of size, morphology or cytolemma) cell adhesion and cell motility in real time.Therefore this mensuration can be measured for cytotoxic assay, proliferation assay, apoptosis, cell adhesion is measured, cell-stimulating is measured, the anticancer compound effect is measured, receptors ligand in conjunction with and signal transduction is measured, cytoskeleton change measure, cellularstructure changes and measures (including but are not limited to the variation of cytolemma size, morphology or composition), cytodifferentiation or dedifferente mensuration, cell adhesion mensuration, cell-cell interaction mensuration, microorganism and environmental toxin analysis, or the like.The real time measure means the cell behavior or the cell situation of mensuration and can estimate continuously with the regular or random timed interval.The cell behavior of mensuration, cell response or cell situation depend on application, in can arrive several minutes in the several seconds that they occur.Cell response between test period can monitoring substantially continuously in the selected time period.Such as, after adding reagent, culture can be monitored every 5 to 15 minutes, continued several hrs by several days.No matter be that the time length that the timed interval between the impedance monitoring and impedance monitoring are measured can be determined by the experimenter with regular or random timed interval monitoring impedance.

Thereby the impedance measuring based on cell of the present invention has avoided because bias or the wrong estimation that the reaction of pair cell unintentionally that one or more time points of selecting when pair cell is taken a sample or measured cause is carried out.In addition, mensuration does not need the pair cell culture to take a sample or adds reagent, thereby can obtain the result more convenient, quickly, has eliminated the mistake that may introduce in many mensuration simultaneously.

The description of pair cell-substrate monitoring and relative unit, system and using method provides in following document: U.S. Provisional Application number 60/379,749, and it was submitted on July 20th, 2002; U.S. Provisional Application number 60/435,400, it was submitted on December 20th, 2002; U.S. Provisional Application 60/469,572, it was submitted on May 9th, 2003; PCT application number PCT/US03/22557, its name is called " Impedance based devices and methodsfor use in assays ", submits on July 18th, 2003; PCT application number PCT/US03/22537, its name is called " Impedance based apparatuses andmethods for analyzing cells and particles ", submits on July 18th, 2003; Application No. 10/705,447, its name is called " Impedance based devicesand methods for use in assays ", submits on November 10th, 2003; Application No. 10/705,615, its name is called " Impedance based apparatuses andmethods for analyzing cells and particles ", submit on November 10th, 2003, at this with being incorporated among the application in the above document about the disclosure of cell-substrate impedance means, system and using method mode with reference.Meeting of the present invention further discloses the details of cell-substrate impedance monitoring technology.

In brief, for measure cell-substrate or cell-electrode impedance with the technology of the present invention, used cell-substrate impedance monitoring device, it is equipped with the microelectrode array of appropriate geometry on the bottom surface in hole (as the hole of microtiter plate), perhaps have similar design, the side in the hole is equipped with electrode in the bottom surface of a plurality of fluid containers (hole).Cell is imported to the hole of device, cell is contacted with electrode surface and adhere to up.The existence of cell, do not exist or the change of character all can influence electrode sensor surface electronic and ionic and passes through.

Impedance between potential electrode can provide about being present in the important information of the cytobiology state on the transmitter.When the cytobiology state changes, can automatic and real-time measure analog electronics read output signal, and can convert thereof into numerary signal to handle and to analyze.In a system of the present invention, can derive automatically and cell index is provided based on the electrode impedance value that measures.The cell index in resulting given hole has reflected: 1) attached to the cell count on this pore electrod surface, 2) the cell attachment state on this pore electrod surface (closely or loose).Therefore, many more attached to the cell of the similar same kind of the physiological status of electrode surface, cell index is big more.Same, cell adheres to well more at electrode surface (thereby for example cellular invasion gets the bigger bigger contact area that has, perhaps cell and electrode surface adhere to closelyr), and cell index is big more.

In one aspect of the invention, provide the method for measuring of carrying out based on cell, it comprises: cell of the present invention-substrate impedance monitoring system a) is provided; B) in one or more hole with the device of cell import system, at least one of one or more hole comprises electrod-array; And d) monitors the cell-substrate impedance at least one hole that comprises electrod-array and cell.

This method can be used to measure cell state, cell state herein includes but not limited to: in cell attachment that (is included on the electrode) in the substrate or adherence state (for example cellular invasion degree, cell attachment area, the tightness degree of cell attachment, morphocytology), and cell growth or vegetative state; Survivaling cell and/or dead cell number in the hole; The variation of cytoskeleton and organize and enter apoptosis again and/or downright bad cell count.The mensuration based on cell of carrying out with aforesaid method includes but are not limited to cell adhesion, apoptosis, cytodifferentiation, cell proliferation, cell survival, cytotoxicity, morphocytology is surveyed, cell is quantitative, the cell quality control, time-dependent cytotoxic feature, the cell-stimulating or the stimulation of IgE-mediation, the receptor-ligand combination, the cell pathology of virus and bacteriotoxin mediation changes and necrocytosis, detect and quantitative neutralizing antibody, the cytotoxic effect that specific T-is cell-mediated, be used to screen and measure the mensuration of ligand-receptor bonded based on cell.

In the preferred embodiment of the present invention aspect this, cell is joined at least two holes of device, each hole all comprises electrod-array, and at least two impedances that comprise the hole of cell and electrod-array of monitoring.

Used cell can be the cell of isolated primary cell or clone from any species in the mensuration.Cell also can be by the cell after engineered.In certain embodiments, in different holes, add dissimilar cells, and the behavior of these cells is compared.

Can monitor impedance with the regular or random timed interval.Though the present invention does not require, preferably monitor the impedance of 3 or more times point.In an embodiment of above-mentioned mensuration based on cell, with well-regulated timed interval monitoring cell-substrate impedance.Can monitor impedance a frequency or on more than a frequency.For example, in certain embodiments, on each time point of monitoring impedance, in a band frequency scope, monitor impedance.Preferably, at least one the frequency monitoring impedance of about 100MHz intermediary, more preferably arrive at least one frequency monitoring impedance between about 2MHz at about 1Hz at about 100Hz.

Fig. 7 has described the result with method monitoring cell proliferation of the present invention.In this experiment, the H460 cell is imported in the hole of 16 aperture apparatus of cell of the present invention-substrate impedance monitoring system, the initial cell number of different hole inoculations is different.Device is engaged with the device operator's console of system, and described device operator's console places and remains on 37 ℃ of temperature and 5%CO ₂In the incubator for tissue culture of atmosphere.With timed intervals of 15 minutes monitoring cells-substrate impedance 125 hours.Cell index is calculated and is shown at each cell inoculation digital display the function of time to provide cell growth (propagation) curve to each time point by system.Cell growth curve has provided exponential phase of growth and stationary phase with the mapping of logarithm (log) ratio.

Fig. 8 has described the cell attachment of real-time monitoring NIH3T3 cell and the result of diffusion.Seed cells on the cell-substrate impedance monitoring device with poly-L-Lysine or fibronectin bag quilt.To install with the device operator's console engage, described device operator's console places and remains on 37 ℃ of temperature and 5%CO ₂In the incubator for tissue culture of atmosphere.By cell attachment and the cellular invasion of the impedance monitoring in measurement cell-substrate impedance monitoring system on the surface of difference bag quilt.Impedance monitoring in real time in per 3 minutes once continues 3 hours.The cell index of each time point is obtained by the impedance monitoring system-computed and as the function construction of time.

Fig. 9 display monitoring Cos-7 cell is subjected to the result of experiment of morphological change behind Urogastron (EGF) side group.Seed cells in the hole of the present invention's 16 hole monitoring devices, described 16 hole monitoring devices engage with the device operator's console of cell-substrate supervisory system.Described device operator's console is placed in and remains on 37 ℃ and 5%CO ₂Incubator in.With cell serum starvation 8 hours, and then stimulate with 50ng/ml EGF.Control cells is not accepted EGF.Impedance was monitored with 3 minutes the timed interval, continued 2 hours, continued 14 hours with timed interval of 1 hour then.Cell index is obtained by system-computed and as the function construction of time.Because film crumple and Actin muscle dynamic response that EGF causes, so in the cell that EGF-handles, observed the initial saltus step of cell index.The arrow mark has shown the point that adds EGF.

D.1. use the influence that comes the test compounds pair cell based on the mensuration of cell

On the other hand, the invention provides the method for measuring based on cell of the influence of studying one or more test compounds pair cells, it comprises: cell of the present invention-substrate impedance monitoring system a) is provided; B) at least one of cell gatherer comprised in the hole of electrod-array; C) in one or more holes that comprise cell and electrod-array, add at least a test compounds; And d) monitoring add before the compound and add compound after the cell-substrate impedance in one or more hole, the change of its middle impedance can provide the information of cell to the reaction of one or more compounds.

Comprise about the information of cell the reaction of one or more compounds, but be not limited to about following information: in cell attachment that (is included on the electrode) in the substrate or adherence state (for example cellular invasion degree, cell attachment area, the tightness degree of cell attachment, morphocytology), cell growth or vegetative state; Survivaling cell and/or dead cell number in the hole; The variation of cytoskeleton and organize and enter apoptosis again or dead cell count.Also comprise any compound-cell interaction of any variation that causes one or more above-mentioned cell state indicators about the information of cell state.For example, if compound combines with cell surface receptor, and described combination causes that morphocytology changes, and the combination of so this compound and acceptor just can be measured by cell-substrate impedance of monitoring.The mensuration based on cell of carrying out with aforesaid method includes but are not limited to cell adhesion, apoptosis, cytodifferentiation, cell proliferation, cell survival, cytotoxicity, morphocytology is surveyed, cell is quantitative, the cell quality control, time-dependent cytotoxic feature, the cell-stimulating or the stimulation of IgE-mediation, the receptor-ligand combination, the cell pathology of virus and bacteriotoxin mediation changes and necrocytosis, detect and quantitative neutralizing antibody, the cytotoxic effect that specific T-is cell-mediated, be used to screen and measure the mensuration of ligand-receptor bonded based on cell.

Used cell can be the cell of isolated primary cell or clone from any species in the mensuration.Cell by the cell after genetic engineering modified (for example also can be, cell has derived from the genetic modification biology, as derive from " gene knockout " biology, perhaps with certain native gene of overexpression or transgenosis, perhaps handled by antisense molecule or reticent RNA by the normal genetic expression of cell by engineered for cell.) in certain embodiments, in different holes, add dissimilar cells, and more described different cell type is to the behavior of one or more compound reactions.

Test compounds can be any compound, comprise small molecules, macromole, molecular complex, organic molecule, inorganic molecule, biomolecules, as, but be not limited to lipid, steroid, carbohydrate, lipid acid, amino acid, peptide, albumen, nucleic acid or their any combination.Test compounds can be synthetic compound, the compound that exists naturally, the derivative or the like of the compound of existence naturally.

In a preferred method of the invention, cell is joined in the hole of at least two cell-substrate impedance monitoring devices that comprise electrod-array, and test compounds is not accepted at least one hole that comprises electrod-array and comprise cell.Can monitor the control wells of not accepting test compounds, and its impedance data and the impedance data of accepting the hole of compound are compared to determine the influence of one or more test compounds pair cells.

Can monitor impedance with the regular or random timed interval.Preferably monitor the impedance of 3 or more times point, at least one described time point is before adding one or more test compounds.In an embodiment of above-mentioned mensuration based on cell, before adding test compounds,, after this monitor with the rule timed interval at least one time point monitoring cell-substrate impedance.For example before adding compound with one or more time interval measurement impedances, and after adding compound, measure with 2 hours, 1 hour, 30 minutes of rule or 15 minute timed interval.Preferably impedance was measured at 3 or more a plurality of time point with the rule timed interval.In this application, The real time measure means that the permission people use various temporal resolutions to measure cell-substrate impedances, for example with the long timed interval as every 1 hour or measured, or with the short timed interval, every 1 minute or measure every several minutes every 2 hours.

Can be at one or more than a frequency monitoring impedance.For example, in certain preferred aspects, monitor impedance in a band frequency scope at each time point of monitoring impedance.Preferably at about 1Hz at least one the frequency monitoring impedance of about 100MHz intermediary, more preferably arrive at least one frequency monitoring impedance between about 2MHz at about 100Hz.

Preferably, will contain the impedance monitoring data that the impedance monitoring data in the hole of cell and test compounds do not contain the hole of compound with containing cell and compare, but this is not essential to the invention.Impedance measurements that for example also may relatively add the previous or a plurality of time points of compound and the impedance measurements that adds one or more time points behind the compound.This relatively can be directly used in the reaction of assessment cell to compound.Also may calculate cell index (or cell number index) with the resistance value that obtains.The method of calculating cell index (cell number index) is herein and at female case application U.S. Patent application 10/705, disclose in 447, will be wherein be incorporated among the application about the disclosure of cell number index and calculating thereof mode with reference at this.The cell index that the cell index that can be relatively calculated by the impedance measurements in the hole of accepting compound and the impedance measurements by control wells calculate is with the influence of assessment compound pair cell.In addition, the cell index that calculates from the impedance measurements in the hole of one or more time points before also can relatively adding behind the compound cell index that calculates from the impedance measurements in the hole of one or more time points and adding compound is with the influence of assessment compound pair cell.In some preferred embodiment, cell index can be used as Cytotoxic indication.

The present invention adds the mensuration of the compound of different concns in the hole of device.Described method comprises: cell of the present invention-substrate impedance monitoring system a) is provided; B) in the hole with at least two each self-contained electrod-arrays in the cell gatherer; C) in comprising the hole of device of cell and electrod-array, at least one adds the test compounds of first kind of concentration; D) to another comprises the test compounds that adds second kind of concentration in the hole of the device of cell and electrod-array at least; And e) monitoring adds before the compound and the cell in one or more hole-substrate impedance afterwards, and the change of its middle impedance can provide about the information of cell to the compound reaction.

Comprise about the information of cell the reaction of compound, but be not limited to: in cell attachment that (is included on the electrode) in the substrate or adherence state (for example cellular invasion degree, cell attachment area, the tightness degree of cell attachment, morphocytology), cell growth or vegetative state; Survivaling cell and/or dead cell number in the hole; The variation of cytoskeleton and organize and enter apoptosis again or dead cell count.Also comprise any compound-cell interaction of any variation that causes one or more above-mentioned cell state indicators about the information of cell state.For example, if compound combines with cell surface receptor, and described combination causes that morphocytology changes, and the combination of so this compound and acceptor just can be measured by cell-substrate impedance of monitoring.The mensuration based on cell of carrying out with aforesaid method includes but are not limited to cell adhesion, apoptosis, cytodifferentiation, cell proliferation, cell survival, cytotoxicity, morphocytology is surveyed, cell is quantitative, the cell quality control, time-dependent cytotoxic feature, the cell-stimulating or the stimulation of IgE-mediation, the receptor-ligand combination, the cell pathology of virus and bacteriotoxin mediation changes and necrocytosis, detect and quantitative neutralizing antibody, the cytotoxic effect that specific T-is cell-mediated, be used to screen and measure the mensuration of ligand-receptor bonded based on cell.

Cell of using in the mensuration and test compounds can be cell and the compounds described in the mensuration of top detection test compounds effect.In a preferred method of the invention, cell is imported in the hole of at least three each self-contained electrod-arrays of device, and test compounds is not accepted at least one hole that comprises electrod-array and comprise cell.The control wells of not accepting test compounds is monitored, and its impedance data and the impedance data of accepting the hole of compound can be compared to determine the effect of one or more test compounds pair cells.

Impedance monitoring can be as tightly carrying out to described in the mensuration that detects the test compounds effect in the preamble.

Preferably, the data that will obtain from the hole middle impedance monitoring that contains cell and different concns test compounds compare.This contrast can be directly used in the reaction that the assessment cell raises to compound concentration.Also may calculate cell index (or cell number index) with the resistance value that obtains.The method of calculating cell index (cell number index) is at this and at female case application U. S. application No.10/705, disclose in 447, wherein is incorporated in the present patent application about the disclosure of cell index and calculating thereof the mode with reference this general.In some preferred embodiment, cell index can be used as Cytotoxic indication.

The cell index that will calculate from the impedance measurements in the hole of accepting the different concns compound compares, with the effect of assessing compound pair cell.In addition, the cell index that can be relatively calculates from the impedance measurements in the hole of the compound that comprises different concns.The dose-response relationship of relatively can deriving thus.In certain preferred aspects, can calculate the IC50 value of the time-dependent manner of the compound of showing cytotoxicity or suppressing concrete cell response by the cell index value.

In an embodiment of present method, the analysis of pair cell toxic reaction comprises the slope that the cell-cytotoxic reaction of derivation time-dependent manner under given compound concentration changes.In another embodiment of this method, the analysis of real-time cell-cytotoxic reaction is comprised that dependent cell-cytotoxic reaction of time is to the higher derivative of time when deriving given compound concentration.

Application is more than a kind of mensuration based on cell of compound

On the other hand, the invention provides a kind of method for measuring of studying two or more compound pair cell influences based on cell.Described method comprises: cell of the present invention-substrate impedance monitoring system a) is provided; B) in the hole with at least two each self-contained electrod-arrays of cell gatherer; C) in comprising the hole of device of cell and electrod-array, at least one adds first kind of test compounds: d) to another comprises and adds second kind of test compounds in the hole of the device of cell and electrod-array at least; And e) monitor at least one hole that comprises cell and first kind of compound and at least one comprises the cell-substrate impedance in the hole of cell and second kind of compound, the change of its middle impedance can provide the information of cell to first kind and second kind compound reaction.

Preferably, cell is compared to observe from the similar of the reaction of two kinds of compounds or in various degree the reaction of the first kind of compound and the time-dependent manner of second kind of compound.In a kind of embodiment preferred of present method, the cell-cytotoxic reaction of time-dependent manner is compared.

Cell of using in the mensuration and test compounds can be cell and the compounds described in the mensuration of top detection test compounds effect.In a preferred method of the invention, cell is imported in the hole of at least three each self-contained electrod-arrays of device, and wherein compound is not accepted at least one hole that comprises electrod-array and comprise cell.The control wells of not accepting test compounds is monitored, and its impedance data can compare with the data in the hole of accepting compound to measure the effect of test compounds pair cell.

Preferably, the impedance monitoring data that will contain the hole of different test compounds compare.In a kind of embodiment preferred, can under multiple dose concentration, carry out impedance monitoring to first kind of compound.In another embodiment, can under multiple dose concentration, measure the cell response of the time-dependent manner of second kind of compound.In another embodiment, can under multiple dose concentration, measure the cell response of the time-dependent manner of first kind of compound and second kind of compound.

In another embodiment of above method, first kind of compound is the compound with known cytotoxic effect mechanism, and second kind of compound is the compound with unknown cytotoxic effect mechanism.If the cell-cytotoxic reaction of the time-dependent manner of second kind of compound is similar to first kind of compound, second kind of compound may have similar cytotoxic effect mechanism to first kind of compound so.

The cell-cytotoxic reaction that several different methods can be used for comparative compound.Can choose wantonly by the resistance value that obtains and calculate cell index (or cell number index).In an embodiment of method described above, based on the cell index value, derive the IC50 of the time-dependent manner of compound, and pass through the cell-cytotoxic reaction of the IC50 curve ratio of their time-dependent manner of comparison than them.If the IC50 curve of two kinds of compounds has similar time-dependent manner trend, then these two kinds of compounds have the mechanism of similar inducing cytotoxic effect.In another embodiment of described method, directly compare the cell-cytotoxic reaction of the time-dependent manner of two kinds of compounds, wherein two kinds of compound concentrations are identical or different.The variation slope (it equals the first order derivative of reaction pair time) of the reaction that can measure by analysis and the slope to time-dependent manner of two kinds of compounds relatively carry out the direct comparison of the cell-cytotoxic reaction of time-dependent manner.In another approach, the more higher derivative of dependent born of the same parents' poison reaction pair time of analysis time.The comparison of this higher derivative can provide more information about the Cytotoxic mechanism of compound inductive.

Application is more than a kind of mensuration based on cell of cell type

On the other hand, the invention provides a kind of method of compound to the cytotoxicity feature of various kinds of cell type that be used to describe, it comprises: cell of the present invention-substrate impedance monitoring system a) is provided; B) with at least one comprises in the hole of electrod-array in first type the cell gatherer; C) with another comprises in the electrod-array hole at least in second type the cell gatherer; D) test compounds is joined at least one hole that comprises first type cell and neutralize that at least one comprises in second type the hole of cell; And e) monitor cell-substrate impedance that the hole of at least one cell that contains first type and test compounds and at least one contain the hole of second type cell and test compounds, the change of its middle impedance can provide cell to first kind and second kind of information that compound reacts.

Preferably, relatively the reaction of first kind and the time-dependent manner of second type cell with the different degree of reacting phase Sihe of the cell of two types of comparisons.In a preferred embodiment of present method, compared the cell-cytotoxic reaction of time-dependent manner.

Used cell type can be the cell of isolated primary cell or clone from any species in the mensuration.In certain preferred aspects, different cell types is the same cell type that derives from Different Individual, and therefore different genotype is arranged.One or more cell types by the cell type after the genetically engineered modification (for example can be, cell has derived from the genetic modification biology, as from " gene knockout " biology, perhaps with certain native gene of overexpression or transgenosis, perhaps handled by antisense molecule or reticent RNA by the normal genetic expression of cell by engineered for cell.) in these cases, genetically modified cell can compare with control cells.In certain embodiments, in different holes, add the reaction of three kinds or more kinds of different cell type and more described three kinds or more kinds of different cell type to one or more compounds.

One or more test compounds of using in the mensuration can be the compounds described in the mensuration of top detection test compounds effect.In a preferred method of the invention, cell is imported in the hole of at least three each self-contained electrod-arrays of device, and wherein test compounds is not accepted at least one hole that comprises electrod-array and comprise cell.The control wells of not accepting compound is monitored, and its impedance data can compare with the data in the hole of accepting compound, to determine the effect of test compounds pair cell.In the preferred embodiment of the invention, at the cell type of each test, all carried out a contrast, wherein test compounds is not accepted in contrast.

Preferably, the data of impedance monitoring that relatively comprise the hole of different cell types.In a kind of embodiment preferred, the different cell types of the multiple dose concentration that is exposed to compound are carried out impedance monitoring.In some embodiments, can use the multiple compound of various kinds of cell type detection.In some embodiments, can use the multiple compound of the multiple concentration of various kinds of cell type detection.

In an embodiment of method, cell-cytotoxic reaction analysis in real time comprises the IC50 value of derivation compound to the time-dependent manner of various kinds of cell type.In another embodiment of method, cell-cytotoxic reaction analysis in real time comprises the slope that the cell-cytotoxic reaction of derivation time-dependent manner under given compound concentration changes.In another embodiment of method, cell-cytotoxic reaction analysis in real time can comprise derivation when given compound concentration dependent cell-cytotoxic reaction of time to the higher derivative of time.

In an embodiment of method, born of the same parents' poison response analysis comprises the IC50 value of derivation compound to the time-dependent manner of various kinds of cell type in real time.In another embodiment, above method can be used for describing the cytotoxicity characteristic of multiple compound to the various kinds of cell type.

In another embodiment of method, born of the same parents' poison response analysis comprises the slope that the cell-cytotoxic reaction of derivation time-dependent manner under given compound concentration changes in real time.In another embodiment of method, cell-cytotoxic reaction analysis in real time can comprise derivation when given compound concentration dependent cell-cytotoxic reaction of time to the higher derivative of time.

Having provided some in the mode of example with reference to the accompanying drawings uses cell of the present invention-substrate impedance system to carry out the example of compound determination.In these examples, the method for calculation of cell index are the same with the cell index method of calculation (A) of description in the application C part.In some accompanying drawing of the application, normalized cell index is drawn.The normalization cell index of point is to calculate by the cell index that the cell index of this time point was put divided by the reference time in preset time.So the reference time normalization cell index of point is 1.

Illustrated as the application, if the condition of adhering to of cell is constant or change very for a short time in using the whole mensuration process of impedance monitoring, cell index is big more so, and the cell count in the hole is just many more.Cell index descends and to show that some cells break away from or because compound effects and dying from substrate surface.The cell index increase shows have more that many cells are attached to substrate surface, also shows the increase of total cell count.

Figure 10 shown expression with the H460 cell of different concns anticarcinogen taxol treatment time-dependent cell exponential curve.In this experiment, the H460 cell is imported in the hole of 16x cell-substrate impedance monitoring device.Device is placed on the device operator's console, and described device operator's console is positioned at and keeps 37 ℃ and 5%CO ₂In the incubator of condition.Culturing cell, and handle with the taxol of different concns in its exponential phase of growth.After processing, by with cell-substrate impedance monitoring system every 15 minutes real-time monitoring cell-substrate impedance 50 hours, monitor the dynamic response of cell to the various dose taxol.The cell index of cell-each time point of substrate impedance monitoring system-computed, and with the function construction of described cell index as the time.For concentration at 67nM to the taxol between the 500nM, the cell index of H460 cell descends gradually after adding compound.But behind the adding compound about 15 hours to 20 hours the time cell index all drop to lower-most point in the time that depends on compound concentration.After this point, the cell index in these holes rises gradually.Cell index with the 33nM compound concentration showed almost constant value at most in about 15 hours after adding compound.Add compound after 15 hours cell index rise gradually.

Figure 11 shown H460 cell that expression is handled with anticarcinogen AC101103 time-dependent cell exponential curve.The H460 cell is imported in the hole of 16X cell-substrate impedance monitoring device.Device is placed on the device operator's console, and described device operator's console is positioned at and keeps 37 ℃ and 5%CO ₂In the incubator of condition.Culturing cell, and in its exponential phase of growth the AC101103 with different concns handles.Handle the back and in cell-substrate impedance monitoring system, pass through to measure about 20 hours of impedance in real time, monitor the dynamic response of cell the AC101103 of various dose every 30 minutes.

Clearly, among the time-dependent manner cell index among Figure 11 and Figure 10 a great difference arranged.For concentration is the compound of 3.125 mcg/ml, 6.25 mcg/ml and 12.5 mcg/ml, and cell index showed respectively about 5 hours, about 15 hours and greater than 20 hours almost constant value.For concentration is the compound of 3.125 mcg/ml and 6.25 mcg/ml, and cell index begins to rise after adding compound about 5 hours and about 15 hours.For concentration is the compound of 25 mcg/ml, and behind the adding compound, cell index takes place progressively and slowly to descend.For concentration is the compound of 50 mcg/ml, exists cell index to keep almost about 10 hours time period of constant, and the stable decline of cell index afterwards.

Figure 12 shows the dynamic drug reaction of the A549 cell of handling with Zorubicin.In every hole of 16X device, inoculate 10,000 A549 cells.Device is placed on the device operator's console, and described device operator's console is positioned at and keeps 37 ℃ and 5%CO ₂In the incubator of condition.Before handling by the impedance of monitoring the rule timed interval, monitoring cell attachment and cell growth in real time on cell-substrate impedance system.When cell grew into exponential phase of growth, Xiang Kongzhong added the Zorubicin of different concns.The solvent that is used for dissolved substance of equal volume is joined some hole in contrast.As shown in the drawing, real time record time and the dependent cell response of drug dose (being calculated as cell index) in cell-substrate impedance monitoring system.

E. monitor the cell-stimulating of IgE-mediation with cell-substrate impedance measuring

The present invention comprises the method for monitoring by the cell-substrate impedance of IgE stimulated cells equally.Present method is based on combined real-time quantitative that the cytoskeleton that causes changes with IgE-Fc (ε) the RI mixture of reactive cell surface by antigen, and wherein the example of reactive cell is, but is not limited to, mastocyte.The electronics that provides is measured and is depended on cytoskeleton kinetics, it be mastocyte to antigenic inner reaction, also be the major portion of mastocyte activation procedure simultaneously, this mensurations does not need to set up report clone or uses other mensuration reagent simultaneously.In addition, be real-time owing to measure, so no matter be that the mastocyte activatory reaction to the IgE-mediation of antigen dependency or antigen dependent/non-dependent can be monitored with same measured.

The mensuration of monitoring IgE stimulated cells described herein-substrate impedance can be used any cell-substrate impedance measurement device, includes but not limited to that female case U.S. Patent application 10/705,447 neutralization is described herein.Available cell-substrate impedance means is meant the surface that is suitable for cell in the inventive method, has cell can settle down device with interactional electrode simultaneously.The measurement of electrode impedance can reflect the state of cell, as cell count, morphocytology or cell adhesion.In preferred embodiments, available cell-substrate impedance means comprises substrate in the inventive method, described substrate comprises one or more electrod-arrays in its surface, each of wherein one or more electrod-arrays all contains two electrodes or electrode structure, and wherein said two electrodes or electrode structure have essentially identical surface-area.In the use, cell-substrate impedance means used in the inventive method can detect the impedance variations of one or more frequencies, and described impedance variations is because cell count, cell size, morphocytology, the variation of quality that is attached to the cell of substrate and is attached to the cell of substrate cause.

The cell of using in the inventive method-substrate impedance means preferably comprises at least one electrod-array that centers on device and the cell of monitoring is provided the fluid container of fluid-tight container.In preferred embodiments, device comprises the container of at least two arrays and two well format, and wherein the array of each device is all centered on by a hole.More preferably, the device that is used for the described screening method of the application comprises at least 8 holes (for example, comprising the device in 16 holes or 96 holes), and wherein most of hole all comprises electrod-array, thereby described mensuration can be carried out in the high-throughput mode.Can choose wantonly and in a plurality of Multiple-Aperture Devices, measure cell (for example, be connected to identical electric impedance analyzer, engage, perhaps connect independent electric impedance analyzer) simultaneously, to improve high throughput ability with the device operator's console that can engage more than a Multiple-Aperture Device.

The system that is used for monitoring cell-substrate impedance comprises that one or more Multiple-Aperture Devices, electric impedance analyzer and device operator's console are described herein and is preferred, but is not essential in the inventive method.

Be used for cell that IgE stimulate to measure and can be separating biological or from any cell of clone from one or more.Cell can be the cell of separating from one or more biological blood or other tissue.Cell is mammalian cell preferably.Cell is mastocyte, basophilic granulocyte or eosinophilic granulocyte preferably, but this is not required in this invention.Cell can be the cell that the expression IgE of genetic engineering modified mistake reacts any kind of path composition.Cell can be by engineered to express or overexpression IgE reaction path composition inadequately.Cell also can be by engineered with the dominant of expressing IgE reaction path composition or the form of other changes, or eliminates the expression (for example use that homologous recombination knocks out, antisense or reticent RNA technology) of IgE reaction path composition.Here said " IgE reaction path composition " comprises that also suspecting is the molecule of IgE reaction path composition.

In the present invention's some embodiments aspect some, the RBL-2H3 rat hypertrophy cell can be used to carry out the research of the signal conduction mechanism of IgE-mediation, can estimate the influence of different inhibitor to the signal transduction pathway that causes mastocyte threshing and medium and discharge simultaneously.RBL-2H3 have much in cultivation, keep and expansion aspect advantage, and use this clone to study the mastocyte activation of IgE-mediation as model system in the lot of documents.Yet method and system of the present invention can be easy to be revised as and be applicable to other interested cell academicly or in the pharmacy.These cells include, but are not limited to the mastocyte and the basophilic granulocyte of mouse bone marrow cells mastocyte, human lung mast cell, human skin mastocyte and other mammalian species.

Following chapters and sections are the mensuration of public use impedance monitoring device, and it is crosslinked that described mensuration can be measured IgE-Fc (ε) RI of the IgE combination that causes mastocyte morphological change and threshing and antigen mediation.Mensuration can be carried out with high-throughput in real time, handles without any need for other reagent or cell simultaneously.

E.1. monitor the method that stimulates the cell-substrate impedance variations that causes by IgE

The cytositimulation that has shown the IgE-mediation can cause the violent morphological change of cell (as mastocyte).For example, the RBL-2H3 mastocyte becomes flat and inoblast morphology from fusiform morphology.Cell of the present invention-substrate impedance monitoring device can be used to survey the variation of the cell-substrate interaction that cell shape changes and caused by the reactive cytositimulation of IgE-mediation.

Method comprises: the device that is used for cell-substrate impedance monitoring that comprises at least one hole a) is provided, and at least one electrod-array is contained in described hole; B) connect described device and electric impedance analyzer; C) transfered cell in the hole of one or more devices that comprise electrod-array; D) in one or more holes that comprise cell, add IgE; E) in one or more holes that comprise cell, add at least a antigen, at least a allergen or at least a IgE linking agent; And e) the one or more cell-substrate impedances that comprise the hole of cell of monitoring.

In the method for the invention, the device that is used for cell-substrate impedance monitoring is the device that comprises substrate, and described substrate contains one or more electrod-arrays on its surface, and each electrod-array is all surrounded by the fluid container of well format.Each of one or more electrod-arrays all contains two electrodes or electrode structure, wherein said two electrodes or electrode structure have essentially identical surface-area, wherein, when being connected in electric impedance analyzer, described device can detect the impedance variations of one or more frequencies, and described impedance variations is because cell count, cell size, morphocytology, the variation of quality that is attached to the cell of substrate or is attached to the cell of substrate cause.

Cell can be any to the reaction of IgE stimulation or the possible interesting cell of reaction.Preferred cell is mastocyte, eosinophilic granulocyte, basophilic granulocyte or genetically modified cell.In some preferred embodiment, cell is the Mammals source.

IgE can be before adding antigen, allergen or IgE linking agent, add afterwards or simultaneously.The concentration that joins the IgE in the hole can be from about 10ng/ml to about 1 μ g/ml.Preferably, do not accept IgE in the hole of a device that comprises electrod-array and cell so that at least one control wells to be provided, but this not essential to the invention.

Antigen, allergen or linking agent can be antigen, allergen or linking agents known or that suspect.The concentration that joins in the hole can be from about 1ng/ml to about 1 μ g/ml.Can accept antigen, allergen or the linking agent of different concns in the different holes.Perhaps for the multiple purpose, antigen, allergen or the linking agent of same concentrations can be accepted in a plurality of holes.Preferably, do not accept antigen, allergen or linking agent in the hole of a device that comprises electrod-array and cell so that at least one control wells to be provided, but this not essential to the invention.

Present method can be by using the morphological change that cell-substrate resistance technique is estimated and quantitative mastocyte stimulates IgE and antigen cross-linking to cause owing to IgE.Preferably monitor impedances in two or more stages of experiment.For example, in one embodiment, before Xiang Kongzhong adds IgE and monitor impedance afterwards.In another embodiment, before Xiang Kongzhong adds antigen, allergen or linking agent and monitor impedance afterwards.In another embodiment, impedance also can Xiang Kongzhong add IgE and antigen, allergen or linking agent before and monitor afterwards.Impedance also can be monitored after Xiang Kongzhong adds IgE and before Xiang Kongzhong adds antigen, allergen or linking agent, and monitors after Xiang Kongzhong adds antigen, allergen or linking agent.In some preferred embodiments, impedance is monitored before Xiang Kongzhong adds IgE, after Xiang Kongzhong adds IgE and before Xiang Kongzhong adds antigen, allergen or linking agent, monitor, and after Xiang Kongzhong adds antigen, allergen or linking agent, monitor.

Can monitor the impedance of two or more time points.Preferably, the impedance of at least three time points of monitoring.The impedance of at least three time points in two or more stages of preferably, monitoring mensuration.The impedance of each time point can or be monitored more than a frequency at one.The impedance monitoring data that obtain can be used for estimating the morphological change that is stimulated the cell that causes by IgE.For example can compare and add IgE in each hole, and/or add before antigen, allergen or the linking agent and impedance afterwards before with afterwards.Can relatively accept the hole of IgE and not accept the impedance data of the control wells of IgE.Can relatively accept the hole of antigen, allergen or linking agent and not accept the impedance data of the control wells of antigen, allergen or linking agent.Impedance data can be used for calculating cell index, and it can be used to compare.

Be a kind of exemplary rules below:

(1) mastocyte of interpolation predetermined number in the hole of the 16-hole that is attached to electric impedance analyzer or 96-porocyte-substrate impedance measurement device.

(2) make cell attachment and growing 16-20 hour, write down the IgE of 10ng/ml simultaneously, and continue to use as mentioned above cell-substrate impedance monitoring system log (SYSLOG) cell response to 1 μ g/ml final concentration.

(3) IgE stimulated the back 16-20 hour, changed substratum into serum free medium.Make cellular-restoring after 30 minutes, stimulate with antigen then.Continuation is monitored cell to antigenic reaction in the impedance monitoring system.

E.2. screening can be regulated the method for cell to the compound of IgE irritant reaction

The present invention also comprises the method for one or more cells of one or more compounds affect to the effect of the irritant reaction of IgE-mediation of estimating.Described method comprises: the device that is used for cell-substrate impedance monitoring that comprises at least one hole a) is provided, and electrod-array is contained in described hole; B) connect described device and electric impedance analyzer; C) transfered cell in the hole of one or more devices that comprise electrod-array; D) in one or more holes that comprise cell one or more, add at least a test compounds; E) in one or more holes that comprise cell and at least a test compounds, add IgE; E) in one or more holes that comprise cell and at least a test compounds, add at least a antigen, at least a allergen or at least a IgE linking agent; And e) the one or more cell-substrate impedances that comprise the hole of cell and at least a test compounds of monitoring.

In the method for the invention, the device that is used for cell-substrate impedance monitoring is the device that comprises substrate, and described substrate contains one or more electrod-arrays on its surface, and each electrod-array is all surrounded by the fluid container of well format.Each of one or more electrod-arrays all contains two electrodes or electrode structure, and described two electrodes or electrode structure have essentially identical surface-area.When being connected in electric impedance analyzer, described device can detect the impedance variations of one or more frequencies, and described impedance variations is because cell count, cell size, morphocytology, the variation of quality that is attached to the cell of substrate or is attached to the cell of substrate cause.

Preferably, the device that is used for the inventive method is the part of cell described herein-substrate impedance monitoring system.

Cell can be any cell that detectable reaction is arranged that IgE is stimulated.Preferred cell is mastocyte, eosinophilic granulocyte, basophilic granulocyte or genetically modified cell.In preferred embodiments, cell is the Mammals source.

IgE can be before adding antigen, allergen or IgE linking agent, add afterwards or simultaneously.The concentration that adds the IgE in the hand-hole can be from about 10ng/ml to about 1 μ g/ml.Randomly, do not accept IgE so that control wells to be provided in the hole of a device that comprises electrod-array and cell.

Antigen can be antigen, allergen or linking agent known or that suspect.The concentration that joins in the hole can be from about 1ng/ml to about 1 μ g/ml.Can accept antigen, allergen or the linking agent of different concns in the different holes.Perhaps for the multiple purpose, antigen, allergen or the linking agent of same concentrations can be accepted in a plurality of holes.Randomly, comprise in the hole of a device of electrod-array and cell and do not accept antigen, allergen or linking agent so that control wells to be provided.

Test compounds can be any compound, comprise small molecules, macromole, molecular complex, organic molecule, inorganic molecule, biomolecules, as, but be not limited to lipid, steroid, carbohydrate, lipid acid, amino acid, peptide, albumen, nucleic acid or their any combination.Test compounds can be synthetic compound, the compound that exists naturally, the derivative or the like of the compound of existence naturally.The structure of test compounds can be known or unknown.In an experiment, can measure one or more compounds.Multiple compound can make up and detect.Use method of the present invention can measure the different concns of given compound.

The appearance of combinatorial chemistry makes it possible to generate big and various compound library, and it can be used to screen the mastocyte activation inhibitor of potential IgE-mediation.Can in cell of the present invention-substrate impedance monitoring system, measure with high-throughout mode, with the SCREENED COMPOUND storehouse to find this potential inhibitor.

Test compounds can be the compound that conducts with the signal that disturbs the IgE-mediation by Fc (ε) the RI acceptor of expressing on mastocyte and the basophil cellular surface.For example, test compounds can be the antibody at IgE, or contains the recombinant protein of all or part of Fc (ε) RI antigen binding domains, can detect its specific combination IgE and stop IgE and the interactional ability of endogenous Fc (ε) RI.Test compounds can also be the material standed for of micromolecular inhibitor compound, and it can be incorporated into the IgE-binding pocket of Fc (ε) RI and blocks Fc (ε) RI and IgE interaction of molecules.Test compounds also can be siRNA, can detect them and reduce the ability of Fc (ε) RI at the mastocyte surface expression.

Also can in born of the same parents, suppress the compound that IgE stimulates the cell response that causes to identify by the filler test compound.For example, can the filler test compound stimulate the kinases that works in the cell signaling that causes or the inhibitor of lipase to identify at IgE.Method for example of the present invention can be used for screening the compound of arrestin kinase c, SRC, Syk or PLC (γ).

Preferably, compound was added in the hole before adding IgE, but also can and add before the antigen after adding IgE, or added after adding IgE and antigen.In the embodiment that IgE and antigen add simultaneously, test compounds can after add but preferably adding after adding IgE and antigen.

Present method can be estimated the reaction of the compound pair cell that causes with quantitative IgE by using cell-substrate resistance technique, for example, but is not limited to mastocyte.Impedance is preferably monitored in two or more experimental phases.Depend on testing sequence, impedance is preferably monitored add IgE in one or hole after, and preferably before in one or hole, adding IgE and monitor afterwards.Preferably after adding test compounds, add IgE and monitor impedance before and afterwards.Can also after add test compounds and IgE, add antigen and monitor impedance before and afterwards.In some preferred embodiments, can after Xiang Kongzhong adds test compounds and before adding IgE, monitor impedance; After Xiang Kongzhong adds IgE and before adding antigen, allergen or linking agent, monitor impedance; With after Xiang Kongzhong adds antigen, allergen or linking agent, monitor impedance.

The impedance of preferred two or more time points of monitoring.Preferably, the impedance of at least three time points of monitoring.Preferably, the impedance of at least three time points in two or more stages during monitoring is measured.The impedance of each time point can or be monitored more than a frequency at one.The impedance monitoring data that obtain can be used for the evaluation test compound to stimulated the influence of the morphocytology variation that causes by IgE.For example can compare in each hole and to add before the compound and afterwards, and/or add before the IgE and afterwards, and/or add before antigen, allergen or the linking agent and impedance afterwards.Can relatively accept the hole of test compounds and not accept the impedance data of the control wells of test compounds.Impedance data can be used for calculating cell index, and it can be used for comparing.Such comparison can be used for identifying and can suppress cell (as mastocyte) irritates reaction to IgE compound.For example, can calculate one or more from the resistance value that records and suppress the IC50 of cell the compound of the reaction of IgE stimulation.

Be a kind of exemplary rules that are used for screening compound below:

(1) mastocyte of adding predetermined amount in the hole of 16-hole or 96-porocyte-substrate impedance measurement device, described device is the part of cell impedance monitoring system.

103. adhering to and grow with impedance monitoring system monitoring cell.

104. one or more potential target inhibitor (anti--IgE antibody, reorganization Fc (ε) RI antigen binding domains fragment or micromolecular compound) that make cell and progressive concentration are one period regular time of incubation in advance.

105. use the specific IgE of used clone, and using system, electronic monitoring stimulates the instantaneous morphocytology that causes to change by IgE.The peak that writes down under the inhibitor concentration condition that increases progressively by cell relatively reacts estimates inhibition degree and definite IC-50.

In another example of the inventive method, mastocyte or other target cell are dispensed in 96 aperture apparatus with liquid treatment platform or multichannel pipettor.Adhering to and grow with as above method monitoring cell.In order to estimate the influence of inhibitor to the signal conduction of the mediation of IgE only, before using IgE with the inhibitor preincubation together of cell and single concentration or multiple concentration.Exist under the drug condition cell to the reaction of IgE with the monitoring of as above method then.Selectively, if estimate the influence of medicine to the antigen cross-linking of IgE-Fc (ε) RI mixture, can be tightly before adding antigen as mentioned above with cell and inhibitor preincubation.

E.3. use cell-substrate resistance technique to confirm to participate in the enzyme and the proteic method of signal transduction pathway, described signal transduction pathway is caused in conjunction with high-affinity Fc (ε) RI acceptor by antigen in the crosslinked existence of IgE-or when not existing.

The intracellular signal conduction path that is stimulated in conjunction with Fc (ε) RI by IgE relates to the activation of key enzyme, such as but not limited to, kinases, Phosphoric acid esterase and Phospholipid hydrolase.The downstream media that these cells react IgE is the potential target in the drug discovery.Yet before the potential inhibitor of these target proteins of screening and enzyme, must confirm the signal conduction that they disturb IgE-to mediate.But DNA or one or more targets of the target protein dominant form that this can suspect by encoding via the mode of transfection, electroporation or virus infection and the siRNA that reduces these protein expressions introduce reactive cell and realize.In addition, antisense reagent also can be used for reducing or removing the expression of the target of suspection.

Described method comprises: the device that is used for cell-substrate impedance monitoring that comprises at least one hole a) is provided, and electrod-array is contained in described hole; B) connect described device and electric impedance analyzer; C) transfered cell in the hole of one or more devices that comprise electrod-array, wherein said cell is genetic modification, changing the function of the target molecule of suspecting, or reduces or eliminates its expression; D) in one or more holes that comprise cell, add IgE; E) in one or more holes that comprise cell, add at least a antigen, at least a allergen or at least a IgE linking agent; And e) the one or more cell-substrate impedances that comprise the hole of cell of monitoring.

Measuring the device of cell-substrate impedance is describing in female case U.S. Patent application 10/705,447 He among the application.The cell of using in the inventive method-substrate impedance means preferably comprises at least one electrod-array that centers on device and the cell of monitoring is provided the fluid container of fluid-tight container.In preferred embodiments, device comprises the container of at least two arrays and two well format, and wherein the array of each device is all centered on by a hole.More preferably, the device that is used for the described screening method of the application comprises at least 8 holes (for example, comprising the device in 16 holes or 96 holes), and wherein most of hole all comprises electrod-array, thereby described mensuration can be carried out in the high-throughput mode.Can choose wantonly and in a plurality of Multiple-Aperture Devices, measure cell (for example, be connected to identical electric impedance analyzer, engage, perhaps connect independent electric impedance analyzer) simultaneously, to improve high throughput ability with the device operator's console that can engage more than a Multiple-Aperture Device.

Genetically modified cell can be the cell that IgE is stimulated any kind that responds.Preferably, use mastocyte, as the RBL-2H3 rat hypertrophy cell.The method of genetically modified cell is well-known in the art, and the expression construct that (for example, the proteic dominant form) of form that comprises the change of the target protein of import to instruct suspecting expressed, import and to instruct expression vector that can reduce or remove the sense-rna expression that target protein expresses and the expression vector that importing can be instructed the reticent rna expression that can reduce or remove the target protein expression.Also can before or after cell is added device of the present invention, antisense or gene silencing reagent be joined cultured cells.

Preferably, also measure the control cells of not carrying out genetic modification with genetically modified cell.

The antigen that is used to measure can be antigen, allergen or linking agent.The concentration that joins in the hole can be from about 1ng/ml to about 1 μ g/ml.

The impedance of preferred two or more time points of monitoring.Preferably, the impedance of at least three time points of monitoring.Preferably, the impedance of at least three time points in two or more stages during monitoring is measured.The impedance of each time point can or be monitored more than a frequency at one.For example can compare and add IgE in each hole, and/or add before the antigen and resistance value afterwards before with afterwards.The hole and the impedance data that comprises the hole of the cell that does not have genetic modification that can relatively comprise genetically modified cell.Impedance data can be used for calculating cell index, and it can be used to compare.This comparison can be identified influences the gene of cell to the reaction of IgE stimulation.

The impedance monitoring data that obtain can be used for estimating genetic manipulation (as expression knock down (knockdown), express knock out or dominant is expressed) pair cell is subjected to IgE to stimulate the influence of back morphological change.Influence cell and be subjected to IgE to stimulate the evaluation of the genetic manipulation of back morphological change can be used for identifying the gene that in the IgE reaction, works, thus and identified gene product.

Below be an example of the present invention's mensuration of confirming target:

(1) provides the cell of the siRNA that contains DNA that the dominant that is useful on target protein expresses or this target protein of target.

(2) with cell transfer in the device of impedance monitoring system, and monitor adhering to and growing of cell as mentioned above.In addition, can in the hole of device, directly add gene and disturb reagent to cell.

(3) cell is stimulated with IgE having antigen or do not exist under the antigenic condition, and use the cell response of impedance monitoring system log (SYSLOG) as mentioned before.This genetic constructs or reagent disturb the ability of the reaction that cell when having antigen irritates IgE make it possible to construct or reagent place at Molecular Identification be the target of potential drug discovery.

E.4. screening decision or influence the crosslinked and method of mastocyte activatory genetic marker subsequently of high-affinity Fc (ε) RI

Known host's genetic background has determined similar antigenic allergic type and intensity.The present invention comprises the method for the cell of comparison different genotype to the reaction of the IgE stimulation of antigen mediation equally.Any of the reaction that the cell of this different genotype stimulates the IgE of antigen mediation and many genetic markers of different genotype cell demonstration is relevant.

Described method comprises: the device that is used for cell-substrate impedance monitoring that comprises at least one hole a) is provided, and electrod-array is contained in described hole; B) connect described device and electric impedance analyzer; C) in the hole of at least one device, import first kind of genotypic cell; D) in the hole of another device at least, import second kind of genotypic cell; E) at least one hole that comprises first kind of genotypic cell with comprise at least one hole of second kind of genotypic cell and add IgE; E) to the one or more holes that comprise first kind of genotypic cell with comprise and add at least a antigen in one or more holes of second kind of genotypic cell; E) monitoring comprises one or more holes and the cell-substrate impedance that comprises second kind of one or more hole of genotypic cell of first kind of genotypic cell; And f) relatively comprises the cell-substrate resistance value in one or more holes of first kind of genotypic cell and comprise the cell-substrate resistance value in one or more holes of second kind of genotypic cell.

Preferably, present method further comprises: make at least a genetic marker and be associated from monitoring cell-substrate resistance value that described first kind of genotypic cell and described second kind of genotypic cell obtain.

The cell of using in the inventive method can be any cell to the IgE reaction, but preferably from the individual isolated mastocyte of difference.Except impedance monitoring, can also be to carrying out the genetic marker analysis from the isolating mastocyte of individuality.Genetic marker includes, but not limited to SNPs, sudden change, alternative RNA splicing variant, gene expression profile and protein expression profiles.With the method for carrying out the genetic marker analysis from individual isolated cells is well-known in the art.

In the mensuration process, IgE can be before adding antigen, add afterwards or simultaneously.The concentration that adds the IgE in the hand-hole can be from about 10ng/ml to about 1 μ g/ml.Randomly, do not accept IgE so that control wells to be provided in the hole of a device that comprises electrod-array and cell.

The antigen that is used for measuring can be antigen, allergen or linking agent.The concentration that joins in the hole can be from about 1ng/ml to about 1 μ g/ml.Antigen can be selected because they are identified allergenic characteristic tendency in group member.

Present method can make genetic marker related with the antigen-IgE reacting phase of cell by using cell-substrate resistance technique, and described cell for example but is not limited to mastocyte.Impedance is preferably monitored in two or more experimental phases.Depend on testing sequence, impedance is preferably monitored add IgE in one or hole after, and preferably before in one or hole, adding IgE and monitor afterwards.Preferably after adding test compounds, add IgE and monitor impedance before and afterwards.Can also after add test compounds and IgE, add antigen and monitor impedance before and afterwards.In some preferred embodiments, can after Xiang Kongzhong adds test compounds and before adding IgE, monitor impedance; After Xiang Kongzhong adds IgE and before adding antigen, allergen or linking agent, monitor impedance; With after Xiang Kongzhong adds antigen, allergen or linking agent, monitor impedance.

The impedance of preferred two or more time points of monitoring.Preferably, the impedance of at least three time points of monitoring.Preferably, the impedance of at least three time points in two or more stages during monitoring is measured.The impedance of each time point can or be monitored more than a frequency at one.For example can compare and add IgE in each hole, and/or add before the antigen and resistance value afterwards before with afterwards.The hole and the impedance data that comprises the hole of the cell that does not have genetic modification that can relatively comprise genetically modified cell.Impedance data can be used for calculating cell index, and it can be used to compare.This comparison can be used to identify the genetic marker that influences the reaction that cell stimulates IgE.

The data that obtain from impedance monitoring can be used for making the genetic marker that is assigned to cell related to the reacting phase that IgE irritates with cell.Identify that the genetic marker that the reactivity of IgE is associated with cell can be used for developing and regulate the therapeutic strategy that IgE reacts.

Exemplary mensuration is as follows:

(1) from two or more allergic individual mastocyte that separates can take place.

(2) determine individual target genetic marker.

(3) cultivate these mastocyte (or in the presence of specific differentiation factor, allow differentiation of stem cells be mastocyte).

(4) with cell transfer in the impedance monitoring device and monitor cell growth.

(5) add IgE (people's), add or do not add allergen simultaneously.

(6) impedance response of quantitative mastocyte.

(7) make genetic marker related with the mastocyte reacting phase.

Can carry out genetic analysis with standard RFLP well known in the art and snp analysis, RNA and protein expression profiles and RNA montage detection method.Preferably, use and measure in the high-throughput mode, so that genetic marker and IgE to be provided the strong dependency of reaction from the cell of very large purpose individuality.

E.4 screen, find and confirm in conjunction with the IgE acceptor to cause the method for crosslinked antigen of acceptor (allergen) or haptens chemistry structure

The present invention also provides evaluation to cause allergic antigen or haptenic method.Described method comprises: the device that is used for cell-substrate impedance monitoring that comprises at least one hole a) is provided, and at least one electrod-array is contained in described hole; B) connect described device and electric impedance analyzer; C) transfered cell in the hole of one or more devices that comprise electrod-array; D) in one or more holes that comprise cell, add IgE; E) in one or more holes that comprise cell, add the antigen of at least a suspection or the allergen or half allergen of at least a suspection; And e) the one or more cell-substrate impedances that comprise the hole of cell of monitoring.

Cell can be any to the reaction of IgE stimulation or the possible interesting cell of reaction.Preferred cell is mastocyte, eosinophilic granulocyte, basophilic granulocyte or genetically modified cell.In some preferred embodiment, cell is the Mammals source.In some preferred embodiments, cell is from the isolating mastocyte of individuality.

IgE can be before adding antigen, allergen or half allergen, add afterwards or simultaneously.The concentration that joins the IgE in the hole can be from about 10ng/ml to about 1 μ g/ml.Preferably, do not accept IgE in the hole of a device that comprises electrod-array and cell so that at least one control wells to be provided, but this not essential to the invention.

Antigen, allergen or half allergen can be antigen, allergen or allergens known or that suspect.The concentration that joins in the hole can be from about 1ng/ml to about 1 μ g/ml.Can accept antigen, allergen or half allergen of different concns in the different holes.Perhaps for the multiple purpose, antigen, allergen or the linking agent of same concentrations can be accepted in a plurality of holes.Preferably, do not accept antigen, allergen or half allergen in the hole of a device that comprises electrod-array and cell so that at least one control wells to be provided, but this not essential to the invention.

Present method can be estimated with quantitative mastocyte because IgE stimulation IgE and the crosslinked morphological change that causes of allergen by using cell-substrate resistance technique.Preferably monitor impedances in two or more stages of experiment.For example, in one embodiment, before Xiang Kongzhong adds antigen, allergen or half allergen and monitor impedance afterwards.In another embodiment, before Xiang Kongzhong adds IgE and further monitor impedance afterwards.In another embodiment, impedance can Xiang Kongzhong add IgE and antigen, allergen or half allergenic before and monitor afterwards.Impedance also can be monitored after Xiang Kongzhong adds IgE and before Xiang Kongzhong adds antigen, allergen or half allergen, and monitors after Xiang Kongzhong adds antigen, allergen or half allergen.In some preferred embodiments, impedance is monitored before Xiang Kongzhong adds IgE, after Xiang Kongzhong adds IgE and before Xiang Kongzhong adds antigen, allergen or half allergen, monitor, and after Xiang Kongzhong adds antigen, allergen or half allergen, monitor.

Can monitor the impedance of two or more time points.Preferably, the impedance of at least three time points of monitoring.The impedance of at least three time points in two or more stages of preferably, monitoring mensuration.The impedance of each time point can or be monitored more than a frequency at one.The impedance monitoring data that obtain can be used for estimating the morphological change that is stimulated the cell that causes by IgE.For example can compare and add IgE in each hole, and/or add before antigen, allergen or half allergen and impedance afterwards before with afterwards.Can relatively accept the hole of IgE and not accept the impedance data of the control wells of IgE.Can relatively accept antigen, allergen or half allergenic hole and not accept the impedance data of the control wells of antigen, allergen or linking agent.Impedance data can be used for calculating cell index, and it can be used to compare.

Exemplary rules are as follows:

(1) mastocyte is inoculated in the hole of impedance monitoring device.

(2) monitor cell with impedance monitoring system electronic ground at one section specified rate in the time.

(3) to the IgE of cell adding to allergen or half allergen specific.

(4) with the reaction of native system monitoring cell.

(5) through after the preset time increase, add special allergen or half allergen of IgE in the step (3).

(6) reaction of cell is monitored in continuation with native system.

Embodiment 1

As an example, we this describe to use cell of the present invention-substrate impedance monitoring systematic survey and monitoring RBL-2H3 cell (ATCC) when existing or not having antigen because the morphological change that the stimulation of IgE-mediation causes.Cell-substrate impedance monitoring system has the device operator's console that can engage 6 16X devices, as shown in Figure 4.It also has electric impedance analyzer, and instructs the software of impedance measurement and record and analysis impedance data.

With RBL-2H3 cell inoculation (as shown in Figure 2) in the chamber of 16X device, and monitor cell adhering to and grow in 37 ℃ of incubator for tissue cultures in real time with 20,000 cells/well with cell-substrate supervisory system.After 22 hours, (Clone SPE-7, Sigma), final concentration is 1 μ g/ml to add the anti-dinitrophenyl of mouse monoclonal (DNP) antibody.Add non-specific mouse IgG in contrast, final concentration 1 μ g/ml.Device is put back into incubator and proceeds record again.Stimulate 24 back hours with indoor substratum sucking-off at IgE, and add 150 μ l fresh cultures to cell.Continue record 1 hour again, add DNP-white protein (Sigma) then, final concentration 1 μ g/ml.Continued record other 4 hours.

As model system, the RBL-2H3 mastocyte is widely used in studying the combination that is mediated by IgE-and stimulates the signal transduction pathway that high-affinity Fc (ε) RI acceptor causes on the film.The threshing of mastocyte is accompanied by morphologic considerable change, and described variation relates to actin cytoskeleton (people such as Pfeiffer, J Cell Biol.1985Dec; 101 (6): 2145-55.).Even without antigenic crosslinked, threshing and morphological change (people such as Oka, Am J Physiol Cell Physiol.2003 Sep 17) that IgE and combining of Fc (ε) RI also cause dose-dependently.As shown in figure 13, the also available impedance system of phenomenon of the dose-dependently of this IgE-mediation is observed.At 22 hours, tightly observing the cell index trace after using IgE had rapid moving.The signal that causes the IgE concentration of change continues about 6 hours.This dynamic variation and the morphological change that arrives with microscopic examination and consistent with the detected threshing of enzymatic determination.Importantly in having the hole of IgG, moving of this trace do not taken place, thereby illustrated that this reaction is that to have an IgE specific.The amplitude dependence at the peak of record is in the concentration of the IgE that is used for irritation cell.The signal that causes the IgE concentration of change continues about 6 hours.

In case the RBL-2H3 mastocyte is by the IgE sensitization, so by using the antigen that can produce IgE just can cause the morphological change of further threshing and Actin muscle mediation.This observes in the RBL-2H3 cell with immunofluorescence microscopy, shows as the formation of film crumple widely and cell top surface [.The dependent mastocyte threshing of IgE-and the morphology dynamic phenomenon of antigen mediation also can be monitored and measure with cell-impedance monitoring system.Added behind the IgE 20 hours, and removed substratum and substitute with fresh culture.The change of substratum can cause non-specific variation instantaneous in the signal equally, and described signal returns to baseline values immediately after 30 minutes.Add the DNP-white protein this moment and can cause sharply moving of record value, its peak appears at about 15-20 minute, and returns to baseline after about 1 hour.Under these conditions, the time length ratio that moves in the sort signal is short a lot (3 hours than 6 hours) when only IgE stimulates.

Embodiment 2

Following embodiment is used for illustrating how real-time electronic cell sensor-based system and method are used for monitoring in real time the mastocyte activation of IgE-mediation.Those that method and apparatus of the present invention is not limited to describe among the embodiment.In fact, within the scope of the invention, also have other special method and apparatus to can be used to monitor the conduction of mastocyte signal and the activatory mensuration of IgE-mediation.

Introduction

This job description use cell-substrate impedance measurement the mastocyte activatory of IgE-mediation is measured.Present method is based on real-time quantitative because the polyvalent antigen of the lip-deep IgE-Fc of mastocyte (ε) RI mixture is assembled the variation of the morphology, cytoskeleton and the cell adhesion that cause.Because electronics is measured readout and is depended on the dynamic (dynamical) variation of morphology, cytoskeleton and adhesion, these all are that mastocyte reacts the antigen intrinsic, and be the essential part of mastocyte activation procedure, so this mensuration does not need to set up report clone or use any other reagent or cell manipulation.Carry out in real time owing to measure, so the mastocyte activatory reaction to the IgE-mediation of antigen dependency and dependent/non-dependent can be monitored in same measured.This mensuration is easy to revise and is applicable to 96 well format, and as described here, can be used for measuring mastocyte activatory pharmacy inhibitor in the high-throughput mode.

Material and method

Cell and reagent

RBL-2H3 clone is bought from American type culture collection (ATCC), and at 37 ℃ and 5%CO ₂Maintain among the DMEM that contains 10% foetal calf serum.(St.Louis MO) buys from SigmaAldrich for the anti-dinitrophenyl IgE of mouse monoclonal antibody (Clone SPE-7) and DNP-HSA.(La Jolla CA) buys from Calbiochem for the specific inhibitor SU6656 of Src-, the specific inhibitor PD98059 of MEK-and the specific inhibitor U73122 of PLC-.Specific inhibitor bisindolylmaleimidesfor of PKC-and Syk inhibitor Piceatannol buy from Sigma.(Eugene OR) buys rhodamine-Phalloidine from Molecular Probes.Lab-Tek chamber slides buys from VWR Scientific.

Cell-substrate impedance monitoring system

Cell-substrate impedance monitoring system comprises 3 integral parts in real time: electric impedance analyzer, device operator's console and one or more 16X microtitration panel assembly.Array of microelectrode sensors is installed on the glass slide with the little manufacture method of lithographic printing, and the slide glass that will contain electrode is assembled into vinyl disc to form 16 holes of containing electrode.The device operator's console of system holds 16X microtitration panel assembly, and electronically any one hole of switch to the transmitter that is used for impedance measurement (impedance) analyser.In the actually operating, the device that will have the cell of cultivation in the hole places the device operator's console, and described device operator's console is positioned at the inside of incubator.Cable connecting arrangement operator's console and transmitter (impedance) analyser.Under the software control of impedance monitoring system, electric impedance analyzer can be selected the hole that will measure automatically and carry out impedance measurement continuously.The impedance data of analyser is passed on the computer, and is analyzed and handled by the software of integrating.

The impedance of electrode in the single hole (electrode member) measurements is depended on geometry proterties, the ionic concn in the hole of electrode and whether cell attachment is arranged on electrode.Do not having under the situation of cell, electrode impedance depends mainly on the ionic environment in electrode/solution interface and tank solution.Exist under the situation of cell, can change the local ion environment of electrode/solution interface, causing impedance to increase attached to the cell on electrode sensor surface.Cell on the electrode is many more, and cell-electrode impedance is big more.In addition, the change of impedance also depends on the morphology of cell and cell attachment in the degree of electrode.

In order to come quantitative cell state based on the cell-electrode impedance that measures, according to the following formula parameter that is called cell index of having derived,

R wherein _b(f) and R _Cell(f) be respectively and do not have cell or the dependent electrode resistance of cell situation lower frequency (composition of impedance) is arranged.N is a number of measuring the used Frequency point of impedance.Therefore, cell index is the quantitative measurment to cell state in the hole that comprises electrode.Under identical physiological condition, adhere to more cell on the electrode and cause bigger R _Cell(f) value, thus bigger cell index value caused.In addition, exist in the hole under the situation of cell of similar number, cell state such as morphologic change will cause the change of cell index.For example the increase of cell adhesion or cellular invasion will add the contact area of maxicell electrode, and this will make R _Cell(f) increase, thereby the cell index value also just increases.

Fluorescence microscopy

The RBL-2H3 cell inoculation to 16 hole Lab-Tek chamber slide, adheres to it and spread 6 hours.With the cell final concentration is that the anti--DNP IgE of 100ng/ml or the non-specific mouse IgG of 100ng/ml stimulate, after 16 hours, the sucking-off substratum, and alternative with fresh culture, with the time shown in the DNP-BSA processing of 100ng/ml, fix with 4% Paraformaldehyde 96 then.Cell with PBS washing 3 times, is sealed with saturatingization of PBS that contains 0.2%TX-100 and at the PBS that is containing 0.5%BSA.Then dyeed 30 minutes, wash 3 times, and manifest and imaging with tritc filter on the Nikon E400 surface illumination fluorescent microscope and Nikon ACT software with PBS with rhodamine-Phalloidine pair cell.

2.5. Hex is measured

Washing is grown in the RBL-2H3 cell on 96 orifice plates and is incubated at Tyrode damping fluid (10mM Hepes, pH 7.4,130mM NaCl, 5mM KCl, 1.4mM CaCl2,1mM MgCl2,5.6mM glucose, and 0.1%BSA), and stimulate with anti--DNP IgE of 100ng/ml, take out supernatant liquor after 2 hours, containing the Tyrode damping fluid lysing cell individual layer of 0.5%TX-100.(1mg/mL) measure hexosaminidase activity in supernatant liquor and the cell monolayer with substrate 4-nitrophenyl-2 acetylaminohydroxyphenylarsonic acid 2-deoxidation-b-D-glucopyranose glycosides (4-nitrphenyl-2acetamido-2-deoxy-b-D-glucopyranoside).Cultivate after 1 hour for 37 ℃, by adding glycine (0.4M, the PH 10.7) termination reaction of two volumes.The absorption of reading 405nm with Molecular Devices ELISA reader (reader).

Impedance measuring

In every hole of 16X microtitration panel assembly, inoculate 20,000 RBL-2H3 cells and monitor with impedance measurement system.Before final concentration IgE shown in adding, allow cell attachment and spreading 5-24 hour.Continuously measured cell-electrode impedance, derive and writing time dependent cell index value.

RBL-2H3 mastocyte activatory impedance monitoring

To cause the startup of signal transduction cascade in the RBL-2H3 mastocyte activation that has the mediation of IgE-under the antigenic situation, thereby cause comprising the threshing of the secretory vesicle of transformation reactions medium such as histamine.In addition, the stimulation of the IgE-mediation by Fc (ε) RI causes the rapid reconstruct of actin cytoskeleton people such as (, 1997) Oliver equally.Because monitoring cell-electrode impedance can provide the information about morphocytology and adhesion parameter, so we wish to determine when having antigen by the impedance response of the RBL-2H3 mastocyte of IgE sensitization in advance.

The RBL-2H3 mastocyte is inoculated into the surface that is integrated with the 16X microtitration panel assembly of microelectronic sensor array in each bottom, hole.Allow cell attachment in the surface of transmitter, use anti--DNP IgE sensitization (Figure 14) after 18 hours.Use the DNP-BSA of final concentration 100ng/ml after general 24 hours to cell, with the oligomerization of inducing IgE-bonded Fc (ε) RI acceptor with induce the mastocyte activation.Use impedance monitoring system monitoring continuously cell-electrode impedance to measure.As shown in figure 14, the increase with instantaneous resistance value has been induced immediately in the application of DNP-BSA, and described resistance value is detectable in using 5 minutes of DNP-BSA, reaches maximum value at 30 minutes, and returns to baseline at about 2.5 hours.The mastocyte activation of IgE-mediation not only causes morphologic great variety (people such as Pfeiffer, 1985), but also increasing 6 integrin-mediated cell adhesion people such as (, 1994) Wyczolkowska, the two all has contribution to the measurement of using impedance monitoring system pair cell-electrode impedance.

The impedance measurement of mastocyte activatory is dynamically relevant with threshing with cytoskeleton

For whether cell-electrode impedance rising of definite IgE-mediation is relevant with the activation of RBL-2H3 mastocyte, the morphological change of IgE-mediation and the release of medium are all monitored.

In order to last method and shown in the time point sensitization and activate the RBL-2H3 mastocyte, fix and dye to manifest actin cytoskeleton (Figure 15 A) with Paraformaldehyde 96 with rhodamine-Phalloidine.As (people such as Pfeiffer, 1985 as described in Figure 15 A and the preamble; People such as O ' Luanaigh, 2002; People such as Powner, 2002), the crosslinked actin cytoskeleton reconstruct that causes time-dependent manner of Fc (ε) the RI acceptor of DNP-BSA-mediation.As far back as 2.5 minutes cells obviously crumple widely just takes place after IgE stimulates, then morphology changes, and it causes cellular invasion and generates [.The peak of cytoskeleton reconstruct appears at IgE stimulated the back 30-45 minute, this and the cell that measures with the impedance monitoring system-electrode impedance reaction peak positive correlation.In contrast, the RBL-2H3 mastocyte with irrelevant IgG sensitization, carries out crosslinked with DNP-BSA simultaneously subsequently.Do not observe tangible cytoskeleton and morphological change.

As RBL-2H3 mastocyte activatory additional markers, IgE stimulates the Hex activity that causes to measure when having antigen cross-linking.Hex is stored in the secretory vesicle, and is the mark of mastocyte threshing.IgE-bonded Fc (ε) the RI crosslinked meeting on the mastocyte surface the causing Hex of known antigens mediation is discharged in the substratum people such as (, 1983) Razin.It is described to press the materials and methods part, and the RBL-2H3 cell with anti--DNP IgE sensitization, is activated by adding DNP-BSA, and measures the Hex activity.The Hex that antigen cross-linking causes depending on experiment increases by 2.5 to 4 times (Figure 15 B).In sum, the increase and the mastocyte of the mastocyte-electrode impedance of IgE-mediation activate distinctive morphological change and threshing positive correlation.Therefore, cell-electrode impedance is measured and can be used as mastocyte activatory readout.

The cell of RBL-2H3 sensitization step-substrate monitoring

The monomer I gE that shows recently high density is not having just can to induce under the situation of antigen cross-linking mastocyte to activate people such as (, 2004) Oka.The increase that we IgE that wishes to determine whether to have only high density just can inducing cell-electrode impedance reaction thus.Stimulate the 16X microtiter plate RBL-2H3 mastocyte that cell-substrate impedance monitoring apparatus surface is grown with the IgE that is increased to 1 μ g/ml amount from 15ng/ml gradually, and its impedance response of monitoring continuously (Figure 16 A).Independent IgE uses the rising of the dose-dependently that causes cell-electrode impedance reaction, and this is dynamically relevant with medium release with morphology.For whether the initial IgE concentration that is identified for sensitized cell influences the reaction of back antigen mediation, with shown in anti--DNP IgE sensitization RBL-2H3 cell of concentration, and be the DNP-BSA incubation of 100ng/ml with final concentration after 16 hours.Shown in Figure 16 B, the reaction of DNP-BSA-mediation is inversely proportional to the initial anti--DNPIgE concentration that is used for sensitized cell.With 1 μ g/ml anti--DNP IgE carries out sensitization and causes with multivalence DNP-BSA the increase of cell-electrode impedance reaction of causing when crosslinked to ignore, and with 15ng/ml anti--cell-electrode (cell-substrate) impedance that sensitization that DNP IgE carries out causes when causing existing DNP-BSA to assemble obviously increases.In addition, if the time increment that will add between high IgE and DNP-BSA is increased to 24 hours, the activation of antigen mediation will take place in cell so.

Therefore the concentration of these description of tests IgE and carry out the IgE sensitization and subsequently the selection of time between the antigenic stimulation all be very important, and must consider.Superiority when these experiments further illustrate on microelectronics cell-substrate impedance transducer array mastocyte aspect the monitoring cell state and reaction at any given time in real time and activate in addition.

The medicine that detects with cell-substrate impedance monitoring system suppresses the mastocyte activatory

The gathering priming signal transduction cascade of IgE-bonded Fc (ε) RI of antigen mediation, this is comprising many signal conductive proteins, activate indispensable signal conductive protein (Turner and Kinet, 1999) as protein kinase, phosphoprotein phosphatase and Phospholipid hydrolase and other activity and function for mastocyte.Therefore we wish to determine and could monitor medicine to some the restraining effect in these signal conductive proteins with mastocyte-electrode impedance measurement.

With the RBL-2H3 cell inoculation in the 16X microtitration panel assembly and with resist-DNP IgE stimulates in advance, with shown in the PKC-specific inhibitor bisindolylmaleimidesfor incubation 1 hour of dosage, then handle (Figure 17) with 100ng/ml DNP-BSA.Bisindolylmaleimidesfor can be monitored by cell-substrate supervisory system in real time to RBL-2H3 mastocyte activatory dose-dependent inhibition.Many other following pharmaceutical agents have also been tested, as to the specific SU6656 of Src family kinase, to the specific U73122 of Phospholipase C, to the specific Piceatannol of Syk Tyrosylprotein kinase with to the specific PD98059 of MEK.Calculated the IC-50 value and as shown in Table I when these medicine inhibitor react at the peak.Remove all activation of the RBL-2H3 mastocyte of dose-dependently ground inhibition antigen mediation of all inhibitor of PD98059.The specific inhibitor U73122 of PLC-demonstrates very strong restraining effect, and the IC-50 value is about 1 μ M.Though the specific inhibitor of Src and Syk needs higher concentration (Table I), they also can suppress the reaction of antigen mediation.The specific inhibitor PD98059 of MEK-is reflected at the effect that test concentrations has minimum to the RBL-2H3 of IgE-mediation.

The elimination of reacting for cell-electrode impedance of confirming the inhibitor mediation suppresses relevant with the mastocyte activatory, has measured the Hex activity of the RBL-2H3 cell that IgE stimulates when having pharmaceutical agent.As shown in figure 18, SU6656, U73122, bisindolylmaleimidesfor and Piceatannol have eliminated the active stimulation by anti--DNP IgE mediation of Hex when having antigen aggregation, and the specific inhibitor of MEK has least action.These find and disclosed data consistent, thereby have shown that the specific inhibitor of Src, PLC, PKC and Syk blocked the threshing of mastocyte of IgE-mediation and activation (people such as Oliver, 1994 fully; People such as Amoui, 1997; People such as Moriya, 1997; People such as Tedeschi, 2000).Generally speaking, result herein is presented at the mastocyte activation of monitoring the IgE-mediation on the microelectrode cell sensor array in real time estimation mastocyte activatory instrument easily is provided.This mensuration is without any need for cell manipulation, as mark, fixing or cracking.By the mastocyte activation positive correlation of proof cell-electrode (cell-substrate) impedance measurement and and medium release measurement IgE-mediation dynamic, verified this mensuration by actin cytoskeleton.In addition, the specific inhibitor of the mastocyte activated channel that characterizes suppressed the mastocyte activation in the past, and as passing through cell-substrate impedance monitoring systematic survey, this has further verified this mensuration.

Compound	Target	IC50±SD(μM)
Compound	Target	IC50±SD(μM)	Bisindolylmaleimidesfor	Protein kinase C	2.9±1.7(N＝3)
SU6656	SRC	5.8±3.5(N＝5)	Bisindolylmaleimidesfor	Protein kinase C	2.9±1.7(N＝3)
SU6656	SRC	5.8±3.5(N＝5)	U73122	Phosphokinase	0.93±0.6(N＝5)
Piceatannol	Syk	1(N＝1)	U73122	Phosphokinase	0.93±0.6(N＝5)

Table I. suppress determining of mastocyte activatory IC-50 by the pharmaceutical agent that cell-the substrate impedance monitoring obtains.

Discuss

The reading of cell-electrode impedance mainly is subjected to three parameter influences: be seeded in cell count, cell shape and cell adhesion on the microelectronics array in the intensity of electrode surface.Because fusion, the rapid rearrangement of actin cytoskeleton and the 6 integrin-mediated adherent enhancing of secretory granules, thereby make the RBL-2H3 mastocyte activation of IgE-mediation be accompanied by increase (people such as Pfeiffer, 1985 of effective surface area; People such as Wyczolkowska, 1994).Strengthen the increase that the morphology that makes up dynamically causes the mastocyte electrode impedance value of the dependent IgE-mediation of antigen with the adhesion interaction of cell and electrod-array.In addition, the medicine inhibitor of the mastocyte activatory signal conductive protein of participation IgE-mediation suppresses the mastocyte activation (Figure 17 and Table I) of IgE-mediation in the mode of dose-dependently.

The mastocyte activatory is familiar with crosslinked the causing based on crucial tyrosine in the activation primitive (ITAM) of immunity receptor tyrosine of IgE-bonded Fc RI of antigen mediation according to existing by Src family kinase Lyn phosphorylation (Turner and Kinet, 1999).The phosphoric acid of ITAM turns to the Syk protein tyrosine kinase stop site is provided, and described kinases causes activation of himself and the phosphorylation of downstream substrate such as Phospholipase C γ (PLC γ) thereof subsequently.PLC γ catalytic film phosphatide is hydrolyzed into inositol 1,4,5-triphosphoric acid (IP ₃) and diacylglycerol (DAG).IP ₃Mobilization is at the calcium of endoplasmic reticulum internal storage, and calcium and DAG cause the generally activation of protein kinase C (PKC) family and other kinases and signal conductive protein.The activation of the kinase pathways of IgE-mediation finally causes the threshing of mastocyte and the release of inflammatory mediator.Therefore, can cause that at the medicine inhibitor of Src, Syk, PLC γ and PKC the mastocyte activatory of the IgE-mediation of dose-dependently suppresses, this can monitor by cell-electrode impedance measurement.Cell-electrode impedance the value and the mastocyte threshing relevant (Figure 18) of passing through the Hex activity measurement of the mastocyte of handling with these medicine inhibitor.Further verified described mensuration with these medicine inhibitor, and showed cell-substrate impedance monitoring system can be used for estimating the activation of mastocyte.

Up-to-date a series of evidences show that the interaction with mastocyte that IgE mediates separately has important biological function, and this has surmounted its effect as just the mastocyte sensitizing agent (Kawakami and Galli, 2002).The application of monomer I gE can be induced mitogen-activated protein kinase (MAPKs) and AKT when two different groups had been presented at the gathering that does not have the antigen mediation phosphorylation and activation, thereby the survival of enhancing mastocyte (people such as Asai, 2001; People such as Kalesnikoff, 2001; People such as Kitaura, 2003).But identical article is also pointed out monomer I gE and can not induced the mastocyte threshing.On the contrary, the nearest disclosed report of another piece points out that the monomer I gE self of high density can induce the mastocyte activation, and need not the connection of antigen mediation.Said by the author, by medium discharge and actin cytoskeleton reset measure by IgE separately the crosslinked mastocyte that causes of mastocyte activation and the antigen mediation of mediation activate as broad as long people such as (, 2004) Oka.According to our result, the monomer I gE of applying high density also can cause the increase of mastocyte-electrode impedance value separately, thus surperficial mastocyte activation (Figure 16).Yet reaction duration is longer than the mastocyte activation of antigen mediation.Though the molecule mechanism with the dependent mastocyte priming reaction of antigen of antigen dependent/non-dependent is still waiting to illustrate, known that monomer I gE monomer can cause important biological respinse.The initial concentration that we have also determined to be used for the IgE of sensitized cell has determined the mastocyte activatory amplitude and the time length of back antigen mediation.Carry out sensitization with the IgE (1 μ g/ml) of high density and cause the dependent mastocyte reaction of low-down antigen, cause the duration of the reaction of mastocyte longer and carry out sensitization, amplitude bigger (Figure 16 B) with the IgE (15ng/ml) of lower concentration.The mastocyte reaction that the mastocyte of high density IgE-sensitization lacks tangible antigen mediation more may be because mastocyte has no chance to form fully its secretory granules again after the activation that is subjected to the IgE-mediation, and this result obtains the support of the following fact: increase with 1 μ g/ml IgE and carry out time increment between sensitization and the cross-linking step, can finally cause the activation of the mastocyte that antigen mediates.

In sum, the mastocyte activation determination of the real-time and unmarked IgE of this paper introduction mediation provides a kind of brand-new and instrument easily for the mastocyte activation of researchist's assessment of pharmacy corporation and scientific research institution and quantitatively IgE-mediation.The suitability that described mensuration is integrated with the 96X microtiter plate of microelectronic sensor to the bottom in the hole becomes it to carry out high throughput analysis and select with the ideal in screening large compound storehouse.In addition, microelectronics cell sensor technology can be used to assess the interaction of other receptors ligand usually.

Here the document of all references comprises patent, patent application and publication, and comprises that the document of quoting in the bibliography all is incorporated herein by reference in full.

Title is only read for helping reader, and does not limit the scope of the invention.

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Claims

1. device of monitoring cell-substrate impedance, it comprises:

A) insulating substrate;

B) two or more are installed on suprabasil electrod-array, and each of wherein said two or more electrod-arrays all comprises two electrode structures; With

C) at least two connection gaskets, each connection gasket all is positioned at the edge of described substrate, wherein, for each of described two or more electrod-arrays, each of described two electrode structures all comprises a plurality of electrode members, and first of described two electrode structures of each of described at least two electrod-arrays is connected in one in described at least two connection gaskets, and each second of described two electrode structures of described at least two electrod-arrays is connected in described at least two connection gaskets another; In addition, wherein, it is uniformly approximate that the electrode resistance of each electrod-array on whole array distributes; In addition, wherein, described substrate has the surface that is suitable for cell attachment or growth; Wherein said cell described suprabasil adhere to or grow can cause between the described electrode structure in each electrod-array can be detected impedance variations.

2. the device described in the claim 1, the electrode structure of each of wherein said two or more electrod-arrays has essentially identical surface-area.

3. the device described in the claim 1, at least two of wherein said two or more electrod-arrays is separately addressable.

4. the device described in the claim 1, first the locational electrode resistance on any one array of wherein said two or more electrod-arrays and the difference of second locational electrode resistance on described any one array are no more than 30%.

5. the device described in the claim 4, the difference of second locational electrode resistance on any one of the locational electrode resistance of first on any one of wherein said two or more electrod-arrays and described two or more arrays is no more than 15%.

6. the device described in the claim 5, the difference of second locational electrode resistance on any one of the locational electrode resistance of first on any one of wherein said two or more electrod-arrays and described two or more arrays is no more than 5%.

7. the device described in the claim 6, the difference of second locational electrode resistance on any one of the locational electrode resistance of first on any one of wherein said two or more electrod-arrays and described two or more arrays is no more than 2%.

8. install described in the claim 1, wherein for each of described two or more electrod-arrays, described first electrode structure links to each other with article one electrode bus, and this electrode bus links to each other by one at least one electric trace and described two connection gaskets at least; And described second electrode structure links to each other with the second electrode bus, and this electrode bus links to each other with in described two connection gaskets another by at least one electric trace at least.

9. the device described in the claim 8, an electrode structure of each of at least two of wherein said two or more electrod-arrays links to each other with a shared connection gasket.

10. the device described in the claim 9, wherein for each of described two or more electrod-arrays, one of described two electrode structures links to each other with connection gasket, and this connection gasket links to each other with another electrode structure at least in described two or more electrod-arrays simultaneously; And another of described two electrode structures links to each other with the connection gasket that does not link to each other with any other electrode structure.

11. the device described in the claim 1, wherein said substrate comprise the silicon-dioxide epitaxial wafer or the polymkeric substance of growing on glass, sapphire, the silicon.

12. the device described in the claim 11, wherein said substrate fabrication becomes flat surfaces.

13. the device described in the claim 12, the electrode member of each of wherein said two or more electrod-arrays has essentially identical width.

14. the device described in the claim 13, the width of wherein said electrode member is greater than 30 microns.

15. the device described in the claim 14, the width of wherein said electrode member is between about 50 microns to about 300 microns.

16. the device described in the claim 15, the width of wherein said electrode member are about 90 microns.

17. the device described in the claim 14, the electrode member of each of wherein said two or more electrod-arrays is evenly spaced apart substantially.

18. the device described in the claim 17, wherein the spacing between the adjacent electrode element is less than 50 microns.

19. the device described in the claim 18, wherein the spacing between the adjacent electrode element is between about 5 microns to about 30 microns.

20. the device described in the claim 18, wherein the spacing between the adjacent electrode element is about 20 microns.

21. the device described in the claim 17, wherein said two or more electrod-arrays comprise circle electrode member on line.

22. the device described in the claim 21, wherein for each of described two or more electrod-arrays, described article one electrode bus comprises the electrode that extends along at least a portion periphery on described each array, described second electrode bus comprises the electrode that extends along at least a portion periphery on described each array, and is not overlapping between wherein said article one electrode bus and the described second electrode bus.

23. the device described in the claim 22, the electrode member in the identical electrodes structure in wherein said two or more electrod-arrays links to each other with the identical electrodes bus.

24. the device described in the claim 13, each array organization of wherein said two or more electrod-arrays is with heart, sinusoidal, interdigitated or castle type.

25. the device described in the claim 8, wherein said at least one electric trace are insulated layer and cover.

26. the device described in the claim 22, wherein said at least one electric trace are placed in second face of substrate.

27. the device described in the claim 1, further comprise a plurality of containers, wherein each container is positioned on the dielectric base with vertical direction, in addition, wherein each container forms fluid-tight container, and be positioned at suprabasil described two or more electrod-arrays each all combine with fluid-tight container.

28. the device described in the claim 27, wherein each container is the tubulose with opposite open end, and the one end contacts with substrate is fluid-tight.

29. the device described in the claim 28, wherein the diameter of the end that contacts with substrate of container is less than the diameter of opposite end.

30. the device described in the claim 28 wherein is being positioned over suprabasil container ends, the diameter of one or more containers arrives between about 20mm at about 1mm.

31. the device described in the claim 28, wherein container forms the porous microtiter plate at the no end together.

32. the device described in the claim 30, wherein the number in the hole of Cun Zaiing is between 2 to 1,536.

33. the device described in the claim 32, wherein the number in the hole of Cun Zaiing is between 4 to 384.

34. the device described in the claim 32, wherein the number in the hole of Cun Zaiing is between 16 to 96.

34. the device described in the claim 28 wherein combines less than all holes and electrod-array.

35. the device described in the claim 31 wherein combines less than all holes and electrod-array.

36. the device described in the claim 34, wherein said two or more electrod-arrays comprise circle element on line.

37. the device described in the claim 36, wherein each combination of two strip electrode buses and described electrod-array, wherein for each electrod-array, a plurality of electrode members of first electrode structure link to each other with article one in the described two strip electrode buses, and a plurality of electrode members of second electrode structure link to each other with second in the described two strip electrode buses.

38. the device described in the claim 37, wherein said two strip electrode buses are positioned on each the periphery of described electrod-array.

39. each bar of the described two strip electrode buses that the device described in the claim 38, each of its described electrod-array that neutralizes link to each other links to each other by one at least one electric trace and described two connection gaskets at least.

40. the device described in the claim 39, second electrode structure that comprises a plurality of electrode members that each of wherein said electrod-array comprises first electrode structure that comprises a plurality of electrode members that links to each other with article one electrode bus and links to each other with the second electrode bus, and described article one electrode bus links to each other with different connection gaskets with described second electrode bus.

41. the device described in the claim 40, wherein first electrode structure of each at least two described electrod-arrays links to each other with shared connection gasket.

42. the device described in the claim 41, wherein in described at least two electrod-arrays each, first electrode structure links to each other with connection gasket, this connection gasket links to each other with the electrode structure of another described electrod-array at least simultaneously, and the connection gasket that any other the electrode structure of electrod-array of second described electrode structure and discord links to each other is continuous.

43. the described device in the claim 42 comprises that further at least one is attached to the printed circuit board (PCB) (PCB) on the described substrate bottom surface.

44. the device described in the claim 43 further comprises the metal clip that contacts with described at least one PCB with described at least two connection gaskets.

45. the device described in the claim 44 comprises further with the metal of at least one PCB contact being connected pin that this connects pin and can be connected on the electric impedance analyzer.

46. the device described in the claim 45 comprises 16 holes.

47. the device described in the claim 45 comprises 96 holes.

48. the device described in the claim 47 comprises 95 electrod-arrays, each electrod-array all is positioned at the bottom in one of described 96 holes.

49. the device described in the claim 1 further comprises the electric impedance analyzer that is electrically connected with described at least two connection gaskets.

50. the device described in the claim 36, wherein said electric impedance analyzer can be measured impedance at about 1Hz in the range of frequency between about 1MHz.

51. a system that monitors cell-substrate impedance, it comprises:

A) one or more porous cells-substrate impedance monitoring device, wherein have at least the bottom of two Kong Zaikong to comprise electrod-array in a plurality of holes of these devices, wherein said electrod-array is independent addressing, in addition, wherein said device can be used for measuring the difference of the resistance value relevant with cell behavior;

B) electric impedance analyzer;

C) comprise the device operator's console of electronic circuit, described device operator's console can engage described device, and described two or more electrod-arrays on the described device optionally are connected with described electric impedance analyzer; With

D) software program, it can be used for controlling described device operator's console, and writes down and analyze the data that obtain from described electric impedance analyzer.

52. the system described in the claim 51, wherein said device operator's console comprise can switch on and off described two or more electrod-arrays each with described electric impedance analyzer between the electronic switch that is connected.

53. the device operator's console described in the claim 51, wherein said software program can instruct described device operator's console to measure one or more resistance value of described two or more electrod-arrays in the rule or the random timed interval.

54. the system described in the claim 51, wherein said electric impedance analyzer can be measured impedance under one or more frequencies.

55. the device operator's console described in the claim 51, wherein said software program can be stored described data.

56. the device operator's console described in the claim 51, wherein said system can show described data.

57. the device operator's console described in the claim 51, wherein said software can based in one or more holes of calculating described porous plate from the described one or more electrod-array measured impedance values that are arranged in described one or more holes at the cell index of one or more time points.

58. the device operator's console described in the claim 56, wherein said system can show the described cell index of described one or more hole at described one or more time points.

59. the system described in the claim 51, wherein said software is programmable, to instruct any one impedance monitoring of described two or more electrod-arrays in the timed interval of selecting.

60. the system described in the claim 55, wherein said software allows input and shows experiment parameter.

61. the system described in the claim 51 further comprises at least one platform, can be used for placing described one or more devices at system's run duration on described platform.

62. a system that monitors cell-substrate impedance, it comprises

A) device described in one or more claims 44;

B) electric impedance analyzer;

C) comprise the device operator's console of electronic circuit, described device operator's console can engage described device and any one of described two or more electrod-arrays on the described device optionally is connected with described electric impedance analyzer; With

63. the system described in the claim 62, wherein said device operator's console comprise can switch on and off described two or more electrod-arrays each with described electric impedance analyzer between the electronic switch that is connected.

64. the system described in the claim 62, wherein said software can instruct described device to measure one or more resistance value of described two or more electrod-arrays in the rule or the random timed interval.

65. the system described in the claim 62, wherein said software program can be stored described data.

66. the system described in the claim 62, wherein said system can show described data.

67. the system described in the claim 62, wherein said software can based in one or more holes of calculating described porous plate from the described one or more electrod-array measured impedance values that are arranged in described one or more holes at the cell index of one or more time points.

68. the device operator's console described in the claim 67, wherein said system can show the described cell index of described one or more hole at described one or more time points.

69. the system described in the claim 62, wherein said software is programmable, to instruct any one impedance monitoring of described two or more electrod-arrays in the timed interval of selecting.

70. the system described in the claim 66, wherein said software allows input and shows experiment parameter.

71. the system described in the claim 62 further comprises at least one platform, can be used for placing described one or more devices at system's run duration on described platform.

72. monitor the method for measuring of cell-substrate impedance, it comprises

The system of claim 51 is provided;

In at least one hole of described system, introduce cell;

Monitor the cell-substrate impedance in described at least one hole.

73. the method described in the claim 72, wherein said monitoring comprise the impedance variations of analyzing three or more time points.

74. the method described in the claim 73, what have three time points at least in wherein said three or more the time points is well-regulated at interval.

75. monitor the method for measuring of the cell-substrate impedance that is caused by one or more test compounds, it comprises

The system of claim 51 is provided;

In one or more holes of the described system that comprises electrod-array, introduce cell;

In described one or more holes, add at least a test compounds; With

Monitor and add before the described at least a test compounds in described one or more hole and the cell-substrate impedance after the described at least a test compounds of adding.

76. the method described in the claim 75 is wherein monitored the impedance of three or more time points.

77. the method described in the claim 75 further comprises analyzing adding before the described at least a test compounds and resistance value afterwards.

78. the method described in the claim 75 further comprises at least one hole and introduces cell, and does not add test compounds to this hole, to generate control wells.

79. the method described in the claim 77, wherein said analysis comprises the calculating cell index.

80. the method described in the claim 77, wherein said analysis can provide following information: the rational variation of cytopathy of the cell-stimulating of the cytotoxicity of the time-dependent manner of cell proliferation, necrocytosis, cell adhesion, apoptosis, cytodifferentiation, cell survival, cytotoxicity, morphocytology, cell concentration, cell quality, compound, IgE-mediation or stimulation, receptor-ligand combination, virus and bacteriotoxin mediation, the existence of neutralizing antibody and amount, specificity T-cell-mediated cytotoxic effect, receptor-ligand bonded exist and avidity.

81. the method described in the claim 79, wherein said cell index can be used as Cytotoxic indication.

82. monitor a plurality of methods for measuring of the cell-substrate impedance that is caused by one or more test compounds, it comprises

The system of claim 51 is provided;

In two or more holes of described system, introduce cell;

The test compounds that adds first kind of concentration at least one hole in described two or more holes of described system adds the test compounds of second kind of concentration in another hole at least in described two or more holes of described system; With

Monitor the cell-substrate impedance in described two or more holes.

83. the method described in the claim 82, wherein said monitoring comprise the impedance variations of monitoring three or more time points.

84. the method described in the claim 83, at least one of wherein said three or more time points are in the test compounds that adds described first kind of concentration in described at least one hole with before the test compounds that adds described second kind of concentration in another hole at least.

85. the method described in the claim 82 is wherein introduced cell and is comprised wherein introduce cell in three or more holes in two or more holes, and in described three or more the holes one is control wells, does not accept test compounds in this hole.

86. the method described in the claim 83, it is well-regulated having three timed intervals between the time point at least in wherein said three or more the time points.

87. the method described in the claim 83 further comprises and analyzes the influence of one or more identical compounds to described cell.

88. the method described in the claim 89, wherein said analysis comprises the calculating cell index.

89. the method described in the claim 89, wherein said analysis can provide following information: the rational variation of cytopathy of the cell-stimulating of the cytotoxicity of the time-dependent manner of cell proliferation, necrocytosis, cell adhesion, apoptosis, cytodifferentiation, cell survival, cytotoxicity, morphocytology, cell concentration, cell quality, compound, IgE-mediation or stimulation, receptor-ligand combination, virus and bacteriotoxin mediation, the existence of neutralizing antibody and amount, specificity T-cell-mediated cytotoxic effect, receptor-ligand bonded exist and avidity.

90. the method described in the claim 88, wherein said cell index can be used as Cytotoxic indication.

91. monitor a plurality of methods for measuring of the cell-substrate impedance that is caused by two or more test compounds, it comprises

The system of claim 51 is provided;

In two or more holes of described system, introduce cell;

In at least one hole in described two or more holes of described system, add first kind of compound;

In another hole at least in described two or more holes of described system, add second kind of compound; With

Monitor described at least one comprise the hole of described first kind of test compounds and described another comprises the cell-substrate impedance in the hole of described second kind of compound at least.

92. the method described in the claim 91, wherein said monitoring comprise the impedance variations of monitoring three or more time points.

93. the method described in the claim 91, at least one of wherein said two or more holes are not accept the control wells of test compounds.

94. the method described in the claim 92, at least one of wherein said one or more time points is before adding at least a test compounds in described at least one hole.

95. the method described in the claim 91, it is well-regulated having three timed intervals between the time point at least in wherein said three or more the time points.

96. the method described in the claim 92 further comprises and analyzes the influence of described one or more compounds to described cell.

97. the method described in the claim 96, wherein said analysis can provide following information: the rational variation of cytopathy of the cell-stimulating of the cytotoxicity of the time-dependent manner of cell proliferation, necrocytosis, cell adhesion, apoptosis, cytodifferentiation, cell survival, cytotoxicity, morphocytology, cell concentration, cell quality, compound, IgE-mediation or stimulation, receptor-ligand combination, virus and bacteriotoxin mediation, the existence of neutralizing antibody and amount, specificity T-cell-mediated cytotoxic effect, receptor-ligand bonded exist and avidity.

98. the method described in the claim 96, wherein said analysis comprises the calculating cell index.

99. the method described in the claim 98, wherein said cell index are plotted the figure to the time.

100. the method described in the claim 98, wherein said cell index can be used as Cytotoxic indication.

101. monitor the method that IgE stimulates the variation of the cell-substrate impedance that causes for one kind, it comprises:

The system of claim 51 is provided;

Introduce cell in one or more holes of described system, each of wherein said one or more holes all comprises electrod-array;

In one or more holes of described system, add IgE;

In one or more holes of described system, add antigen; With

Monitor the cell-substrate impedance at least one hole in described one or more holes of described system.

102. the method described in the claim 101, wherein said cell are mastocyte, eosinophilic granulocyte, basophilic granulocyte or genetically modified cell.

103. the method described in the claim 102, wherein said cell is a mastocyte.

104. the method described in the claim 101, the concentration that wherein said IgE adds arrives between about 1 μ g/ml at about 10ng/ml.

105. the method for claim 101, wherein said cell-substrate impedance is monitored add described IgE in described one or more holes after.

106. the method for claim 105, wherein said cell-substrate impedance are before adding described IgE and monitoring afterwards in described one or more holes.

107. the method for claim 105, wherein said cell-substrate impedance is monitored add described IgE in described one or more holes after and add antigen in described one or more holes before.

108. the method for claim 107, wherein said cell-substrate impedance is to monitor add described IgE in described one or more holes after and add antigen in described one or more holes before and add antigen in described one or more holes after.

109. the method for claim 108, wherein said cell-substrate impedance is add described IgE in described one or more holes before, after in described one or more holes, adding described IgE and before in described one or more holes, adding antigen and after in described one or more holes, adding described antigen, monitor.

110. screening can be regulated the method for cell to the compound of the reaction of IgE stimulation, it comprises:

The system of claim 51 is provided;

In one or more holes of described system, introduce cell;

Add at least a test compounds at least one of described one or more holes of described system, wherein each hole all comprises electrod-array;

In described one or more holes of described system, add IgE;

In described one or more holes of described system, add antigen; With

Monitor the cell-substrate impedance in described one or more holes of described system; With

Whether analysis can regulate the generation that cell stimulates IgE from the described impedance data in described one or more holes to estimate described at least a test compounds.

111. the method for claim 110, wherein said cell are mastocyte, eosinophilic granulocyte, basophilic granulocyte or genetically modified cell.

112. the method for claim 111, wherein said cell is a mastocyte.

113. the method for claim 110 wherein add described IgE in described one or more holes before, adds described one or more test compounds in described one or more holes.

114. the method for claim 110, the concentration that wherein said IgE adds arrives about 1 μ g/ml at about 10ng/ml

115. the method for claim 110, wherein said cell-substrate impedance is monitored add described IgE in described one or more holes after.

116. the method for claim 115, wherein said cell-substrate impedance is monitored add described IgE in described one or more holes before.

117. the method for claim 110, wherein said cell-substrate impedance is monitored add described IgE in described one or more holes after and add antigen in described one or more holes before.

118. the method for claim 117, wherein said cell-substrate impedance is all to monitor add described IgE in described one or more holes after and add antigen in described one or more holes before and add described antigen in described one or more holes after.

119. the method for claim 118, wherein said cell-substrate impedance is add described IgE in described one or more holes before, after in described one or more holes, adding described IgE and before in described one or more holes, adding antigen and after the described antigen of adding in described one or more holes, all monitor.

120. the method for claim 110, wherein said at least a test compounds be can with IgE bonded compound or can with Fc (ε) RI bonded compound.

121. the method for claim 120, wherein said at least a test compounds be can with IgE bonded compound.

122. the method for claim 121, wherein said at least a test compounds be can with IgE bonded antibody.

123. the method for claim 120, wherein said at least a test compounds be can and Fc (ε) RI bonded compound.

124. the method for claim 123, wherein said at least a test compounds be can and Fc (ε) RI antigen binding domains bonded compound.

125. the method for claim 120, wherein said at least a test compounds is the compound that can act in born of the same parents.

126. the method for claim 125, wherein said at least a test compounds is to suppress kinase whose compound.

127. the method for claim 126, wherein said can suppress kinase whose at least a test compounds can the arrestin kinase c, SRC, Syk.

128. the method for claim 125, wherein said at least a test compounds is to suppress the compound of Phospholipid hydrolase.

129. the method for claim 125, wherein said at least a test compounds is to suppress the compound of PLC (γ).

130. the method for claim 110 wherein adds described at least a test compounds at least in two of described one or more holes, and accepts the described at least a test compounds of different concns at least among two of wherein said one or more holes.

131. the method for claim 120 further comprises record cell-substrate resistance value and calculates the IC50 of described at least a test compounds based on resistance value at the bottom of the described cell based.

132. identify and regulate the method for cell to the target of IgE irritant reaction, it comprises:

The system of claim 51 is provided;

Introduce cell in one or more holes of described system, wherein said cell is genetic modification, with the function of target molecule that change to suspect, or reduces or eliminates its expression.

In described one or more holes of described system, add IgE;

In described one or more holes of described system, add antigen; With

Monitor the cell-substrate impedance in described one or more holes of described system, obtaining impedance data, and analyze described impedance data and regulate the target of cell the IgE irritant reaction to identify from described one or more holes.

133. the method for claim 132, wherein said cell are mastocyte, eosinophilic granulocyte, basophilic granulocyte or genetically modified cell.

134. the method for claim 133, wherein said cell is a mastocyte.

135. the method for claim 132 wherein, add described IgE in described one or more holes before, adds one or more compounds at described genetically modified cell in described one or more holes.

136. the method for claim 132, the concentration that wherein said IgE adds arrives about 1 μ g/ml at about 10ng/ml.

137. the method for claim 132, wherein said cell-substrate impedance is monitored add described IgE in described one or more holes before.

138. the method for claim 132, wherein said cell-substrate impedance is monitored add described IgE in described one or more holes after.

139. the method for claim 138, wherein said cell-substrate impedance is monitored add described IgE in described one or more holes after and add antigen in described one or more holes before.

140. the method for claim 139, wherein said cell-substrate impedance is monitored add described antigen in described one or more holes after.

141. the method for claim 140, wherein said cell-substrate impedance is add described IgE in described one or more holes before, after in described one or more holes, adding described IgE and before in described one or more holes, adding antigen and after in described one or more holes, adding described antigen, all monitor.

142. the method for claim 132 wherein adds at least a test compounds at least in two of described one or more holes, and accepts the described at least a test compounds of different concns at least among two of wherein said one or more holes.

143. the method for claim 142 further comprises record genetically modified cell and the cell of genetically modified cell-substrate resistance value not.

144. relatively the different genotype cell is to the method for the reaction of IgE stimulation, it comprises:

The system of claim 51 is provided;

In one or more holes of described system, introduce first kind of genotypic cell, to generate the one or more first genotypic cell hole;

In one or more holes of described system, introduce second kind of genotypic cell, to generate the one or more second genotypic cell hole;

In described one or more first genotypic cell hole and the described one or more second genotypic cell hole, add IgE;

In described one or more first genotypic cell hole and the described one or more second genotypic cell hole, add antigen;

Monitor the cell-substrate impedance at least one of at least one and described one or more second genotypic cell holes of described one or more first genotypic cell holes; With

Cell-substrate resistance value at least one of at least one of more described one or more first genotypic cell holes and described one or more second genotypic cell holes.

145. the method for claim 144 further comprises with described cell-substrate resistance value with from the molecular genetic data that the described first genotypic cell and the described second genotypic cell obtain being associated.

146. the method for claim 145, wherein said molecular genetic data comprise RFLIP data, SNP data, dna marker data, rna expression spectrum data, protein expression profiles data or alternative splicing data.

147. the method for claim 146 further comprises at least one of at least one and described one or more second genotypic cell holes of described one or more first genotypic cell holes adding at least a antigen.

148. the method for claim 144, wherein said at least a antigen are allergens known or that suspect.

149. the method for claim 144, the wherein said first genotypic cell and the described second genotypic cell are from people experimenter.

150. the method for claim 144, wherein said one or more porous cells-substrate impedance monitoring device is positioned in the incubator.

151. identify to cause allergic antigen or haptenic method, it comprises:

The system of claim 51 is provided;

In one or more holes of described system, introduce cell;

In described one or more holes of described system, add IgE;

In at least one of described one or more holes of described system, add to suspect it is antigen or haptenic at least a compound;

Monitor at least one the cell-substrate impedance in described one or more holes of described system, to obtain the cell-substrate impedance data of the described cell of being handled by antigen or haptens; With

Analyze described data to identify antigen or haptens.

152. the method for claim 151, wherein said one or more porous cells-substrate impedance monitoring device is positioned in the incubator.

153. the method for claim 72, wherein said one or more porous cells-substrate impedance monitoring device is positioned in the incubator.

154. the method for claim 82, wherein said one or more porous cells-substrate impedance monitoring device is positioned in the incubator.

155. the method for claim 88, wherein said one or more porous cells-substrate impedance monitoring device is positioned in the incubator.

156. the method for claim 91, wherein said one or more porous cells-substrate impedance monitoring device is positioned in the incubator.

157. the method for claim 101, wherein said one or more porous cells-substrate impedance monitoring device is positioned in the incubator.

158. the method for claim 110, wherein said one or more porous cells-substrate impedance monitoring device is positioned in the incubator.

159. the method for claim 132, wherein said one or more porous cells-substrate impedance monitoring device is positioned in the incubator.