Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
  • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
  • G01N27/403—Cells and electrode assemblies
  • G01N27/414—Ion-sensitive or chemical field-effect transistors, i.e. ISFETS or CHEMFETS
• • 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
  • G01N33/4833—Physical analysis of biological material of solid biological material, e.g. tissue samples, cell cultures
  • G01N33/4836—Physical analysis of biological material of solid biological material, e.g. tissue samples, cell cultures using multielectrode arrays

Definitions

• the present invention relates to an assembly comprising a field-effect transistor having vertebrate cells, particularly cells of mammalian origin, more particularly myocardial cells, fixed thereon, an apparatus for extracellular electrophysiologi- cal recordings comprising the assembly and their use for testing potential pharmaceutically effective compounds, such as compounds having potentially cardiac physiologically effectiveness, or compounds having potential side effects, or potentially toxic compounds.
• Impulse propagation in the cardiac muscle tissue is determined by both active (including ion channels, ion exchangers, and ion pumps) and passive properties (membrane resistance and capacitance, size and shape of individual cells, cell assemblage, topology and density of gap junctions, and the spatial organization of the extracellular space) of the cardiac muscle cells.
• active including ion channels, ion exchangers, and ion pumps
• passive properties membrane resistance and capacitance, size and shape of individual cells, cell assemblage, topology and density of gap junctions, and the spatial organization of the extracellular space
• the presence of spontaneous rhythmic electrical and mechanical activity in cultured myocardial cells is widely used to study cardiac physiology. Electrical activity in tissue culture is usually recorded by intracellular glass micropipettes, patch clamp pipettes, or metal microelectrodes. However, it is difficult to use these techniques for extended periods because of movement, breakage, or cell damage.
• a FET based assembly or an apparatus comprising such a FET based assembly, respectively which enables the recording of the electrical activity in cultured vertebrate cells, to thereby study, for example, cardiac physiology, wherein such an assembly or an apparatus comprising such a FET based assembly, respectively, should not be affected by cell movement, breakage, or damage for extended periods of time.
• a further object of the present invention is to provide a FET based assembly or an apparatus comprising such a FET based assembly, respectively, which can used for testing potential pharmaceutically effective compounds.
• an assembly comprising at least one field-effect transistor comprising a source contact and a drain contact, and one or more layers of vertebrate cells, particularly cells of mammalian origin, more particularly myocardial cells, in contact with an non- metallized surface region of the field-effect transistor between the source con ⁇ tact and the drain contact.
• the non-metallized surface region covers at least a portion of a channel of the field-effect transistor, the conductivity of which can be influenced via field-effect by the layer(s) of vertebrate cells.
• the non- metallized surface region corresponds to at least a portion of a non-metallized gate region of the field-effect transitor.
• a monolayer of myocardial cells are fixed onto the non- metallized surface region of the field-effect transistor.
• the fixed cells are preferably present in serum-containing medium.
• the myocardial cells are beating rat cardiac muscle cells.
• the cell density ranges, for example, from 10 4 - 10 6 cells/cm 2 .
• the field-effect transistor is a n- or p-channel silicon field-effect transistor.
• the non-metallized surface region corresponds to least a part of a gate-oxide surface region of the silicon field-effect transistor in this case.
• the field-effect transistor comprises at least one lll-V or ll-V semiconductor heterostructure.
• a lll-V semiconductor high electron mobility transistors (HEMT) can be employed with the non-metallized surface region either being formed on the semiconductor surface between the source contact and drain contact or on an optional protective oxide surface.
• the size of the non-metallized gate regions of the field effect transistors ranges from 28x9 ⁇ m 2 to 10x1 ⁇ m 2 .
• the field-effect transistors comprising the non-metallized surface regions are arranged in a matrix with spaced apart centers, preferably a 4x4 matrix with the centers 200 ⁇ m apart.
• an apparatus for extracellular electrophysiological recordings comprising the aforementioned assembly, and optionally means for amplifying electrical signals detected at the recording terminals of the assembly, for example source and drain contacts, and/or means for monitoring the electrical signals, e.g. a computer connected to the electronic set-up.
• Such an apparatus with an additional means for measuring time-delays between the electrical signals can be advantageously used to determine the characteristics of a burst pattern of signals in the layer/layers of vertebrate cells, e.g. myocardial cells.
• the aforementioned assembly as well as the apparatus mentioned above can be advantageously used for testing potential pharmaceutically effective compounds, such as compounds having potentially cardiac physiologically effectiveness, or compounds having potential side effects, or pontentially toxic compounds.
• potential pharmaceutically effective compounds such as compounds having potentially cardiac physiologically effectiveness, or compounds having potential side effects, or pontentially toxic compounds.
• a combination comprising at least two of the aforementioned different types of compounds can also be tested by the assembly as well as the apparatus mentioned above.
• the assembly or the apparatus, respectively, according to present invention can be used as an "biochip-sensor" for on-line monitoring whether potential compounds induce irregularities of pulse or whether the cardiac beat loses the rhythm by bringing in contact with such potential compounds or, otherwise, which compounds are capable of restoring the cardiac beat after the cardiac cells have been artificially set in arhythmic condition, for example by addition of calcium.
• the origin, the direction and the velocity of burst pattern between the cells can be determined for the evaluation of potential cardiacs.
• Fig. 1 is a schematic drawing showing the measurement setup used for the electrical recordings from rat cardiac myocytes.
• Fig. 2 shows the electrical recordings from rat cardiac myocytes after 4 days in culture, wherein the measurements have been performed simultaneously with FET (lower trace) and an intracellular microelectrode (upper trace).
• the source- drain current l DS , the effective gate voltage V G determined from the transfer characteristics of the FET are depicted in the lower trace and the membrane voltage V M measured by the impaled microelectrode is depicted in the upper trace.
• Fig. 3 shows the expansion of the action potentials in Fig . 2 recorded with an microelectrode (A) and an FET (B).
• the lower traces show the simulated curve using the equivalent circuit shown in Fig. 6: (C) using only the highpass filter elements, (D) without and (E) with active membrane elements.
• Fig . 4a shows the equivalent circuitry for the explanation of the measured signals.
• the model consists of the capacitance of the gate oxide C G in the junction and the seal resistance R Jt the capacitance C M and resistance R M of the membrane, as well as the contribution of the current due to active ion channels.
• Fig. 4b shows an equivalent circuitry presented in the four-pole configuration used for the determination of the effective gate-source voltage V by applying the intracellular signal V M .
• Fig. 5 shows the modulus and phase difference of the spectral transfer functions h ⁇ ) used for the modelling of the extracellular signals recorded with FET: A) highpass filter elements, B) including passive membrane elements and C) including active and passive membrane elements.
• Fig . 6 shows the simultaneous recording from cardiac myocytes with an intracellular microelectrode (A) and an FET (B) .
• the lower traces show the simulated curves applying the equivalent circuit from Fig. 6 (highpass filter elements (C), without (D) and with (E) active membrane elements) .
• Fig. 7 shows the largest signals recorded from the heart muscle cells with an FET.
• Fig. 8a shows the spontaneous extracellular activity from a monolayer of cardiac muscle cells recorded kom 4 different FETs.
• Fig. 8b shows the position of the FETs which were used for the recordings.
• Field-effect transistor (FET) arrays used in this example were fabricated using standard silicon planar technology.
• the array consisted of 1 6 p-channel FETs with non-metallized gate regions corresponding to non-metallized surface regions.
• the size of the gates or gate regions ranged between 28x9 ⁇ m 2 down to 10x1 ⁇ m 2 and were arranged in a 4x4 matrix with the centers 200 ⁇ m apart.
• These chips were mounted on standard 28 DIL ceramic chipcarriers (NGK Spark Plug Co., LTD, Japan), wire-bonded and encapsulated using a silicone polymer (Sylgard 1 84 and 96-083, Dow Corning) . Together with a glass ring fixed onto the chip carrier the encapsulated device forms a small culture dish.
• Cardiac myocytes were prepared following a technique which has been described in detail in Bhatti et al., J. Mol. Cell. Cardiol. 1 989, 10, 995. Briefly the hearts were removed from 1 to 3 day old rats, finely minced and dissociated and plated onto the recording devices in serum containing medium. Prior to plating the FET arrays were cleaned with 25% sulfuric acid, washed with Milli-Q water, sterilized with 70 % ethanol and coated with fibronectin for about 1 h.
• the FET array was connected to a 1 6-channel preamplifier mounted under a microscope.
• the offset currents arising from the driving conditions of all 1 6 channels were compensated and recorded signals were amplified by a gain of 1 00.
• Up to four selected channels were monitored using a computer connected to the electronic setup.
• simultaneous extracellular and intracellular recordings were made using glass micro electrodes filled with 3 M KCI and mounted in a holderpreamplifier headstage (Luigs & Neumann, Germany).
• the headstage signal was amplified using a whole cell amplifier (List electronic, Germany) which could be connected to the computer and an oscilloscope.
• the temperature was kept constant at 37 °C using a heater pad fitted to the FET preamplifier.
• Fig . 1 the principal setup for the described experiment is schematically shown.
• Fig. 2 shows typical recordings from cardiac muscle cells performed simultaneously with an intracellular microelectrode (upper trace) and an FET (lower trace) . Intracellular recordings were made from cells grown several ⁇ m away from the recording site of the FET. A more detailed view of the recordings is shown in Fig. 3. The fast rising of the intracellular voltage V M (microelectrode, Fig.
• the extracellular voltage V j (f) at the gate can be simulated by a convolution of the intracellular voltage V M (t) by means of the Fourier transform of the transfer function ( ⁇ ) .
• the equivalent circuitry for determination of the transfer function is shown in Fig. 4.
• Fig. 6 shows the typical shape of these signals recorded simultaneously with an intracellular microelectrode (A) and an FET (B) .
• A intracellular microelectrode
• B FET
• the first part of the extracellular signal contributes only about 1 /3 of the total amplitude due to the slower rise time.
• the second part is more pronounced and lasts almost as long as the intracellular signal does.
• the simulated curve shows a very good agreement with the measured data (Fig. 6E) .
• Fig. 7 shows the largest signal recorded in this example.
• the shape of the extracellular signal is almost identical to intracellular signals (Fig. 6A) .
• the capacitive current is negligible and current flow is determined mainly through the ratio R R j .
• This kind of signal can be explained by assuming that the seal resistance R d of the junction is in the range of the membrane resistance R M in the junction. This can be explained either by assuming that the seal resistance R d is a factor 1 000 larger than expected or by the fact that the mem- brane resistance R M is small compared to the usual membrane resistance.
• Fig. 8 shows spontaneous extracellular activity from a monolayer of cardiac muscle cells recorded from 4 different FETs. The position of the FETs from which recordings were made is shown in Fig. 8b. All the signals are similar in shape as described in Fig. 3. The amplitude of the signals varies between the different FETs probably because of the variable strength of the coupling. Due to the electrical connections between cells in the layer, the cell with the highest repetition rate of action potentials determines the beating frequency of the whole layer. From the time delay between the recordings of the action potentials at the various sites, the origin, the direction and the velocity (ca 0.2 m/s) of the burst pattern assuming an isotropic spreading of the excitation between the cells can be estimated.

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• 1999
  • 1999-07-23 AU AU51640/99A patent/AU5164099A/en not_active Abandoned
  • 1999-07-23 JP JP2000561490A patent/JP2002522028A/ja active Pending
  • 1999-07-23 EP EP99936603A patent/EP1099110A1/de not_active Withdrawn
  • 1999-07-23 WO PCT/EP1999/005298 patent/WO2000005574A1/en not_active Application Discontinuation
  • 1999-07-23 CA CA002338456A patent/CA2338456A1/en not_active Abandoned

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