WO2013083270A2 - Procédé et dispositif à microstructure permettant la mise en contact électrique de cellules biologiques - Google Patents

Procédé et dispositif à microstructure permettant la mise en contact électrique de cellules biologiques Download PDF

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Publication number
WO2013083270A2
WO2013083270A2 PCT/EP2012/005018 EP2012005018W WO2013083270A2 WO 2013083270 A2 WO2013083270 A2 WO 2013083270A2 EP 2012005018 W EP2012005018 W EP 2012005018W WO 2013083270 A2 WO2013083270 A2 WO 2013083270A2
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Prior art keywords
lipid membrane
microaperture
compartment
electrolyte
electrode
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German (de)
English (en)
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WO2013083270A3 (fr
Inventor
Jan Behrends
Gerhard Baaken
Srujan Kumar DONDAPATI
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Universitaetsklinikum Freiburg
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Universitaetsklinikum Freiburg
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    • 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

Definitions

  • the present invention relates to a method and a microstructure device for electrically contacting biological cells and other lipid membrane-bound lipid membrane particles such as e.g. Lipid vesicles or cell organelles.
  • ICAD Ion Channel Active Drugs
  • electrophysiology using the patch-clamp technique provides an extremely precise and meaningful analysis method for the ion channel function.
  • a glass pipette is filled with an opening diameter of about 1 ⁇ with an electrolyte solution and placed their opening on the surface of the cell membrane of a living cell.
  • an electrically sealed connection between the glass pipette and the cell membrane is produced when the pipette tip is attached. This makes it possible to measure currents through individual ion channels, which are located in the membrane patch directly below the pipette tip.
  • An ion channel protein in a cell membrane forms a pore through the cell membrane with specific conductivity for certain ions, ion currents through this pore can be detected, for example, with the voltage-clamp technique.
  • the voltage between two compartments is kept precisely constant via a feedback amplifier separated by this cell membrane (or another membrane or separation layer).
  • the current supplied to keep the potential constant is proportional to the ion current flowing through the membrane or separation layer. Due to the constancy of the voltage, this current can be converted directly into the conductivity according to Ohm's law.
  • the classical patch-clampA / eriahren is methodologically relatively complex and slow. Therefore, in recent years, chip-based (planar), automated patch-clamp methods have been developed with increased throughput.
  • the cell to be treated is sedimented over the microaperture of a support substrate surface, the microaperture forming the upper edge of a microcavity or microhole in that support substrate
  • a gigaseal between the cell membrane and the insulating carrier substrate can be produced analogously to the classical patchc-amp technique by further increasing the negative pressure or by applying larger voltage pulses (eg 800 V for 0.01 -10 ms) or by adding pore-forming peptides such as nystatin or amphotericin B, the membrane patch (patch) can be perforated above the microaperture and so a whole-cell recording can be realized.
  • larger voltage pulses eg 800 V for 0.01 -10 ms
  • pore-forming peptides such as nystatin or amphotericin
  • An electrode is disposed in each of the chambers, and a lipid membrane is spanned over the aperture which spans the aperture.
  • This object is achieved by the method according to claim 1 and the microstructure device according to claim 9.
  • Preferred embodiments of the method are objects of the subclaims 2 to 8, which are achieved in particular by preferred embodiments of the microstructure device according to claim 9.
  • the method according to the invention for electrically contacting at least one lipid membrane particle, in particular a biological cell comprises the steps of: providing a microstructure device which has at least one carrier substrate whose upper side has at least one microaperture having a diameter which is smaller than the diameter of the at least one first lipid membrane particle to be measured, at least one first compartment arranged below the microaperture, at least one second compartment arranged above the microaperture, at least one first electrode, which in the
  • At least one first compartment Contact with the at least one first compartment is arranged, at least one second electrode, which is arranged in contact with the at least one second compartment, and at least one electrical control device;
  • the lipid membrane particle having a membrane contact portion having a first membrane side with which the lipid membrane particle rests on the lipid membrane and is supported over the support substrate, and a second membrane side opposite the first membrane side; Generating an ohmic contact between the at least one electrolyte in the first compartment and the electrolyte present on the second membrane side of the at least one lipid membrane particle by generating at least one voltage pulse between the at least one first and the at least one second electrode by the at least one control device.
  • the described electrical contact can be formed more reliably than would be possible, for example, by spontaneous fusion.
  • this contact can be preferably without mechanical action on the lipid membrane of a lipid membrane particle, for example by inducing mechanical shocks between clamped lipid membrane and lipid membrane particles, or chemical action, eg the use of Fusion proteins.
  • lipid membrane particles can be contacted electrophysiologically with an increased throughput and, in particular, measured in the "whole-cell-recording" method, in particular by integrating a plurality of microapertures on a carrier substrate, which enables the parallel contacting and measuring of a multiplicity of lipid membrane particles ,
  • the lipid membrane is preferably a bilipid layer (double lipid layer) which is the typical basic constituent of natural biological (cell) membranes.
  • a "self-supporting" bilipid membrane (“BLM”) can be artificially produced. This is done, for example, by “brushing"("paintingmethod”,”painting") a solution of the first solvent (eg hexane, heptane, octane, nonane, decane, hexadecane or other alkanes, or a mixture of one or more of these substances) and
  • Such a lipid membrane may be further prepared by vesicle fusion on top of the carrier substrate or by the Langmuir-Blodgett / Langmuir-Schäfer technique or other methods
  • Lipid membranes are often used as models of natural membranes and serve, for example, as an environment for the investigation of membrane channel proteins that transport charges between two compartments separated by the membrane To effect lipid membrane particle over the microaper
  • lipids in particular preferably phospholipids, in particular lipids suitable for forming membrane-like double lipid layers, as are mentioned, for example, in the document "Baaken et al.” Or in US 2009/0167288 A1 has in particular molecular dimensions, may in particular be between 1 nm and 100 nm .
  • the spanned lipid membrane may have other constituents.
  • such a lipid membrane can also be a natural, biological lipid membrane which is applied over the at least one microaperture. This can be done by using a substantially planar patch of the membrane of a biological cell or by using a complete, treated or untreated biological cell which rests on the at least one microaperture.
  • a solvent namely as an electrolyte, in particular as a first or second electrolyte, which is placed below and / or above the membrane (molecular layer), in particular aqueous salt solutions in question, in particular physiological saline solutions, the electrophysiological measurement of Molkül harsh, eg lipid membrane and charge-transporting pores contained therein, eg channel proteins.
  • Suitable solvents in particular for carrying out measurements by means of the voltage clamping technique, are disclosed, for example, in the document "Baaken et al.” Or US 2009/0167288 A1
  • the first electrolyte and the second electrolyte can have different compositions or can have essentially the same composition.
  • the first electrolyte may have substantially the same composition as the interior of the lipid membrane particle
  • a lipid membrane particle is preferably a biological cell, but may also be an artificial or natural lipid vesicle or other lipid membrane structure, eg, a cell organelle or a cell fragment
  • the lipid membrane of the lipid membrane particle are preferably channel proteins that allow ionic conduction from one side to the other side of this lipid membrane, especially between the interior and exterior of the lipid membrane particle by treating the lipid membrane particle, eg a cell of a certain type, in its natural state.
  • channel proteins or other substances may also be introduced or introduced artificially.
  • any lipid membrane particle is suitable for the method according to the invention, which can form a gigaseal with the lipid membrane over the microaperture, in particular can fuse, so that the membrane tightly connected via the microaperture forms an electrical barrier suitable for measuring ion channel currents on the order of ⁇ to fA through this membrane.
  • the ion currents to be measured are preferably in the range between 1 ⁇ and 1 fA, preferably between 500 nA and 10 fA.
  • the lipid membrane particle may also be a composite of other lipid membrane particles, e.g. a cell network or a vesicle network.
  • lipid membrane particles e.g. a cell network or a vesicle network.
  • a plurality of lipid membrane particles, in particular cells are added to the second electrolyte, in particular before, during or after this electrolyte has been arranged in the at least one second compartment.
  • the lipid membrane particles are substantially single, that is essentially not as a cell composite. This separation can be carried out mechanically by trituration (gentle trituration of cell networks in rapid fluid streams at bottlenecks) or in particular enzymatically, for example by using trypsin.
  • the positioning of the lipid membrane particles, in particular cells, above the at least one microaperture can take place uncontrolled, for example by using a cell suspension of high density, so that a lipid membrane particle sediments with high probability above a microaperture.
  • a positioning device for positioning individual lipid membrane particles may also be provided. This positioning device may be provided by the microstructure device and in particular may be part of it.
  • the positioning device may comprise guide sections for mechanically guiding cells, for example micro-channel elements or micro-channel elements.
  • the positioning device may in particular be an electrical control device designed as a positioning device, which controls, in particular, the first and the second electrode.
  • the control device is designed to carry out a positioning method in which an inhomogeneous alternating electric field is generated by applying an alternating electric field between the first and second electrodes via the microaperture.
  • the microaperture which in particular has a field line course in which the field lines of the microaperture are "compressed" in the region of the microaperture, which is caused by the voltage drop across the microaperture
  • This can be achieved in particular by alternating electric fields with frequencies in the range between 100-1000 kHz and with field strengths around 10-120 V / cm.
  • the at least one electrical control device is designed to at least automatically perform a voltage pulse at a predetermined time interval t1 after a positioning process for positioning the at least one lipid membrane particle on the at least one microaperture is completed, preferably t1_u ⁇ t1 ⁇ t1_o, and the lower limit t1_u respectively preferably e selected from the set of times ⁇ 0s, 1s, 2s, 3s, 5s, 10s, 60s ⁇ , and the upper limit t1_o each preferably selected from the set of times ⁇ 5s; 10s; 30s; 60s; 200s; 300s ⁇ .
  • a lipid membrane particle is placed or positioned concentrically above a microaperture.
  • the lipid membrane particle is preferably positioned on the microaperture, it being possible that at least the lipid membrane is arranged between lipid membrane particles. It is also possible that the surface of the carrier substrate has a further coating.
  • the at least one voltage pulse has a duration T which is preferably between 0.1 ms and 500.0 ms, preferably between 1 ⁇ and 10.0 s, preferably between 10 ps and 10.0 s, preferably between 100 ps and 10 , 0 s, preferably between 1 ms and 10.0 s, preferably between 10 ms and 10.0 s, wherein in particular the range between 0.1 ms and 500 ms has proven to be particularly efficient in experiments.
  • the voltage pulse is preferably a direct current voltage (DC) between the at least one first electrode and the at least one second electrode.
  • more than one voltage pulse is carried out, preferably a number of successive voltage pulses in a time interval t2 between 1 and 100 voltage pulses, preferably between 1 and 10 voltage pulses, preferably t2_u ⁇ t2 ⁇ t2_o, and the lower limit t2_u each preferably selected from the set of times ⁇ 0s, 1 ⁇ , 10ps, 100ps, 1ms, 10ms, 100ms ⁇ , and the upper limit t2_o each preferably selected from the set of times ⁇ 100ps, 1ms, 10 ms, 100 ms, 1 s, 10 s ⁇ . If a plurality of voltage pulses are used, they are preferably applied periodically.
  • the ratio between pulse duration and period can be 0 and 1 and is preferably between 0.1 and 0.5.
  • the duty cycle is 0.5.
  • a plurality of voltage pulses are applied in a non-periodic pattern or in a random sequence.
  • the at least one voltage pulse can also be applied as an alternating voltage, which in particular can have a rectangular or sinusoidal oscillating profile or can be sawtooth-shaped.
  • a sawtooth-shaped course may have one or more sawtooth-shaped voltage profiles, in particular may be periodic or non-periodic, wherein the sawtooth-shaped voltage curve may each have a slower increase than drop in voltage.
  • the duration of the increase may be between 5 s and 120 s.
  • the frequency of this alternating voltage can be, for example, between 1 kHz and 10 kHz, the amplitude for example between 100 mV and 1000 mV, in particular with a minimum distance of the first and the second electrode between 1 mm and 10 mm.
  • the at least one voltage pulse has an amplitude V_A whose magnitude is preferably between 100 mV and 1000 kV; more preferably between 100 mV and 2000 mV, which has proven to be particularly efficient in experiments, in each case in particular at a minimum distance of the first and the second electrode between 1 mm and 10 mm.
  • V_A amplitude whose magnitude is preferably between 100 mV and 1000 kV; more preferably between 100 mV and 2000 mV, which has proven to be particularly efficient in experiments, in each case in particular at a minimum distance of the first and the second electrode between 1 mm and 10 mm.
  • an electrical measurement is carried out on the at least one lipid membrane particle between the at least one first and the at least one second electrode by the at least one control device, wherein the measurement of the detection of ion currents between the interior of the lipid membrane particle and serves the second electrolyte.
  • This measurement is preferably carried out in a "whole-cell" configuration on a biological cell or another particle delimited by a lipid membrane, and this measurement was preferably carried out in a "voltage-clamp” measuring method in which, in particular, the voltage between the at least a first electrode and the at least one second electrode is kept constant and thereby the currents between the electrodes are decoded, which in particular correspond to the ion currents through channel proteins and in particular in the range from 0.1 femtoamps (fA) to 1 nanoampers (nA)
  • fA 0.1 femtoamps
  • nA nanoampers
  • another electrical measuring method can also be used.
  • the electrical measurement is carried out at a time interval t3 after the ohmic contact between the at least one first electrolyte and the electrolyte of the aperture-side remote lipid membrane of the particle over the Mikroapertur, in particular the interior of the at least one lipid membrane particle, preferably t3_u ⁇ t3 ⁇ t3_o, and the lower limit t3_u each preferably selected from the set of times ⁇ 0s, 1ps, 10ps, 100ps, 1ms, 10ms, 100ms, 1s, 10s, 60s ⁇ , and upper limit t3_ each preferably selected from the set of times ⁇ 100 ps, 1 ms, 10 ms, 100 ms, 1 s, 10 s, 60 s, 300 s, 10 min, 20 min ⁇ .
  • the time of producing the ohmic contact in particular by observing the electrical resistance or another suitable for the detection of the ohmic contact electrical Size between the first and second electrodes are detected.
  • the at least one electrical control device is designed to automatically start this electrical measurement in this time interval t3 after the generation of this ohmic contact, in particular without requiring user activity. This has the advantage that the measurement can be automated and the throughput can be increased in such a method or a device designed to perform it.
  • a method for electrically contacting at least one, at least two, or in particular a plurality of lipid membrane particles is provided that is electrically measured by the at least one control device, in particular at the same time or temporally overlapping or chronologically successively measured electrically, whether over a Mikroapertur, in particular at which particular time T1, the ohmic contact is made to a superposed lipid membrane particles.
  • this electrical measurement is automatically carried out by the at least one control device.
  • the at least one control device is preferably designed to carry out this method, for example by means of suitably provided programming or suitably designed microelectronics of the at least one control device.
  • the electrical measurements can be performed with high throughput.
  • the microstructure device preferably has at least one, preferably at least two and preferably a multiplicity N of microapertures, which are preferably each formed by a microcavity and preferably each have an individual first electrode.
  • each microaperture is associated with an individual second electrode; but it can also be a total of a smaller number of second electrodes are provided as the first electrode.
  • a single second electrode may also be provided for all individual first electrodes.
  • the direction designation "top” or “upward” in the present case denotes the direction of the normal vector which points away from the microcavity plane from the plane of the microaperture. This direction is also defined as the direction of the positive z-axis of a Cartesian coordinate system (the origin of this coordinate system is preferably not determined hereby).
  • the direction designation "down” or “down” designates in the present case the direction of the normal vector pointing into the microcavity from the plane of the microaperture (also referred to as the direction of the negative z-axis of this coordinate system).
  • the plane of the microaperture is the plane that is coplanar with the surface framed by a planar microaperture (microaperture surface).
  • the electrical control device preferably has at least one printed circuit board and preferably components with integrated circuits (ICs), which are preferably arranged on at least one printed circuit board.
  • the control device preferably has programmable electrical circuits.
  • the control device preferably has a signal processing device with which the at least one measurement signal is detected by which the ohmic contact of at least one microaperture is observed.
  • the signal processing device is preferably designed for processing analog signals, which is referred to as an analog signal processing device.
  • the signal processing device to a digital data processing device, in particular a computing unit (CPU) or a microprocessor device, a data bus, data storage, one or more interfaces for device internal or external data transmission, and / or one or more signal connections, such as wired or wireless having other electrical facilities.
  • the control device is designed to digitally detect the at least one electrical measured value, in particular to process digitally, in particular to evaluate digitally and in particular to store digitally.
  • the control device preferably has a measured value memory, in particular measured data memory, for storing the at least one measured value.
  • a measured value memory in particular measured data memory
  • the measurement data memory is preferably housed in a physically rewritable memory device, e.g. RAM, FLASH memory, EEPROM, but can also be arranged in other memory devices.
  • the at least one control device has at least one program data memory in which a program code can be stored.
  • the program code is preferably designed to perform a function of the control device, and in particular configured to use the at least one electrical measurement value, and to evaluate this.
  • This function may comprise the steps of controlling the method for monitoring the gigasel via at least one microaperture and / or the generation of the at least one voltage pulse and / or the performance of at least one electrical measurement at a microaperture.
  • the program code is designed to effect, in a first step, at least one voltage pulse between the first and the second electrode, with which an ohmic contact between the first electrolyte and the interior of the above the lipid membrane and the microaperture arranged lipid membrane particle can be generated, and in particular a second step, which is provided in particular for the automatic execution by the at least one control device at a predetermined time interval after the first step, to carry out an electrical measurement between the first and the second electrode, which is used for the detection of ion currents between the interior of the lipid membrane particle and the second electrolyte is used.
  • this adaptation may be, for example, the loading of software or programming a programmable microprocessor device, but already has all means to actually fulfill this function by, for example, the required program code or already has the required software, in particular in the form of a firmware of the microstructure device or measuring device.
  • the means for performing this function include in particular an evaluation device.
  • means for carrying out this function, in particular the evaluation device, for example, correspondingly configured electrical circuits may have, for example, an analog signal representing the measured value, evaluate and compare for example by means of a comparator circuit with a reference signal (reference value).
  • control device preferably has an electrical evaluation device, which may comprise electrical circuits and / or suitable program code.
  • an electrical evaluation device may comprise electrical circuits and / or suitable program code.
  • it can be detected by the evaluation device whether and / or when a measured electrical variable falls below or exceeds a predetermined reference value, as a result of which the presence of an ohmic contact can be detected.
  • Such an electrical quantity may be the resistance between the at least one first electrode and the at least one second electrode.
  • the ohmic contact is preferably defined so that the measured resistance R is greater than the preferably predetermined reference value R Ref, which is in each case preferably between 0.1 and 2 gigaohms and preferably between 0.5 or 1 gigaohm, and in the control device can be stored.
  • R Ref preferably between 0.1 and 2 gigaohms and preferably between 0.5 or 1 gigaohm
  • Such electrical quantity may also be the electrical current, the voltage or an impedance.
  • a microaperture, over which a lipid membrane is stretched preferably has a resistance R_LM in Range of preferably greater than 1 gigaohm, or more preferably R LM> 10 gigaohms, the latter range in particular indicating the presence of a stable lipid bilayer over the microaperture. Destruction of this stretched lipid membrane would lead to resistances R_D in the range ⁇ 20 megohms, in particular R_D ⁇ 10 megohms.
  • t4 t3
  • the first electrode is preferably arranged in the first compartment, which is preferably formed by a microcavity integrated in the carrier substrate.
  • the carrier substrate has a first layer, within which the electrode is at least partially or substantially completely arranged.
  • a layer may be a layer in the carrier substrate or a coating of the carrier substrate. Such a layer is preferably at least partially or completely substantially planar. If the electrode is arranged completely in the first layer, this preferably means that no portion of the electrode protrudes from the first layer or does not penetrate the imaginary main planes, which envelop the layer, for example, upwards and downwards.
  • the carrier substrate may be at least a layer, in particular more than two layers or a plurality of layers, wherein the microcavity may be arranged in one of these layers, a plurality of these layers or each of these layers or may not be arranged.
  • the microcavity may extend through at least one or more of these layers and / or the first electrode may be arranged in a further layer, in particular below this layer lying at least one layer.
  • the diameter of the cavity in a first electrode layer may be greater than the diameter of the cavity in an overlying layer.
  • the first and / or second layer of the carrier substrate is preferably a coating of the carrier substrate, which preferably forms its upper side and is preferably hydrophobic, preferably consists of a photosensitive layer, in particular an epoxy resin or photoresist, or has these.
  • the coating preferably consists of a polymer, preferably of epoxy resin or preferably of a photoresist, eg SU8, or comprises such a material.
  • This photoresist is preferably SU8, the dried layers of which are hydrophobic.
  • SU-8 is a commercially available photoresist from Microchem Corp., USA, and belongs to the group of negative resists. Like most resists, SU-8 consists of the three components base resin, solvent and photosensitive component. These are particularly suitable for producing the microstructure device, since in particular many bilipid layers form on these resists particularly reliably and since they can be processed photolithographically, for example to produce microstructures, and are chemically relatively inert.
  • the coating of the carrier substrate may further preferably comprise polytetrafluoroethylene (PTFE) or consist of this material.
  • PTFE polytetrafluoroethylene
  • the microstructure device has at least one feed device, by means of which the electrode is electrically contactable via a feed line distance.
  • one (or each) electrode is assigned at least one supply device.
  • a feeder device may comprise or consist of a metallic conductor, which may consist for example of gold, titanium, nickel-chromium, platinum, or silver or has one or more of these materials.
  • the supply line device is preferably arranged completely or at least partly in the first layer.
  • the electrode preferably forms at least part of the inner wall of the microcavity.
  • the microcavity is preferably designed such that the inner volume of the microcavity is limited by the area framed by the microaperture and the inner wall (or several inner walls) of the microcavity.
  • the at least one inner wall of the microaperture is preferably formed from lateral inner walls and a bottom wall.
  • the bottom wall of the microcavity is preferably formed completely or at least partially from the contact side of the electrode.
  • the electrode can also be at least partially or completely surrounded by the internal volume of the microcavity.
  • the electrode may be partially self-supporting.
  • this first layer is disposed substantially below or at least partially below the microcavity. This makes possible, in particular, the simple production of the electrode, which is preferably arranged below the microcavity.
  • the carrier substrate has a second layer within which the microcavity is at least partially or substantially completely disposed.
  • the microcavity is at least partially, preferably with less than half or a quarter of its internal volume, also arranged in the first layer.
  • the carrier substrate is preferably formed at least in sections or substantially completely planar.
  • the carrier substrate has one or more microstructures, that is, spatial highlights and / or pits with small dimensions, e.g. these microcavities e.g. a few nanometers, a few micrometers, a few tens of micrometers or a few hundred micrometers.
  • microstructures can be e.g. produce by known optical lithographic process in which structures defined by means of optical masks layers on a support substrate are applied and partially removed again.
  • the carrier substrate preferably has an upper side, which is preferably at least partially or substantially completely planar.
  • the upper surface preferably has at least one microaperture, preferably a number N of microapertures, where N is in each case preferably between 2 and 2000, greater than 2000, preferably between 2 and 400, between 4 and 100, between 4 and 50 or between 4 and 20 ,
  • N is in each case preferably between 2 and 2000, greater than 2000, preferably between 2 and 400, between 4 and 100, between 4 and 50 or between 4 and 20 .
  • the lipid membrane may in particular be a double lipid membrane, that is to say consist of two individual layers of lipid layers arranged one above the other, wherein a single layer consists in particular of self-assembled molecules.
  • a molecular membrane may be made artificially, in particular, a bilipid layer of lipid molecules may be prepared by painting, by vesicle fusion or Langmuir-Blodgett / Langmuir-Schäfer technique or related methods.
  • Such artificial lipid membranes are often used as models of natural membranes. They serve, for example, as an environment for the investigation of membrane proteins which, for example, transport charges through the membrane between two compartments separated by the membrane.
  • Such a membrane can also be a natural, biological membrane applied over the at least one microaperture. This can be done by using a substantially planar patch of the membrane of a biological cell, or by using a complete, treated or untreated biological cell which rests on the at least one microaperture, in particular the thickness of the molecular layer molecular dimensions, may in particular be between 1 nm and 100 nm.
  • a microaperture is understood to mean the open cross-section, e.g. through an opening, e.g. a recess or a hole, in a -particularly planar- surface of the top of the carrier substrate results.
  • the aperture is therefore in particular preferably planar insofar as the substrate surface is planar.
  • the shape of the microaperture contour is preferably circular, ellipsoidal, triangular, quadrangular or polygonal.
  • the maximum, minimum or average diameter of the individual microaperture is preferably less than 1000 ⁇ m and is preferably between 5 nm and 500 ⁇ m, preferably between 500 nm and 500 ⁇ m, preferably between 5 nm and 1 ⁇ m, preferably between 1 ⁇ m and 250 ⁇ m, preferably between 2 pm and 50 pm, preferably between 2 pm and 10 pm, or between 5 pm and 150 pm.
  • a molecular layer over the microaperture can be generated, which is not possible in macroscopic apertures with diameters of several millimeters in the rule.
  • microapertures can be achieved by selectively removing a photosensitive layer, e.g. Photoresist, which is mounted on the top of the carrier, be generated by optical lithography, such.
  • a photosensitive layer e.g. Photoresist
  • the microaperture can also form the edge of a hole that extends from the top to the back of the carrier substrate, for example by chemical etching or by irradiation be achieved with laser or other high-energy radiation.
  • Nanoapertures in particular with diameters between 5 nm and 1000 nm, can, for example, by applying a further layer, for example silicon oxide or silicon nitride or Graphene, or polymers, such as polyimide, or polyelectrolytes are formed on the carrier substrate already containing microapertures, if the further layer either already contains nanometer-sized holes.
  • a further layer for example silicon oxide or silicon nitride or Graphene, or polymers, such as polyimide, or polyelectrolytes are formed on the carrier substrate already containing microapertures, if the further layer either already contains nanometer-sized holes.
  • such nanoapertures can also be produced by using conventional nanostructuring methods such as FIB (focussed ion beam), e-beam (electron beam lithography) or wet-chemical or dry etching methods.
  • the arrangement of the number N of microapertures preferably corresponds to an array, preferably a periodic lattice, in which the position of the microapertures or the microaperture centers can be described by one or a few lattice parameters.
  • the arrangement in a periodic grating has advantages in the design of a parallelized sensor system in which many identical measuring points are to be created.
  • the microapertures may also be arranged in a non-periodic or incompletely periodic pattern.
  • the carrier substrate is preferably made of glass or has glass. However, it may also consist of or may at least comprise a semiconductor material, e.g. Si / Si02. Other materials are also possible.
  • the upper side of the carrier substrate preferably has a coating. This is preferably hydrophobic, but may also be hydrophilic. The advantage of a hydrophobic top surface is that many types of bilipid layers form on such surfaces particularly reliably.
  • a “hydrophobic" boundary layer is understood to mean a layer on which a water droplet has a contact angle of at least 70 °, preferably at least 80 °, 85 ° or 90 °, preferably between 80 ° and 130 ° or between 90 °
  • the present definition of the term "hydrophobic” may be broader than is commonly used in the literature, where the term most often refers to contact angles greater than 90 °.
  • Such contact angles can be easily determined by means of commercially available contact angle measuring devices or by evaluation of light microscopic cross-sectional images of the drops (room temperature, standard conditions).
  • the contact angles are each preferably between 70 ° and 0 °, 80 ° and 0 °, 85 ° and 0 ° or 90 ° and 0 °.
  • a carrier substrate has at least one microcavity, or preferably an array of microcavities, one or each microcavity being open at the top and terminating in one of said microapertures in the top of the carrier substrate.
  • the molecular membrane to be measured can then be formed such that it covers at least one microaperture or several microapertures.
  • a microcavity is a depression in the top whose depth can be the same size as a possible called microaperture diameter, or can be deeper or less deep.
  • Each cross section through the depression in a plane parallel to the plane of the microaperture) preferably has the same cross section as the microaperture, which opens the microcavity upwards.
  • the microcavity can in particular be cylinder-like or cuboidal. But it can also be hollow cone (dull) shaped or have another shape with variable cross-section.
  • the measuring arrangement or the microstructure device preferably has at least one sensor device, which in particular has a sensor for electrophysiological examinations on the molecular layer, in particular bilipid layer.
  • the sensor device may comprise this first electrode in the first compartment, which is arranged on this side of the microaperture on the carrier substrate, and may further comprise at least one further electrode (counter electrode) on the other side of the microaperture, which is arranged in the first compartment in the electrolyte above the lipid membrane.
  • An electrode is preferably a redox electrode, preferably a "second type" redox electrode, preferably a calomel electrode or, more preferably, an Ag / AgCl electrode.
  • the electrode is preferably a non-polarizable electrode which facilitates easy transition of the ionic charge carriers in the electrolyte into electronic ones Charge carriers in the metal are allowed, preferably Ag / AgCl electrodes, preferably in combination with chlorine ion-containing measurement solutions (electrolyte).
  • This sensor device is preferably designed for carrying out the voltage clamping technique, by means of which the smallest currents in the nanoampere range and below can be measured, especially in the picoampere range, eg using a voltage-clamp amplifier or a patch-clamp amplifier (eg an axopatch 200B, Axon Instruments, Foster City, CA, operated in resistive feedback mode.)
  • the sensor device may comprise an array of sensors which may be arranged in the carrier substrate or on the surface thereof or partially automated measuring device, in particular a robot system, in particular with automatically controlled electrolyte transfer devices for the automatic transfer of an electrolyte, in particular of the first and / or the second electrolyte to at least one microaperture, in particular to for automatic measurement at a plurality N of microapertures, wherein preferably 2 ⁇ N ⁇ 1000, and preferably 3 ⁇ N ⁇ 100.
  • the microstructure device and / or its at least one control device and / or a program code of the at least one control device is designed to perform the inventive method partially or completely.
  • the microstructure device used in carrying out the method according to the invention is preferably a microstructure device according to the invention. Further preferred embodiments of the method according to the invention can be derived from the description of the microstructure device according to the invention. Further preferred embodiments of the microstructure device according to the invention can be derived from the description of the method according to the invention.
  • Fig. 1a shows a first embodiment of the invention
  • Microstructure device in a schematic, vertical cross-section, with spanned lipid membrane and arranged above cell before a
  • Fig. 1b shows the microstructure device of Fig. 1a after a voltage pulse has been generated.
  • Fig. 2a shows a second embodiment of the invention
  • Microstructure device with a variety of microapertures.
  • Fig. 2b shows a third embodiment of the invention
  • Microstructure device with a variety of microapertures.
  • FIG. 3 shows a flow chart of an embodiment of the invention
  • FIG. 4a shows current-voltage measurements measured with a microstructure device according to FIG. 1, wherein an RBL cell is arranged on a bilipid layer over the microaperture and the interior of which is electrically contacted with the first electrolyte.
  • FIG. 4 b shows current-voltage measurements of a reference measurement by means of a classic patch clamp on a BL cell, as used in the example of FIG. 4 a.
  • FIG. 1a shows the microstructure device 1 for electrical measurement on lipid membrane particles, in particular on biological cells 30.
  • the microstructure device 1 has a carrier substrate 2 with a top 3 for supporting a lipid membrane 20 and a cell 30 above this lipid membrane.
  • the upper side 3 has a microaperture 4, which has a diameter of 10 ⁇ m, which is smaller than the diameter of a lipid membrane particle to be measured, in particular a biological cell 30.
  • the microstructure device 1 has a first compartment 5 arranged below the microaperture 4 for receiving a first electrolyte 6 and a second compartment 7 arranged above the microaperture 4 for receiving a second electrolyte 8 containing the cell 30.
  • the first compartment 5 is designed as a microcavity 5, in which the inner side walls of the compartment open out into the microaperture.
  • the microcavity has essentially the same horizontal cross-section over its entire height as the microaperture and is therefore also referred to as microcuvette 5.
  • the microcuvette 5 is here in particular hollow cylindrical.
  • the microstructure device 1 has a first electrode 11, which is arranged to make electrical contact with the first electrolyte 6 in contact with the at least one first compartment 5.
  • the first electrode 11 is an Ag / Ag-Cl
  • the first electrode which is arranged with respect to the z-axis concentric with the microaperture.
  • the first electrode preferably has at least half the minimum diameter of the microaperture.
  • the first electrode has the same diameter as the microaperture, or a larger diameter, the latter being able to be achieved by modifying the shape of the microcavity.
  • the relatively large area of the first electrode has the advantage that only a relatively low current density is produced at the surface of the electrode itself in the case of the relatively large currents which can occur when the surge is applied. As a result, the electrode is more stable and a subsequent electrical measurement more reliable.
  • the microstructure device 1 further has a second electrode 12, which is arranged to make electrical contact with the second electrolyte 8 in contact with the at least one second compartment 7.
  • the second electrode 12 may be movably disposed opposite to the second compartment 7 to facilitate the addition of the second electrolyte.
  • the lipid membrane 20 is a bilipid layer.
  • a bilipid layer generally refers to a bilayer consisting essentially of two superimposed lipid monolayers arranged by self-assembly in polar solvents such as water.
  • the lipid membrane particle here a cell, has an inner space 31 which is filled with an electrolyte, here a cytoplasm.
  • the lipid membrane particle has a lipid membrane 32 serving as a shell with a membrane contact section 33, which has a first membrane side 34, with which the lipid membrane particle rests on the lipid membrane and is supported above the carrier substrate, and a second membrane side 35 opposite the first membrane side.
  • the lipid membrane 32 has channel proteins 36, by means of which an electrical conduction of ions between the inside and the outside of the lipid membrane particle is possible.
  • the microstructure device 1 has an electrical control device 15, which controls the voltage difference between the first and the second electrode, or the Current between these electrodes controls, and which is designed so that in a first step, at least one voltage pulse between the first electrode 1 1 and the second electrode 12 is generated, wherein the voltage pulse, an ohmic contact between the first electrolyte 6 and the interior 31 of the lipid membrane particle 30 arranged above the lipid membrane 20 and the microaperture 4, and in particular in a second step, which is provided in particular for automatic passage through the at least one control device 15 at a predetermined time interval after the first step, an electrical measurement between the first Perform electrode 11 and the second electrode 12, which serves for the detection of ion currents between the interior of the lipid membrane particle and the second electrolyte.
  • Fig. 1a shows the situation of the cell-positioned microstructure device before a voltage pulse is generated.
  • 1 b illustrates the situation after the voltage pulse has been carried out, which leads to an electrically tight connection of the interior 31 of the cell with the first electrolyte 6, so that ion currents can be measured by the ohmic contact of the first electrolyte with the cytoplasm.
  • the seal is likely to be due to a fusion of the planar lipid membrane 20 and the cell membrane 32 in a region 21 along the edge of the microaperture. This fusion is believed to occur through self-assembly of the lipid molecules.
  • the lipid membrane and membrane contact portion 33 is at least partially perforated immediately above the microaperture, and probably completely removed (as shown in Fig. 1b), as was also demonstrated in the case of a bilipod layer in Fig. 4b.
  • a gigaseal is created between the two membranes, which is measurable by the microstructure device.
  • the current through channel proteins 36 can be measured, which essentially form the only electrical feedthroughs between the interior 31 plus the electrolyte 6 and the exterior of the cell 30 (the second electrolyte 8).
  • FIG. 2 a shows a microstructure device 60 in which a plurality of microcavities 65, in particular microcuvettes 65, are provided in a carrier substrate 62 each open into a Mikroapertur 64. Each of these microcuvettes has a first electrode 11 'which substantially completely covers the bottom of the microcuvette. Above a microaperture 64, an individual second compartment 7 'with an electrolyte 8' is arranged in each case. In each compartment, an individual second electrode 12 'can be arranged. These electrodes are individually controllable by the conduit means 69.
  • the control device 15 ' is designed to individually control the pairs of the first electrode and the second electrode for each microcavity and, in particular, to control them in parallel in time.
  • each measuring station consisting of microcavity, microaperture and first and second electrodes can be individually controlled, in particular measured or monitored (current and / or voltage and / or resistance), the at least one voltage pulse can be individually controlled, and a subsequent electrical measurement can be done individually.
  • an increased working throughput of the microstructure device is made possible.
  • FIG. 2 b shows a microstructure device 80, in which a plurality of microcavities 85, in particular microcuvettes 85, are provided in a carrier substrate 82, each opening into a microaperture 84.
  • Each of these microcuvettes has a first electrode 11 "which essentially completely covers the bottom of the microcuvette
  • a second common compartment 7" with an electrolyte 8 "in which a common second electrode 12" can be arranged
  • These electrodes 11 are each individually actuated in a time-shifted manner
  • the control device 15" is designed to individually control the pairs of the first electrode and the second electrode for each microcavity and, in particular, to control them in a time-offset manner.
  • each measuring station consisting of microcavity, microaperture and first and second electrodes can be controlled individually, in particular measured or monitored one after the other (current and / or voltage and / or resistance), the at least one voltage pulse can be controlled individually one after the other, and a subsequent electrical measurement can be carried out individually in chronological succession.
  • these functions can also be carried out simultaneously in time.
  • a microstructure device can have several Have groups of measuring stations that are independently and or jointly operable.
  • a method 100 according to the invention may in particular comprise the steps of providing a microstructure device according to the invention, providing at least one first electrolyte in the at least one first compartment and forming a lipid membrane on top of the carrier substrate, the lipid membrane comprising the at least one Microaperture spans (101); Checking the electrical resistance between the first and second electrodes, which should in particular be 5-30 gigaohms (102); Providing at least one second electrolyte in the at least one second compartment; Providing at least one lipid membrane particle above the at least one microaperture and above the lipid membrane in the at least one second compartment and; in particular renewed checking of the electrical resistance between the first and second electrodes, which in particular should continue to be 5-30 gigaohms (103); Generating an ohmic contact between the at least one first electrolyte and the electrolyte present on the second membrane side of the at least one lipid membrane particle by generating at least one voltage pulse between the at least one first and the at least one
  • Epifluorescence photographs show a fluorescent dye-labeled bilipid layer (99.5% diphytanoylphosphocholine, 0.5% La-phosphatidylethanolamine N- (lissamine rhodamine B sulfonyl)) generated by coating in electrolyte (150 mM KCl) over an MEC.
  • electrolyte 150 mM KCl
  • the microaperture is spanned by a bilipid layer which is destroyed upon electroporation, as evidenced by the lack of fluorescence inside the microaperture (dark circle).
  • the homogeneous wetting of them surrounding SU8 surface with the phospholipid remains stable, which is recognizable by the fluorescence in this area.
  • the odell cells used were RBL-1 or RBL-2H3 (rat basophilic leukemia) cells. These constitutively express a K + -selective ion channel which, due to a block of intracellular (poly) cations (eg, Mg2 +, spermine), exhibits a very characteristic current-voltage relationship, at membrane potentials that are positive from the electrochemical K + equilibrium potential (EK). is occluded, while at potentials that are more negative than EC, it increasingly conducts. Thus, the channel causes a so-called anomalous or inward rectification of the membrane, a very characteristic phenomenon that can not be mimicked, for example, by nonspecific conductivities. This is crucial for demonstrating successful electrical contacting of cells via MECs.
  • a K + -selective ion channel which, due to a block of intracellular (poly) cations (eg, Mg2 +, spermine), exhibits a very characteristic current-voltage relationship, at membrane potentials
  • the model cells have a diameter of about 20 pm, 6-10 pm MECs were used for the contacting experiments.
  • the MECAs were wetted with electrolyte solution which corresponds in composition to the intracellular pipette solution in classical patch-clamp measurements (130 mM KCl, 10 mM NaCl, 2 mM MgCl 2, 4 mM CaCl 2, 10 mM HEPES, 10 mM EDTA, pH 7.4 ).
  • a phospholipid layer was applied, resulting in the formation of a stable bilipid layer over the MECs (resistances> 10Gohms).
  • Figures 4a and 4b show how, in these cases, by applying a series of voltage jumps, the characteristic current-voltage relationship of the inwardly rectifying K + conductivity of an RBL cell can be detected. Such discharges were stable for up to 30 minutes or one hour. It is believed that the electroporation of the bilipid layer leads to destabilization of the directly lying cell membrane, so that an electrical access to the interior of the cell is formed. With a certain probability, this leads to the electrically induced "fusion" of the two membranes, at least in such a way that an electrically dense connection of cell and lipid membrane for the measurement of transmembrane ion currents is possible ..
  • FIG. 4a shows the current-voltage relationship at an MEC in the configuration as shown in FIG. It corresponds to the characteristic, inwardly rectifying behavior of the membrane of an RBL cell.
  • the invention makes it possible to produce an externally dense electrical access to the interior of the lipid membrane particle, in particular the cell, as Prerequisite for electrophysiological measurements, without the need for mechanical or chemical action on the cell membrane.
  • This makes it possible, for the first time, to use platforms such as the Microelectrode Cavity Array developed by the inventors for measurements which, for reasons of higher integration density and better measurement resolution, omit microfluidic connection of the first compartment connected to the active, first electrode.
  • This opens up the possibility of increasing the throughput of electrophysiological measurements on cells (now several hundred measurements per day) by a factor of 10 to 100.
  • the present invention can provide a reliable and stable platform for high throughput electrophysiology and can be inexpensively marketed.
  • the microstructure device according to the invention and the method according to the invention can each be used for measuring ion currents through the membrane of the lipid membrane particle to be measured, in particular as a function of the predetermined concentration of pharmacologically active substances in the second electrolyte.

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Abstract

L'invention concerne un procédé de mise en contact électrique d'au moins une particule à membrane lipidique, en particulier d'une cellule biologique, qui comprend les étapes consistant : à produire un dispositif à microstructure comprenant au moins un substrat support, dont la face supérieure est pourvue d'une micro-ouverture d'un diamètre inférieur à celui de la particule à membrane lipidique à mesurer, au moins un premier compartiment placé au-dessous de la micro-ouverture, au moins un deuxième compartiment placé au-dessus de la micro-ouverture, au moins une première électrode qui se trouve en contact avec le premier compartiment, au moins une deuxième électrode qui se trouve en contact avec le deuxième compartiment, et au moins un dispositif de commande électrique; à placer au moins un premier électrolyte dans le premier compartiment; à réaliser une membrane lipidique sur la face supérieure du substrat support, la membrane lipidique recouvrant ladite au moins une micro-ouverture; à placer au moins un deuxième électrolyte dans le deuxième compartiment; à placer au moins une particule à membrane lipidique au-dessus de la micro-ouverture et au-dessus de la membrane lipidique dans le deuxième compartiment, la particule à membrane lipidique présentant une zone de contact avec la membrane, qui présente un premier côté membrane, par lequel la particule à membrane lipidique repose sur la membrane lipidique et s'appuie au-dessus du substrat support, et un second côté membrane opposé au premier côté membrane; à établir un contact ohmique entre le premier électrolyte et l'électrolyte présent contre le second côté membrane de la particule à membrane lipidique par production d'au moins une impulsion de tension entre lesdites première et deuxième électrodes par l'intermédiaire dudit au moins un dispositif de commande. L'invention concerne également un dispositif à microstructure pouvant être utilisé à cet effet.
PCT/EP2012/005018 2011-12-06 2012-12-05 Procédé et dispositif à microstructure permettant la mise en contact électrique de cellules biologiques Ceased WO2013083270A2 (fr)

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