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WO2008111047A1 - Micropointes pour utilisation en interaction électrique avec des cellules - Google Patents

Micropointes pour utilisation en interaction électrique avec des cellules Download PDF

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Publication number
WO2008111047A1
WO2008111047A1 PCT/IL2008/000313 IL2008000313W WO2008111047A1 WO 2008111047 A1 WO2008111047 A1 WO 2008111047A1 IL 2008000313 W IL2008000313 W IL 2008000313W WO 2008111047 A1 WO2008111047 A1 WO 2008111047A1
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WO
WIPO (PCT)
Prior art keywords
micro
nail
cell
membrane
electrical
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
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PCT/IL2008/000313
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English (en)
Inventor
Micha Spira
Shlomo Yitzchaik
Joseph Shappir
Carmen Bartic
Gustaaf Borghs
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Interuniversitair Microelektronica Centrum vzw IMEC
Yissum Research Development Co of Hebrew University of Jerusalem
Original Assignee
Interuniversitair Microelektronica Centrum vzw IMEC
Yissum Research Development Co of Hebrew University of Jerusalem
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Publication date
Application filed by Interuniversitair Microelektronica Centrum vzw IMEC, Yissum Research Development Co of Hebrew University of Jerusalem filed Critical Interuniversitair Microelektronica Centrum vzw IMEC
Publication of WO2008111047A1 publication Critical patent/WO2008111047A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • 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

Definitions

  • This invention is generally in the field of bio-molecular electronics, and relates to an electronic device utilizing micro-nails for electrical interaction with living cells.
  • Interaction between neurons and electronic devices has been in existence for several decades for a plurality of purposes. During the past decades, these interactions were usually achieved by inserting an electrode assembly (single electrode or an array of electrodes) into the neurons, or patching an electrode on to the cell's membrane (patch electrode), or placing an electrode assembly in the vicinity of the neurons' membranes, so as to detect extracellular voltage changes.
  • an electrode assembly single electrode or an array of electrodes
  • the detection electrode assembly can also be used for the stimulation of neurons.
  • Another international publication WO 2004/109282 also assigned to the assignee of the present patent application, concerns a tight physical linkage between the membrane of cells and the surface of the substrate supporting electronic devices by constructing a surface having nanometer to micrometer scale protrusions in the form of "micro-nails” or micro mushroom like structures. The interaction is achieved by wrapping (engulfment) of the cell's membranes around the micro-nails without disturbing the integrity of the membrane (i.e. the micro-nails do not enter the membrane).
  • the micro- nails are defined as having cellular-internalization (engulfing) promoting properties either due to the morphology and dimensions of the protruding micro- nail, or due to the inherent physical and chemical properties of the micro-nail material from which the micro-nail is formed or is coated by (such as metal), or due to the immobilizing of moieties onto the micro-nail which promote cellular internalization.
  • cellular-internalization promoting properties refers to chemical or structural properties of the micro-nail which induce its engulfment by the cell membrane by wrapping of the cell's membrane around the micro-nail providing an increased seal resistance and a tight physical contact between the cell, the nail and the device surface.
  • the seal resistance being the resistance formed between the neurons membrane and the transistors gate, depends on the dimensions of the space formed between the transistors gate surface and the membrane facing the gate.
  • Theoretical considerations, as well as experimental considerations show that the larger seal resistance, the better the electrical coupling between a cell and an electronic device.
  • the signal to noise ratio (SNR) can be greatly improved by increasing the seal resistance formed between the neurons membrane and the transistors gate. This is illustrated in Fig 1, showing as an example a floating gate (FG) transistor structure placed in the vicinity of a neuron, enabling measurement of the seal resistance R sea ⁇ .
  • FG floating gate
  • FIG. 2A-2D shows intracellular voltages traces Pl and voltages P2 recorded at the floating gate (FG) of the floating-gate transistor.
  • curve Al corresponds to the field potential (FP) profile before a mechanical pressure is applied
  • curves A2 to A5 correspond to increase in the mechanical pressure
  • curves A6 and A7 - to a condition when the mechanical pressure is released.
  • curves A2 and A3 the increased pressure led to increase in the FP amplitude, while not causing a change in FP shape.
  • Fig. 2D illustrates the FG voltage under mechanical pressure, in this example, a fire polished micro-electrode tightened the cell body onto the chip surface.
  • Fig. 2C illustrates the ' cell body after releasing the pressure. The increase of the cell diameter under the applied pressure is emphasized by circles. Fig.
  • 2D illustrates a normalized intracellular recording P3 superposed on the FP P4 (corresponding to curve A3).
  • the mechanical pressure applied onto cultured neurons increases the area of contact and reduces the distance (gap) between the plasma membrane and the sensing pad.
  • the pressure leads to increased conductance of the plasma membrane facing the sensing pad probably by creating reversible micro- injuries in the membrane or activation of ionic channels within the cell's membrane (curves A4-A6, P2). This is associated with a change in the shape of the FP (from biphasic to mono-phasic) and a dramatic increase in the coupling coefficient. While such mechanical manipulation can be used to analyze the relations between the planar dimensions of the contact area, the seal resistance, and the electrical coupling, such mechanical manipulation cannot be translated into a useful procedure to regulate the seal resistance.
  • Patch clamp traditionally uses a glass pipette, with an open tip diameter of about one micrometer. The interior of the pipette is filled with a solution. A metal (Ag/AgCl) electrode in contact with this solution conducts the electrical changes to a voltage clamp amplifier. The patch clamp electrode is pressed against a cell membrane and suction is applied to the inside of the electrode to pull the cell's membrane inside the tip of the electrode.
  • the suction causes the cell to form a tight seal with the electrode (a so-called “gigaohm seal” or “giga-seal", since the electrical resistance of that seal is in excess of a gigaohm).
  • the patch clamp enables recording of single channels or when the membrane is ruptured and while forming a gigaseal, macroscopic currents from the cells (whole cell configuration).
  • using the patch clamp technique in the whole cell configuration (classical, or the on-chip versions) is some times problematic, as the cytoplasm is perfused and altered by the solution of the patch electrode. This in turn brings changes in the cell biochemistry, affects the cytoskeleton and the function of ion channels, subsequently patched cells degenerate.
  • the present invention solves the above problems by providing a novel approach, which is based on the findings that a tight physical linkage (interaction) can be formed between cells and an external (electrical) device by providing in such device a surface having nano- to micro scale protrusions in the fo ⁇ n of "micro-nail” mushrooms like structures, at least a region of said micro-nail having cellular- interaction promoting properties so that at least one protrusion is insertable into the cell's cytoplasm, allowing formation of giga-seal between the stalk of the nail and the cell's membrane.
  • a tight physical linkage can be formed between cells and an external (electrical) device by providing in such device a surface having nano- to micro scale protrusions in the fo ⁇ n of "micro-nail” mushrooms like structures, at least a region of said micro-nail having cellular- interaction promoting properties so that at least one protrusion is insertable into the cell's cytoplasm, allowing formation of giga-seal between the stalk of
  • cellular-interaction promoting properties refers to one or both of the following: engulfhient (tight wrapping) of the membrane around the micro-nail as a step preceding penetration/insertion (by current or pressure) thus allowing such penetration by physically breaking the cell's membrane (e.g. microperforating); and adapting the micro-nail for chemically breaking the membrane allowing penetration of the micro-nail (for example by the use of enzymes on the micro-nail that can make holes/microperforations in the membranes), and then closing of the membrane around the micro-nail.
  • engulfhient tilt wrapping of the membrane around the micro-nail as a step preceding penetration/insertion (by current or pressure) thus allowing such penetration by physically breaking the cell's membrane (e.g. microperforating); and adapting the micro-nail for chemically breaking the membrane allowing penetration of the micro-nail (for example by the use of enzymes on the micro-nail that can make holes/microperforations
  • the micro-nail may be insertable into the cytoplasm by one of the following: engulfhient of the micro-nail around a region having cellular interaction properties, which include also the breaking of the cellular membrane (such as for example by enzymatic activity) and penetration into the cytoplasm and closure of the membrane again around the micro-nail; and/or engulfhient of the micro-nail by the cellular membrane following a second step of breaking the cell's membrane (for example by application of a short duration current or of pressure) and allowing a part of the micro-nail (the head and a part of the stalk) to penetrate through the plasma membrane into the cytoplasm.
  • the properties for promoting a cellular interaction of the micro-nail are denoted in the micro-nail by immobilizing thereto biological moieties which promote such interaction.
  • the micro-nail may have a uniform structure.
  • the micro-nail is composed of two chemically distinct regions: a head portion, and a base portion.
  • the cellular-interaction promoting properties are a property of the head portion, although during the process of interaction many times both the head and the base portion are internalized by the cell.
  • promoting means increase of the probability of interaction of a micro-nail with said properties (metal/biological molecules attached) as compared to micro-nails without these properties (non-metal/lacking molecules).
  • cellular-interaction-promoting biological moieties refers to any biological molecule, complex of biological molecules, or fragments of biological molecules, which increase the probability of a component, coated therewith or attached thereto, to be internalized by the cell.
  • these biological moieties are divided into the following groups: hydrolytic enzymes that facilitate degradation of extracellular matrix, thus "cleaning" the debris coating the cell to be adhered and increasing the probability of interaction; molecules that recognize plasma membranes components located on the external surface of the plasma membrane of cells, which enable an intimate and tight recognition interaction between the molecules and the plasma membrane of the cell, and thus promote interaction; or a combination of both.
  • Hydrolytic enzymes may be any enzymes which degrade at least one extracellular component such as polysaccharides degrading enzymes, proteinases, and lipid degrading enzymes.
  • these hydrolytic enzymes may damage in the long run cells attached to the surface-substrate of the invention
  • these enzymes are connected to the micro-nail through a spacer which is biodegradable, so that the time span of their activity is short, and after a while they are spontaneously detached from the surface-substrate.
  • the term "molecules that recognize plasma membrane components" refers to any member of pair forming group, the other member of which is a plasma membrane component, the membrane component may be a protein, a lipid, a polysaccharide, a glycoprotein, etc.
  • Such molecules are ligand of plasma membrane receptors (or receptor binding parts of said ligand), receptors that recognize plasma membrane components; lectins that bind to plasma membrane glycoproteins; antibodies that recognize plasma membrane antigenic components (either proteins or non-proteins) or binding fragments of said antibodies; integrins that recognize short linear amino acids present in extracellular proteins, or a combination of two or more of these proteins.
  • the surface also contains an additional "adhesion molecule " which tightens the connection between the cell and the surface.
  • the adhesion molecules preferably should be present either in the base portion of the micro-nail, or on the region of the surface surrounding the base portion of the protruding micro-nail.
  • the adhesion molecules may be in the form of a charge monolayer, such as a monolayer of polylysine, which is known to promote adhesion of neurons to a substrate.
  • the micro-nail has a channel running along its longitudinal axis (from the base of the micro-nail to its head), thereby enabling at least one of chemical and electrical interactions between the micro-nail and the cell's cytoplasm through the channel.
  • the interaction(s) may be by liquid/pressure transfer from the micro-nail to the cell or from the cell to the micro-nail (e.g. positive or negative liquid/pressure interaction).
  • the micro-nail channel may penetrate the membrane of the cells for two purposes: (1) agents of interests (hormones, growth factors, agonists, antagonists, drags) may be injected via the micro-nail in the vicinity of the cells (by current- induced injection of charged agents, or by pressure), allowing careful control of the microenvironment of single cells and enabling manipulation of the cells growth patterns, survival, secretion etc; (2) material(s) can be withdrawn from the cell's cytoplasm; (3) "whole cell" patch clamp can be performed, using suction to place the membrane of the cell in tight connection with the channel opening forming a gigaohm seal.
  • agents of interests hormones, growth factors, agonists, antagonists, drags
  • material(s) can be withdrawn from the cell's cytoplasm
  • "whole cell” patch clamp can be performed, using suction to place the membrane of the cell in tight connection with the channel opening forming a gigaohm seal.
  • the solid micro-nail(s) is/are inserted inside the cells by breaking the cell membrane, allowing a part of the micro-nail (the head and a part of the stalk) to penetrate through the plasma membrane into the cytoplasm, and allowing the membrane to close around the micro-nail forming a giga-seal between the plasma membrane and the micro-nail.
  • the protrusions of the surface are internalized into the cell and the cell membrane is wrapped around the surface and by this the cell is adhered with a strong physical tight contact onto the surface forming a gigaseal.
  • the insertion of the micro-nails inside the cells' allows sensing and recording at least one of electrical and chemical events inside the cells, as well as electrical stimulation from inside the cells.
  • the micro-nail channel penetrates the cell membrane to inject agents of interests (proteins, peptides, drugs, DNA, RNA etc) into the cell' cytoplasm, either from inside the cell membrane or from the outside.
  • agents of interests proteins, peptides, drugs, DNA, RNA etc
  • the micro-nails may be coupled to an injecting element enabling injecting agents (either inside the cells or outside the cells) by current or pressure, or may be coupled to a vacuum system enabling to place the membrane of the cell in tight connection with the micro-nail channel opening enabling patch clamp recording.
  • This injecting/suctioning element allows for simultaneous injection of agents into a plurality of cells (not necessarily neurons) through the micro-nail channels, optionally checking the effect of the addition of these agents on the electrical parameters recorded through the same micro-nails used for injection, or allow patch clamp recording from a plurality of cells.
  • the micro-nails may be connected to at least one external electronic circuits via conducting lines (e.g. thin Film), (such as multi electrode array systems) or may be fabricated as part of transistor structure, such as floating gate electrode of MOS transistor (depletion type).
  • conducting lines e.g. thin Film
  • MOS transistor floating gate electrode of MOS transistor
  • Such transistor can be fabricated as described for example, in example 3 of WO 2004/109282 assigned to the assignee of the present patent application; a Floating Gate Depletion Type (FGDT) can be fabricated as described for example, in example 4 of WO 2004/109282, incorporated herein by reference.
  • FGDT Floating Gate Depletion Type
  • the above two types of connections may serve to record and stimulate cells from the inside of the cell membrane as the micro-nails are inserted through the membrane.
  • Such a structure comprising a plurality of micro-nails inserted into a plurality of cells, allows simultaneous recording and/or stimulation from a plurality of the cells e.g. hundreds of cultured cells (such as neurons muscles and non-excitable cells and cell lines) or from tissue cultures (such as brain slices).
  • the recording may be of "pure” electrical events, as well as recording of chemical status, translated to electrical recording in an "electrochemical sensing" configuration.
  • at least a region of said at least one micro-nail may be functionalized by chemical moieties (proteins, enzymes, ligands, antibodies etc) that may change at least one electrical property of the micro-nail (e.g. conductance, capacitance, surface potential, dipole movement) upon association (binding/complexation) with other biological moieties inside the cells.
  • each micro-nail may record changes in intracellular levels of compounds of interest from a plurality of cells simultaneously.
  • the micro-nails may sense levels of a protein kinase of interest, as well as modifications of existing proteins, such as phosphorylation, de-phosphorylation.
  • the sensing may be done by the binding of the agent of interest, or by one modified form of the agent of interest (for example the phosphorylated form), or by the chemical moieties with which the micro-nail is functionalized.
  • the binding changes an electrical property of the micro-nail such as resistance or capacitance, surface potential or dipole movement.
  • the chemical functionizing moieties are one member of an "affinity pair" such as one of: receptor/ligand; receptor/ion; antibody /antigen; enzyme/substrate; nucleic acid /complementary sequence.
  • the micro-nail channel penetrates the cell membrane to record electrical/chemical status as indicated above, or to stimulate the cells from inside.
  • the micro-nail channel may penetrate the membrane by one of the following mechanism:
  • the micro-nail channel may be used to inject iontophoretic perforating agents, which transiently perforate the membrane. This (together with the formation of a giga-seal between the membrane and the micro-nails stalk) allows the micro-nail to be ohmically linked to the cell interior through the plasma membrane and thus without actually mechanically breaking the membrane to be used as an intracellular electrode.
  • the micro-nail channel may be used to apply negative pressure (vacuum) of a magnitude and duration which are sufficient to break the membrane adjacent to the channel opening (allowing insertion of the micro-nail) but not killing the cell.
  • the present invention in its yet another aspect discloses a method for recording electrical or chemical input and/or stimulating a plurality of cells from inside the cells, the method comprises: placing the plurality of cells on a surface having a plurality of micro-nail structures protruding from the surface; at least a region of said micro-nails having cellular interaction promoting properties; said micro-nails being electrically coupled to a current injecting element; providing conditions allowing tight enwrapping of the cell membrane around said region of the micro-nail; injecting a current, by the current injecting element, through the micro- nails, the current having parameters so as allow insertion of a region of the micro- nail through the membrane and into the cytoplasm without substantially damaging the viability of the cells; thereby placing the micro nail or a portion thereof in the cytoplasm; applying at least one of the following procedures: recording electrical parameters from inside the cell through the micro-nails; electrochemical recording of the level and/or chemical state of biological agents inside the cells; electrically stimulating the cells through the micro
  • the cells may be any type of cell of interest, especially for the electrochemical sensing, as the method of the invention allows for the sensing of the level of various biological agents (proteins, peptides, nucleic acid sequences, small molecules), or chemical state for example achieved by enzymatic modification of existing biological agents (phosphorylation, dephosphorylation, methylation, demethylation etc) from inside the cells.
  • the cells are typically (but not exclusively) excitable cells such as neurons, muscles endocrine cells and others (of any species).
  • the present invention concerns a surface with at least one, preferably with a plurality of micro-nails structures, protruding from the surface; the micro-nails electrically coupled to a current injecting and recording elements; at least a region of said micro-nails having cellular interaction promoting properties; and at least a region of said micro-nails having moieties that change, upon binding or complexing to a target biological agent, at least one of their electrical parameters.
  • the parameters of the micro-nail that change upon the binding/complexing are either resistance or capacitance, surface charge or dipole movement which is due to changes in surface potential.
  • the moieties causing this change are moieties that are one member of an "affinity forming group" i.e. one of a ligand /receptor; receptor/ion; antibody/antigen; enzyme/substrate, nucleic acid sequence/ complementary sequence etc.
  • the present invention provides a method of manipulating a plurality of cells comprising: placing the plurality of cells on a surface having a plurality of micro-nail structures protruding from the surface; at least a region of said micro-nails having cellular interaction promoting properties; each micro-nail having a channel running parallel to its longitudinal axis, the micro-nail channels forming a flow interaction between the micro-nail base and the micro-nail head, providing conditions allowing engulfment of the cell membrane around said region of the micro-nail; providing conditions for manipulating the cells through said micro-nails.
  • the manipulation of the cells may be performed externally by one of the following:
  • agents outside the cell and in the vicinity of the membrane can be injected using current or pressure according to their nature and in such a case the micro-nails are coupled to a current generating, or a pressure generating microfluidics element.
  • the agents are typically those that external manipulated cells such as hormones, growth factors, as well as receptors' agonists and antagonists.
  • the cells membrane may be "sucked" to the micro-nails (to the opening of the channel) for whole cell patch clamp purposes.
  • the micro-nails are coupled to a microfluidics vacuum system enabling the patch clamping, to a system for electrical recording, and optionally to a system capable of injecting agents.
  • the manipulation of the cells is internal mainly for injecting agents of interest into the cells.
  • the agents may be biological agents (proteins, peptides, antibodies DNA, RNA), chemical molecules such as drugs, as well as dyes and agents used for imaging.
  • conditions are provided (such as current/pressure) for expelling the agent from the micro-nails' channel to the cells' cytoplasm.
  • the cells in accordance with the "injection" method may be any type of cell as injection of agents to a plurality of cells may be applicable also for non- excitable cells.
  • the present invention further provides a surface with at least one, preferably a plurality of micro-nails structures protruding from the surface; at least a region of said micro-nails having cellular interaction promoting properties each micro-nail having a channel running parallel to its longitudinal axis, the channels forming a flow interaction between the micro-nail base and the micro- nail head.
  • the micro-nail channel is electrically coupled with an element for injecting agents from the micro-nails (by current/pressure application) and /or with a vacuum generating element for patch clamp purposes.
  • flow/pressure interaction refers to the fact that liquid medium can flow (under proper conditions such as current or pressure) from the micro- nails base to its head or vice versa.
  • the micro-nail channel may be a priori filled with the agent to be injected.
  • the present invention further concerns a device comprising the above surface electrically coupled to a current injecting element.
  • the micro-nail channel is formed may be electrodes or may be fabricated as parts of a transistor gate as will be explained below.
  • the electrode may be gold micro-nail electrodes or made of other metals (not attached to transistors gate) and may be coupled to amplifiers pulse generator and microfluidic systems.
  • the micro-nail can be made from one or more electrically conductive material, e.g. metal, e.g. gold, platinum, etc., for example the stalk being made from one such material (e.g. gold) and the head from another such material (e.g. platinum).
  • gold electroplating is performed.
  • the radius of the micro-nail head is a direct result of the gold electroplating duration.
  • the stalk of the micro-nail is formed. If the electroplating is stopped at this stage, a micro-nail in the form of a rod is obtained.
  • the micro-nail head in the shape of a hemisphere is starting to build up with radius increasing with electroplating time.
  • the top part of the micro-nail stalk directly under the micro-nail head can be changed by photo-resist post baking temperature and time.
  • the current injected should not damage significantly the viability of the cells as determined for example by a small percentage of cell death and narcosis following the current injection, by return of the membrane potential to normal physiological potential with minutes following current injection and micro-nail interaction.
  • the suction applied should be such that it does not substantially damage the viability of the cells.
  • the micro-nails may be arranged in a spaced-apart relationship such that each electrode is operable as a base for a single or many micro-nails associated with on a single or many electronic components or may be distributed as specially constructed clusters, which can be internalized by a single cell.
  • each micro-nail structure is present on a single gate electrode.
  • the micro-nail channel is adapted to form a cell- communicating part of an electrode.
  • cell communicating part refers to the part of the electrode that is in physical contact and in electrical communication (for sensing and/or recording and/or stimulating purposes) with a cell.
  • the micro-nail channel may be a part of a transistor gate electrode.
  • the electrode is an electrode which is intended to communicate with cells having electrical properties, or having physiological responses to electricity, such as neurons, muscle cells, and cells of secreting glands.
  • the electrode may be a regular electrode or a gate electrode.
  • the surface- substrate is adapted to form the cell communicating part of the electrode the base portion of each micro-nail should be electrically isolated from its surrounding so as to decrease "shunting" to the electrolyte containing solution in which the cell is present.
  • the micro-nail may be a polysilicon rod (being an integral part of the polysilicon gate electrode) which is isolated from its surrounding by an internal oxide layer.
  • Both the regular electrode and the gate electrode described as above may form a part of an electrical device for electrical communication with a cell, preferably a cell which is either electrically active or a cell having physiological response to electricity, such as muscle cells, neurons and cells of secreting glands.
  • electrical communication refers to any relationship between an electrode or cells which may be selected from the following: detection of the presence of current in cells by the electrodes or detection of current changes; detection of changes in potential on plasma membranes of cells or detection of changes in potential on plasma membranes of cells providing current to cells; applying an electric field to cells; a combination of two or more of the above.
  • Electrical communication using such a device may be carried out for a variety of purposes, such as for basic research purposes; for the construction of biomedical devices; especially those which need a functional link between nerves or muscles and electric components of an external device such as robotic prosthesis visual and hearing aids, artificial secreting elements, for example in order to provide amputees with robotic prosthesis that is controlled by neurons or muscles; in order to restore vision after retinal or optic nerve damage, in order to electrically stimulate cells of secreting glands to secrete required components, etc.
  • the electrical device is configured and operable as a sensor.
  • the electrode surface morphology is designed in a novel, rough form, consisting of protrusions projecting from the electrode surface.
  • These protrusions are also termed “micro-nails”, and they comprise a rod- or stem-like base portion and may also comprise a head portion.
  • the micro-nails length is preferably ranging between tens of nanometers to thousands of nanometers.
  • the diameters of the micro-nails at the base portion range between tens to hundreds of nanometers.
  • the structure, dimensions and density of the micro-nails can be optimized to maximize the electrical and chemical coupling between the hybrid components, namely the transistor and the living cell.
  • an electrical device for interaction with cells comprising at least one micro-nail structure protruding from a surface, at least a region of said micro-nail structure having cellular- interaction promoting properties and being at least partially insertable into cytoplasm of the cell by breaking the cell membrane, the micro-nail comprising a channel running along a longitudinal axis of the micro-nail, thereby enabling at least one of chemical and electrical interactions between the micro-nail and the cell through said channel.
  • the method comprises coupling to the cell an electrical device, which comprises at least one micro-nail structure that protrudes from a surface and has cellular-interaction promoting properties, said coupling comprising inserting at least a part of said at least one micro-nail into the cell by breaking the cell membrane, thereby allowing said at least part of the micro-nail to penetrate into the cytoplasm and allowing the membrane to close around the micro-nail forming a gigaseal between the membrane and the micro-nail.
  • the method comprises applying a current or pressure interaction with the cell via a channel running along a longitudinal axis of the micro-nail, thereby enabling at least one of the following procedures: recording electrical parameters from inside the cell through the at least one micro- nail, electrically stimulating the cell through said at least one micro-nail, injecting at least one agent of interest into the cell's cytoplasm, and withdrawing material from the cell's cytoplasm.
  • the method may comprise electrochemical recording of at least one of a level and the chemical state of biological agents inside the cell.
  • the method may also comprise sensing at least one of biological agents' level, and/or chemical state of existing biological agents from inside the cell.
  • the method comprises injecting at least one agent into the cell through said channel in the micro-nail.
  • FIG. 1 schematically illustrates a neuron-transistor electrical model and the seal resistance formed in between the two;
  • Fig. 2A-D illustrates an application of mechanical pressure onto the cell body of a cultured neuron which increases the seal resistance and the electrical coupling between the neuron and the transistor;
  • Fig. 3A illustrates a phagocytosis;
  • B an envisioned engulfment of a micro- nail;
  • CI an envisioned of engulfment of a micro-nail by a cell;
  • CII an envisioned insertion of the micro-nail into the cell in which the plasma membrane adheres to the micro-nails stalk forming a giga-seal;
  • D an engulfment of a functionalized latex bead by cultured neuron;
  • E and F electron micrographs depicting engulfed gold micro-nails by neuron (E), and a human cardiomyocyte (F);
  • Fig. 4A-B illustrates different types of gold micro-nails;
  • Fig. 5 illustrates a schematic circuit of the sense electrode with the micro- nail
  • Fig. 6 illustrates the realization of various types of micro-nails on CMOS devices ( ⁇ ⁇ and ⁇ -types);
  • PPP phagocytosis promoting peptides
  • HE hydrolytic enzymes
  • C giga-seal forming layer, NHS-functionalized phospholipids
  • Fig. 8 illustrates tethering and induction of nail's head interaction: (A) engulfment of the micro-nail; (B) giga-seal formation following the electrode "buzzing in” the neuron; and;
  • Fig. 9 illustrates an example of a micro-nail channel cross-section.
  • Fig. 3 illustrating the use of particle interaction by cells to internalize fabricated micro-protrusions (micro-nails).
  • Phagocytosis is a highly conserved cell mechanism to internalize large particles (0.5 ⁇ m) such as food, cellular debris, or signaling molecules.
  • the main steps leading to the interaction of a particle by phagocytosis are illustrated in Fig. 3 A.
  • the primary step that triggers phagocytosis is the interaction between the extracellular domains of a receptor molecule and the molecules presented on the target surface. This interaction tethers the target surface to the membrane.
  • Additional receptors are recruited to the target leading to increased contact between the target and the plasma membrane and initiate the extension of the plasma membrane around the particle.
  • Signal from the cytoplasmic domain of the engaged receptors recruit cytoskeletal elements that nucleates around the particle. Actin filaments together with myosin generate the mechanical force to drive the process of particle engulfment into the cell.
  • the plasma membrane surrounding the particle is pinched off from the plasma membrane. The detached plasma membrane that contains the particle is then free to move in the cytosol and fuse with endosoms.
  • a comparison between a typical phagocytosis and the technique of the present invention in which an intimate contact is formed between the neuron and the protruding micro-nails reveals critical differences:
  • phagocytosis the particle is totally engulfed by the cell.
  • the process of the present invention requires the cell to close the membrane over the particle and form a sealed vacuole (which contains the particle, Fig. 3 AIV).
  • the interaction of the micro- nail requires that the cell "swallow" the head of the micro-nail but do not “finish off' the process of vacuole formation (Fig. 3 B) resulting in wrapping on the cell membrane of the cell around the micro-nail.
  • the inventors of the present invention have demonstrated using a Scanning Transmission Electron Microscope (STEM) the effect of phagocytose, as illustrated in Fig. 3 E and F. Quantitative evaluations suggest that the seal resistance formed by a neuron that engulfs a micro-nail is increased significantly.
  • STEM Scanning Transmission Electron Microscope
  • the present invention enables to insert the micro-nail through the plasma membrane, into the cytoplasm (Fig 3 CI to CII), while maintaining the viability of the cells and thus provide a new tool to intracellularely record and stimulate hundreds of neurons as well as to inject agents into the cells through a micro-nail channel.
  • the micro-nail may penetrate the membrane by current injection by applying alternating current in the range of about 10 to 10.0 microamps for duration of 10ms to lsec at approximately 1000Hz. It should be noted that insertion of sharp (micrometer) glass microelectrodes into cells is done on routine basis by electrophysiologists for several decades.
  • micro-nails are formed using gold electroplating.
  • the micro-nails may be connected to external electronic circuits via conducting lines (e.g. thin film), (such as multi electrode array systems) or may be fabricated as part of transistors, such as floating gates of MOS transistor (depletion type). Silicon chips with properly designed MOS transistors can be used.
  • An additional switch transistor may connect the floating gate of the MOS transistor to an external circuit as illustrated in Fig. 5. During current injection, the transistor is switched on by applying voltage to its gate.
  • the transistor is switched off resulting in insulating from the rest of the system in the giga-ohm level
  • Differential functionoiialization of the head, stalk and base may be achieved by different surface chemistries by forming the three parts of the micro-nail with different materials .
  • a silicon nitride layer is deposited over the substrate and the micro-nail may be formed through an opening in the resist and in the underlying nitride layer.
  • a silicon oxide layer is deposited over the entire area followed by a Reactive Ion Etching (RIE) step which removes the oxide everywhere except on the nail stalk where the oxide is overlapped by the gold head.
  • RIE Reactive Ion Etching
  • a schematic cross section of this structure is presented in Fig. 6.
  • the micro-nail structure is therefore made of three different materials: Au-nails head, SiO2 around the nails stalk, and Si3N4 on the substrate surface.
  • an oxide layer may coat the micro-nail stalk and base, and a
  • RIE by oxygen may differentiate between the surface chemistries of the stalk and base.
  • the micro-nail heads ( ⁇ ,. ⁇ . ⁇ types) are exposing bare gold surfaces. These Au surfaces are assembled with phagocytosis-promoting peptides (PPP) and/or hydrolytic enzymes (HE) that facilitate engulfment of the nails by the cell.
  • PPP phagocytosis-promoting peptides
  • HE hydrolytic enzymes
  • the micro-nail stalks are exposing bare gold surfaces (in ⁇ -type) or oxide layer (in ⁇ - types) to be functionalized with giga-seal forming layer. Such a layer is designed for integration on the molecular level with the neurons' membrane following the insertion of the micro-nail into the cell.
  • the devices' base are composed of oxide ( ⁇ ⁇ types) or nitride ( ⁇ type) layer and are tailored with molecular layers that enhance neuron cell adhesion.
  • Fig. 6 illustrates schematically the proposed routes towards functionalization (without conflicting surface chemistries) of the various device areas and geometries with biologically-active molecular layers according to the various device types:
  • ⁇ -type Step 1 anchors a phospholipids layer on both Au surfaces (stalk and head). Applying RIE to the nail's geometry, the head shadows the stalk from the reactive ions and thus only the head surface is etched.
  • Step 2 self-assembles the cell's adhesion peptide layer on the base-surface.
  • Step 3 assembles PPP and BDE on the naked Au head's surface.
  • Step 1 anchors a phospholipids layer on both SiO2 surfaces (stalk and base). Applying RIE to the nail's geometry, again, the head shadows the stalk from the reactive ions and thus only the base surface will be etched.
  • Step 2 self- assembles the cell's adhesion peptide layer on the base-surface takes place.
  • Step 3 assembles PPP and HE on the naked Au head's surface.
  • Step 1 self-assembles the cell's adhesion peptide layer on the Si3N4 base- surface.
  • Step 2 self-assembles a phospholipids layer on the SiO2 stalk surface.
  • Step 3 assembles PPP and HE on the naked Au head's surface.
  • Fig.7A illustrating Au micro-nails surface assembled with Phagocytosis Promoting Peptides (PPP) having integrin-ligand motifs. Integrins typically recognize short linear amino acid sequences in extracellular matrix (ECM) proteins, one of the most common being Arginine-Glycine- Aspartate (RGD).
  • ECM extracellular matrix
  • RGD Arginine-Glycine- Aspartate
  • the PPP having a cysteine terminal amino-acid are capable for direct coupling with gold surfaces (or maleimide functionalized surfaces) connected to oligolysine spacer connected further to RGD repeating units.
  • Fig.7B illustrates Au micro-nails surface assembled with Hydrolytic
  • HE Enzymes
  • hydrolytic enzymes that facilitate degradation of the ECM are expected to allow intimate physical contact between the micro-nail head's surface and receptor molecules located on the external plasma membrane's surface. Peptides that recognize plasma membrane receptors and tether it will be covalently linked with the hydrolytic enzymes (HE) to the head's surface via a terminal cysteine amino- acid.
  • the HE includes polysaccharide-degrading enzymes (e.g., sialidase or neuraminidase and hyaluronidase).
  • the enzymatic hydrolysis is directed towards saccharides of the ECM that won't attack the peptides and lipids in this system.
  • the recognition of plasma membrane receptors by PPP anchored to the gold hemisphere of the micro-nails induces engulfment of the micro-nail.
  • the insertion of the micro-nails head through the plasma membrane and into the cell is achieved using the routine and well established procedures to insert sharp ( ⁇ 1 ⁇ m diameter) glass microelectrodes into cells.
  • sharp ⁇ 1 ⁇ m diameter glass microelectrodes
  • Classically the insertion of a micropipette into a cell is facilitated by passing alternate current through the electrode ("buzzing the electrode in" alternating current for a duration of 10ms to lsec, at approximately 1000Hz, 10-100 microamps) that is pressed against the cells membrane.
  • Successful long term recordings and stimulation of a neuron by an intracellular sharp electrode requires that a high resistance seal is formed between the plasma membrane and the glass electrode (see Fig. 8).
  • lipids with carbonyl functional group e.g., N-hydroxysuccinyl (NHS) ester
  • NHS N-hydroxysuccinyl
  • Fig. 7C illustrates the coupling of a NHS- lipid with an aminoalkyl functionalized stem's surface forming imide linkage.
  • the adhesion monolayer is covalently linked to the device base and practically has the largest area of the device.
  • adhesion peptide one can use cysteine terminated oligolysine or even one of the PPP.
  • Anchoring to the oxide layer is conducted as described for the PPP.
  • a hetero-binuclear coupling agent, containing active-ester and maleimide groups, can selectively react with the nitride leaving the maleimide-group ready for substitution with thiol-group of the peptide.
  • Fig. 9 illustrating an example of a micro-nail channel.
  • the micro-nail channel runs along the longitudinal axis of the micro- nails, enabling flow of liquid, agents from the base of the micro-nail, to the head of the micro-nail and outside of the nail, either to the cytoplasm or the vicinity of the cell membrane as the case may be.
  • one of the openings of the micro-nail channel (at the base or head of the micro-nail) is placed in a liquid medium containing the agent to be injected, which enters the micro-nail channel through capillary force.
  • the channel can also be used for material withdraw from the cell's cytoplasm.

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Abstract

L'invention concerne un dispositif électrique pour interaction avec des cellules. Le dispositif comprend au moins une structure de micropointe saillante depuis une surface, où au moins une région de la structure de micropointe a des propriétés favorisant l'interaction cellulaire. La micropointe comprend un canal passant le long d'un axe longitudinal de la micropointe, permettant ainsi au moins une des interactions chimique et électrique entre la micropointe et le cytoplasme de la cellule à travers ledit canal.
PCT/IL2008/000313 2007-03-09 2008-03-09 Micropointes pour utilisation en interaction électrique avec des cellules Ceased WO2008111047A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012160565A1 (fr) 2011-05-26 2012-11-29 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Dispositif électronique biomoléculaire et procédé d'utilisation

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19841337C1 (de) * 1998-05-27 1999-09-23 Micronas Intermetall Gmbh Verfahren und Vorrichtung zur intrazellulären Manipulation einer biologischen Zelle
US20020053915A1 (en) * 2000-07-07 2002-05-09 Weaver Charles David Electrophysiology configuration suitable for high throughput screening of compounds for drug discovery
WO2004109282A1 (fr) * 2003-06-10 2004-12-16 Yissum Research Development Company Of The Hebrew University Of Jerusalem Dispositif electronique permettant de communiquer avec des cellules vivantes

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19841337C1 (de) * 1998-05-27 1999-09-23 Micronas Intermetall Gmbh Verfahren und Vorrichtung zur intrazellulären Manipulation einer biologischen Zelle
US20020053915A1 (en) * 2000-07-07 2002-05-09 Weaver Charles David Electrophysiology configuration suitable for high throughput screening of compounds for drug discovery
WO2004109282A1 (fr) * 2003-06-10 2004-12-16 Yissum Research Development Company Of The Hebrew University Of Jerusalem Dispositif electronique permettant de communiquer avec des cellules vivantes

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012160565A1 (fr) 2011-05-26 2012-11-29 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Dispositif électronique biomoléculaire et procédé d'utilisation
US9629995B2 (en) 2011-05-26 2017-04-25 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Biomolecular electronic device and process of use

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