Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/483—Physical analysis of biological material
  • G01N33/487—Physical analysis of biological material of liquid biological material
  • G01N33/48707—Physical analysis of biological material of liquid biological material by electrical means
  • G01N33/48728—Investigating individual cells, e.g. by patch clamp, voltage clamp
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
  • G01N27/02—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating impedance
  • G01N27/04—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating impedance by investigating resistance

Definitions

• the present invention relates to an electrophysiological measuring arrangement as well as to an electrophysiological measuring method, in particular using the electrophysiological measuring arrangement according to the invention.
• electrical currents and / or electrical voltages are impressed and / or measured between a measuring electrode and a counterelectrode, which are arranged between the biological object.
• the measured currents and / or voltages should provide information about the underlying physiological processes, in particular transport processes, conformational changes and the like.
• comparatively very low signal strengths are present.
• high sealing resistances that is to say the lowest possible residual electrical conductivities, are necessary over the membrane itself or in the contact or contact region between the membrane of the biological object to be examined and the aperture wall.
• the object of the invention is based solved in an electrophysiological measuring arrangement according to the invention with the features of independent claim 1. Furthermore, the object underlying the invention in an electrophysiological measurement method according to the invention with the features of independent claim 10 is achieved. Advantageous developments are described in the dependent claims.
• the present invention provides an electrophysiological measuring arrangement with a carrier, which at least in its interior with or from a carrier material having a specific composition - for example, certain concentrations of material or ion species - and with an aperture region in the region of the carrier - ie with an area that at least one aperture o- has the measuring aperture or forms - for the controlled sealing or sealing attachment of a biological object, for example a cell, a cell organelle, a vesicle, a liposome, a natural or artificial membrane, for example a lipid bilayer, or the like or a fragment thereof ,
• the aperture region is formed with at least one aperture and with a wall region which forms the aperture as an aperture inner wall and surrounds the aperture.
• an electrophysiological measuring arrangement with an aperture region, which is designed for sealing or sealant attachment, that is to say for attaching with high sealing resistance with respect to a biological object to be examined, at or in at least one Wall region which forms and surrounds an aperture of an aperture region, to modify and in particular to increase the concentration of at least one material or ion species, compared to the concentration of the material or ion species outside the modified region, ie in the underlying material of the support outside the aperture region, in particular in the middle of the carrier and / or inside the carrier.
• the change in concentration and in particular concentration increase then lead via a corresponding chemical and / or electrical interaction to the fact that the sealing or sealing attachment of a biological object to be examined can be controlled and in particular promoted, in particular the probability of attachment of the biological object to be examined at the measuring aperture and / or the sealing resistance between the biological object to be examined and the measuring aperture are increased, compared with the situation in which the concentration modification according to the invention does not exist in an otherwise identical electrophysiological measuring arrangement or compared to the special case fully doped wafer, where the function of promoting or stabilizing the gigaseal is given, but the properties of the chip is inferior to the ability to measure.
• Biological objects that are subjected to an examination can be cells, cell organelles, oocytes, bacteria or their combinations or fragments, in each case in the broadest sense. Also artificial or partially artificial essentially biological structures are conceivable, for example in the form of vesicles, liposomes, micelles, membrane fragments or the like, in which proteins are incorporated and / or deposited in a natural or artificial manner.
• selected biological objects to be examined are attached with an improved sealing resistance and then measured so that a better signal-to-noise ratio is established and the addition is mechanically stabilized, e.g. resulting in a longer measurement time span and improved reliability in terms of measurement results.
• the at least one material species or ionic species may be selected from the group consisting of protons (H + ), halide ions, in particular fluoride ions (F), doubly charged ions, in particular doubly charged metal cations, preferably Be 2+ , Sr 2+ , Having Ca 2+ and Mg 2+ .
• the invention is not limited to this species, in principle all material species are conceivable, for example, to cope with certain specific surface situations of a biological object to be measured, for example in the presence of glycocalyx structures or in other situations high sealing resistances and mechanically stable deposits achieve.
• the increase in the concentration of the at least one material or ion species may be spatially limited locally to the area of the wall area in such a way that the material of the carrier is at least in an area outside the area Apertur Schemes has no increased concentration of at least one material or ion species.
• the carrier to be provided may also be referred to as base, substrate or base substrate.
• the provision of such a substrate or such a carrier mechanically stabilizes the measuring arrangement and in particular the arrangement of the arranged biological object to be examined and allows a macroscopic subdivision of the measuring arrangement with respect to the electrolyte bath to be grounded in the sense of dividing a measuring cuvette or Wet cell in compartments with measuring and counter electrode.
• a respective wall region forming an aperture can also be integrated in the inner wall of the hole in combination with the electrode arrangement.
• one or more apertures with corresponding wall areas with respect to the upper side may then be formed protruding or emphasizing.
• they may also be everted flush inwardly at the top to protrude on the underside of the carrier or substrate; However, this is not mandatory and can be omitted with appropriate thickness of the membrane.
• the extent of the respective infeed or protrusion influences the inner wall of the respective wall area and thus the available exchange rate. Acting surface with the membrane of the biological object.
• the choice of the degree of protrusion or invagination makes it possible to adapt it to the respectively available measurement objects, for example with regard to their shape or number in the measurement solution.
• the carrier may be formed as - in particular planar - plate element with front or top and back or bottom. Other geometries are conceivable. Alternatively, it may be departed from the plate shape in which e.g. the shape of a pipette, e.g. in the sense of a classic patch pipette.
• the wall region which forms an aperture can be designed in the manner of a lateral surface or as a combination of lateral surfaces.
• the shell of a cylinder, a prism, a truncated cone and / or a truncated pyramid can be gripped back, with a corresponding wall thickness.
• the shape of the wall region for forming the aperture there are therefore numerous possibilities. These can be selected depending on the shape and the other - e.g. mechanical, geometric and / or electrical properties of the biological objects to be examined.
• the wall region forming an aperture can be formed by an edge region, so that the aperture is formed as a planar hole in the underlying substrate, and the concentration-modified region with or out of the material with modified concentration in the edge region embedded in the planar hole to promote attachment and sealing, thereby increasing the sealing resistance.
• An aperture forming wall portion may be formed with or from material selected from the group of materials including glass, quartz glass, silicon, carbon and their combinations and derivatives. With regard to the choice of material, the properties of the underlying biological objects can also be taken into account, for example with regard to the surface structure or surface charge of the membrane outer side and / or the membrane inner side of the biological objects, for example, to provide a particularly intimate adhesion and thus the To further increase the sealing resistance of the seal.
• the diameter of the aperture and in particular the inner diameter of one or the wall region forming the aperture can have a value in the range from about 0 .mu.m to about 50 .mu.m, preferably in the range from about 1 .mu.m to about 50 .mu.m.
• One or the aperture forming wall portion may have a height or depth in the range of about 0 ⁇ to about 20 ⁇ over the top or bottom of the carrier or substrate.
• a measuring electrode can be provided in the region or within the aperture or in a region on the rear or underside of the carrier or substrate.
• a counter-electrode may be provided outside the aperture and in the region on the front or top side of the carrier.
• Measuring electrode and counter electrode are preferably arranged on opposite sides of the carrier or substrate or the measuring aperture, in any case so that when attaching a preferably biological object to be measured, this is arranged between the electrodes and the formation of a suitable seal this practically electrically separates, preferably with a very high resistance, ideally greater than 1 GQ.
• the basic structure of the electrophysiological measuring device thus includes in particular the provision of a measuring electrode arrangement and a counter electrode arrangement, between which an electric current and / or an electrical voltage can be measured, between the measuring electrode arrangement and the counter electrode arrangement just the biological object to be measured
• the residual conductivity ie the conductivity between the membrane of the biological object and the wall region of the aperture
• Currents and / or voltages as being of the characteristic th membrane of the biological object can be considered, for example, by transport processes, charge shifts within or over the membrane, by substrate binding or dispensing or the like.
• an electrophysiological measurement method is provided. This is carried out in particular using an electrophysiological measuring arrangement according to the present invention.
• an electrophysiological measuring arrangement for controlling and, in particular, for promoting the sealing or sealing attachment of a biological object to be measured at an aperture of an aperture region, at least a region of an inner wall of an aperture-forming wall region is controlled with respect to at least one material or ion species with a correspondingly suitable and opposite concentration the at least one material or ionic species is formed in the carrier outside the aperture region and in particular in the middle of the carrier and / or in the interior of the carrier increased concentration.
• a key aspect underlying the electrophysiological measurement method according to the invention is the controlled modification and in particular increasing the concentration of at least one material or ion species at least on or in the wall forming the aperture, thereby interacting with the biological object and / or the electrolyte environment promoted a sealing or sealendes attaching and the sealing resistance is increased.
• the focus is placed on the - especially lateral - localization of the modification of the concentration of the at least one material or ion species on the area of the aperture.
• the wall region is formed with a region made of a material which essentially corresponds at least in its composition to the carrier material outside the aperture region, but has a locally modified, in particular locally increased, concentration with respect to at least one material species, in particular ion species.
• the local concentration change and in particular concentration increase then leads, via a corresponding chemical and / or electrical interaction, to the fact that the sealing or sealing attachment of a biological object to be examined can be controlled and promoted, in particular the probability of attachment of the biological object to be examined the measuring aperture and / or the sealing resistance between the biological object to be examined and the measuring aperture are increased, compared with the situation in which the local modification of the concentration according to the invention does not exist in an otherwise identical electrophysiological measuring arrangement.
• the concentration of the at least one species of material or ion may also be modified to a certain depth, eg, about 10 nm, from the surface of the inner wall of the aperture in this alternative aspect of the present invention. But there are also other local limited layers Deep or - especially in lateral locality - also a continuous implantation of the aperture wall conceivable.
• an electrophysiological measuring method is again provided.
• this is carried out using an electrophysiological measuring arrangement according to the alternative view of the present invention.
• the inner wall of an aperture-forming wall region is or are controlled with respect to at least one material or ion species with a correspondingly suitable and opposite to the concentration of the at least one material or ion species Ion species formed in the carrier outside the aperture region locally increased concentration.
• this measurement method according to the invention can therefore also a direct or indirect influence of molecules - the electrolyte environment and / or the membrane to be attached - near the wall by the locally modified concentration come into play.
• a key aspect underlying this electrophysiological measurement method according to the invention is the controlled local modification and in particular increasing the concentration of at least one material or ionic species on or in the aperture forming wall region, thereby interacting with the biological object and / or the electrolyte environment promoted a sealing or sealendes attaching and the sealing resistance is increased.
• measuring signals can also be measured, in particular in a capacitive manner, in order, for. B. single-channel activities to determine.
• FIGS. 1B-1D show diagrammatic and sectional views of different cross-sectional shapes of an aperture and of the respective underlying wall region.
• FIGS. 2A-2D show, in a schematic and sectional side view, details of various embodiments of the formation of the modified concentration at or in the wall region of an aperture.
• FIGS. 3-7 show, in an analogous manner as in FIG. 1A, a schematic and sectional side view of various embodiments of an electrophysiological measuring arrangement according to the present invention, in which a respective aperture is formed by wall regions corresponding to lateral surfaces of different geometric bodies.
• 8 shows an electrophysiological measuring arrangement according to an embodiment of the present invention, which is designed in the manner of a so-called patch pipette.
• FIGS. 9A-9E show, in a schematic and sectional side view, various aspects of the use of the electrophysiological measuring arrangement according to the invention.
• Figures 10-12 show in schematic and sectioned side view details in the attachment of a biological object to a respective embodiment of the electrophysiological measuring arrangement.
• the patch-clamp technology is applied to e.g. Perform ion channel analyzes for drug testing.
• the manual patch-clamp method and its refinements e.g. on single cells - electrical currents and voltages are measured which - e.g. of ion channels - are caused in the membranes of biological cells. Due to the growing importance of electrophysiological examinations and the time and effort involved in their implementation, a great demand has arisen for automated electrophysiological measurement techniques and in particular for planar patch clamp and other automated patch clamp or APC systems.
• a cell as a biological object O is possibly sucked.
• a small, limited part of the cell membrane M which is referred to as a patch, is thereby drawn in with a slight negative pressure.
• the measuring surface or, in the case of a whole-cell measurement according to FIG. 9D the cell interior is electrically sealed in the mega to gigaohm region toward the outside of the cell.
• the cell-attached configuration also allows current measurement on individual ion channels in the cell membrane.
• the gigaseal rate and the sealing resistance of the manual patch clamp and the APC systems are a measure of the quality of the possible ion channel measurements. To date, a 100% gigaseal rate is not possible.
• the training of the Gigaseal depends on many factors that are not accessible to active influence. So there is no real control of the gigaseal in the appendage and measurement process.
• the present invention is based on the recognition that negative or positive charges on or in the carrier 12 and in particular on or in the inner wall I ii of a measuring aperture 14, e.g. also a patch pipette, support the training of a Gigaseal and can improve the Gigaseal itself.
• a targeted and e.g. e.g.
• the local charge density - by means of positive or negative charges - on or in the inner wall I ii of the measuring aperture 14 modified accordingly, ie in particular increased and it will be the processes for establishment and / or stabilizing a gigaseal influenced by direct and possibly local modification on or in the surface, eg by material implantation in the aperture wall, e.g. by means of a plasma.
• the control of the charge density on or in the pipette wall or aperture wall 1 1 is effected by the modification and in particular by increasing the concentration of a material or ion species at least in a region 20 of the wall portion 1 1 of the aperture 10, so that at least in the Meßaperturwand 1 1 and there locally in region 20, the material 12 "of the support 12 has a modified and in particular increased concentration with respect to at least one material or ion species, compared to the concentration of the corresponding species in the material 12 'of the support 12 outside the region 20 and thus outside of the aperture region 10, in particular compared with the concentration in the interior of the carrier 12 and / or of the carrier 12 in the middle.
• the described effects and advantages are achieved according to the invention.
• the invention accordingly provides, inter alia, the targeted and optionally spatially resolved or local modification or increase in the concentration of ions or protons, at least in or on the inner wall of the measuring aperture, thereby positively influencing processes for establishing the Gigaseal. This can not be guaranteed when using conventional physiological measuring buffers.
• the present invention accordingly relates to an electrophysiological measuring arrangement 100 and to an electrophysiological measuring method in which the sealing or sealing attachment of a biological object O to be examined to a support 12 of the measuring arrangement 100 can be controlled and in particular conveyed by at least on or in a region 20 of a Wall portion 1 1, which forms an aperture 14 of an aperture region 10, a material 12 "is provided which the material 12 'of the carrier outside the aperture region 10 and in particular the material of the carrier 12 in the interior 12i or in its spatial means at least in his Substantially corresponds to composition, but with respect to at least one material or ionic species - possibly locally - increased concentration, so that by this - possibly locally - increased concentration of or in the Aperturinnenwand I ii of the aperture 14, the sealing system
• the carrier 12 of the so-called aperture area 10 is incorporated.
• This has at least one aperture 14, namely e.g. in the manner of a through hole, which completely penetrates the carrier 12 in its thickness or layer thickness direction, namely in the direction from the upper side 12a to the lower side 12b.
• a portion 70 of the electrolyte bath 40, 60, 70 is also provided.
• the wall region 11 or the aperture wall 11 is not completely modified in terms of concentration.
• the concentration-modified region 20 and the aperture wall 11 or the wall region 11 do not completely coincide in this embodiment. Rather, the concentration modification of the at least one material or ion species and thus the concentration-modified region 20 extend only up to a depth ⁇ of about 10 nm, namely viewed from the inner wall 11 of the aperture wall 11.
• FIGS. 1C and 1D show a square as the base area, so that spatially a vertical square prism results or a prism with a base in the manner of an oval, quasi a square or rectangle with rounded corners, such as this is shown in Fig. 1 D.
• the concentration modification of the at least one material or ion species and thus the concentration-modified region 20 extend only to a depth .DELTA. Of approximately 10 nm, specifically from the inner wall I.sub.i of the aperture wall 11.
• the lateral locality of the concentration-modified region 20 and thus restriction to the aperture region 10 are and in particular canceled on the aperture wall 1 1. Rather, here are the entire top 12a of the support 12 and the aperture inner wall I ii concentration-modified to a depth ⁇ of about 10 nm in their concentration of at least one material or ion species.
• FIGS. 3 and 4 show, analogously to FIG. 1A and likewise in schematic and sectional side view, other embodiments of the measuring arrangement according to the invention, in which there is a difference in that, according to FIG. 3, the wall region 11 for the aperture 14 is flush with the Top 12a of the substrate 12 closes and only protrudes beyond the bottom 12b of the substrate 12, so that a total of a kind of invagination from the top 12a inwardly to the compartment 60 for the aperture 14 is formed.
• a portion of the wall portion 1 1 of the aperture 14 rises from the top 12 a of the substrate 12 in the compartment 40, on the other hand, however, a portion of the wall portion 1 1 from the bottom 12 b of the substrate 12 in the compartment 60th into it.
• the heights above the upper side 12a and above the lower side 12b, about which the wall region 11 for the aperture 14 each rise, may be identical. This is not mandatory. In Fig. 4 they are designed differently.
• the cross section or diameter along the extension direction of the wall portion 1 1 perpendicular to the top 12a and perpendicular to the bottom 12b of the substrate 12 is constant in its course.
• FIG. 8 shows, in a schematic and sectional side view, an embodiment of the electrophysiological measuring arrangement 100 according to the invention, in which the aperture area 10 is formed by a wall area 11, which is designed overall in the manner of a patch pipette.
• 2A to 2D show in more detail section X from FIG. 1A and various modifications with regard to the design of modified region 20 with material 12 "having the modified or increased concentration of the at least one material or ion species.
• FIGS. 9A to 9E show, in a schematic and sectional side view, various measuring principles that can be used in the electrophysiological measuring arrangement 100 according to the invention and the corresponding electrophysiological measuring method according to the invention.
• a biological object O in this case, for example, a cell sucked and approximated to the aperture 14.
• the approaching mechanism may also be done in another manipulative manner, for example by means of a separate pipette, a laser pin or the like.
• the cell is deposited without any destruction and completely on the measuring aperture 14. If a measurement is carried out in this state, this is called a cell-attached mode.
• a membrane spot can be torn out of the membrane of the biological object O, so that according to FIG. 9C the torn-out membrane patch, a so-called patch, in the sealed or seamed state in FIG the measuring aperture 14 remains and actually forms the measuring object O.
• this measurement mode is also called Inside-Out mode.
• the cell O can be opened in its entirety by a renewed negative pressure, for example in the manner of a pressure surge, so that a transition takes place from the so-called cell-attached mode to the whole-cell mode according to FIG Fig. 9D, in which the measuring electrode 30 has direct access to the entire cell interior. That is, the cell interior is open to compartments 70 and 60, but substantially isolated from compartment 40. From the situation according to FIG. 9D, namely the whole-cell mode, it can be achieved by mechanical pulling that in turn a membrane fragment is torn out of the membrane of the biological object.
• FIG. 10 shows in detail the geometrical situation which results in a sealed or sealing attachment of a biological object O to be measured between its membrane M and the measuring aperture 14 and in particular the inner wall l Oi, I ii of the wall region 1 1 is present.
• the foremost portion of the wall portion 11 is formed by the concentration modified portion 20 with or from the modified concentration material 12 ".
• FIG. 10 again shows a whole-cell mode in which the entire cell O, with its interior, is open to the measuring electrode 30 and to the electrolyte compartments 60 and 70.
• This embodiment of the electrophysiological measurement arrangement 100 according to the invention is designed here in the manner of a patch pipette.
• the sealing resistance between the cell membrane M of the biological object O to be measured and the inner wall 10i, 11 of the wall area 11 of the measuring aperture 14 is improved in that the concentration-modified area 20 is made with or from the material 12 "with modified concentration is provided and interacts with the outside cell membrane M of the biological object O.
• the modification of the concentration in the concentration-modified region 20 it is possible according to type and strength to take into account the different situations on different surfaces of membranes M, be this cell membrane, membranes of organelles or membranes of artificial objects. This has not been possible so far and offers a possibility to promote an investment and a seal controlled or, once a seal is made to stabilize.
• the substrate is quasi occupied by a layer of cells O '.
• concentration-modified region 20 provided with the invention in the aperture wall 11 with or with modified concentration material 12 "
• a single cell O of the ensemble of cells O ' is to be measured in whole-cell mode analogous to the situation according to FIG. 9D with improved sealing or sealing attachment of an electrophysiological measurement accessible.
• FIG. 12 shows an arrangement of the measuring arrangement 100 according to the invention, in which the wall region 1 1 forming an aperture 14 is formed by an edge region, so that the aperture 14 is formed as a planar hole in the underlying substrate 12 and thereby the concentration-modified region 20 with or from the material 12 "forms the edge region with a correspondingly modified concentration of at least one material or ion species in order to promote the attachment and to increase the sealing resistance.
• electrolyte compartment electrolyte compartment, electrolyte bath to the top 12a of the support 12 60 electrolyte compartment, electrolyte bath to the back 12b of the support 12 70 electrolyte compartment, electrolyte bath in the lumen of the aperture 14

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• 2013
  • 2013-04-26 DE DE102013007295.6A patent/DE102013007295B4/de not_active Expired - Fee Related
• 2014
  • 2014-03-27 WO PCT/EP2014/000834 patent/WO2014173488A1/de active Application Filing
  • 2014-03-27 EP EP14716237.4A patent/EP2989456A1/de not_active Withdrawn
  • 2014-03-27 US US14/786,855 patent/US20160103116A1/en not_active Abandoned
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