EP2092319A1

EP2092319A1 - Apparatus and method for measuring biological and electronic characteristics of a sample

Info

Publication number: EP2092319A1
Application number: EP07817752A
Authority: EP
Inventors: Sven Ingebrandt; Norbert Wolters; Günter Wrobel; Herbert Bousack; Andreas Offenhäusser
Original assignee: Forschungszentrum Juelich GmbH
Current assignee: Forschungszentrum Juelich GmbH
Priority date: 2006-11-24
Filing date: 2007-10-31
Publication date: 2009-08-26
Also published as: DE102006055990A1; WO2008061489A1

Abstract

The invention relates to an apparatus and a method for measuring biological characteristics of a sample, the sample being in contact with the gate of a field-effect transistor. According to the invention, an AC voltage is applied to the sample. The nature of the sample then governs the extent to which charges are transferred to the gate. The presence of such charges can be tested using the field-effect transistor, for example using a source-drain current. The invention can be used to measure the response of a biological sample to an AC voltage at zero current and therefore very much more sensitively than by using impedance measurements based on the prior art, in which small currents are to be measured. It is possible to examine single cells or else portions of cells, the prior art having been able to attain a sufficiently large signal only on cell complexes containing a large number of cells.

Description

Apparatus and method for measuring biological and electronic properties of a sample

The invention relates to an apparatus and a method for measuring biological and electronic properties of a sample.

State of the art

Biological properties of cellular samples are measured by electrical means, inter alia, with impedance sensors. In this case, a small alternating current flows between a reference electrode and a working electrode. Overgrowing the working electrode with a biological sample changes the AC impedance of the system (impedance) and thus the current flowing through the device. This change is measured. Such a measuring arrangement is known for example from US Pat. No. 5,187,096.

Disadvantageous with a progressive miniaturization of the electrodes and the samples, the currents to be measured smaller and smaller. Therefore, higher measurement frequencies are necessary, in which again parasitic effects of the supply lines and external disturbances gain more and more influence. In that case, the signal-to-noise ratio for examining individual cells or even parts thereof may not be sufficient.

Task and solution

It is therefore the object of the invention to allow the measurement of biological and electronic properties of a sample by electrical means with a better signal-to-noise ratio than is possible in the prior art.

This object is achieved by a device according to the main claim and a method according to the independent claim. The use of a device according to the invention for carrying out the method is the subject of a further subsidiary claim. Further advantageous embodiments will be apparent from the dependent claims. Subject of the invention

The device according to the invention comprises a field effect transistor with contacting means for contacting the sample with the gate of the field effect transistor.

As a field effect transistors are, for example, non-metallized open-gate field effect transistors, in particular so-called ion-selective field effect transistors (ISFETs), metallized or non-metallized floating gate field effect transistors, metallized or non-metallized nano-field effect transistors, carbon nanotube field effect transistors or metallized or non-metallized Nanowires suitable.

As a contacting means, any electrically conductive element is suitable, which is connected to the gate of the field effect transistor and can be brought into contact with the sample. For this purpose, aqueous measuring solutions or hydrogels or polyelectrolytes are preferably used. In particular, the gate electrode itself can be provided as a contacting means, on which the sample can be applied directly.

According to the invention, stimulation means are provided for applying a voltage to the sample. These stimulation means advantageously comprise a reference electrode introduced into a liquid or gelatinous contacting agent, such as a chlorinated silver wire (Ag / AgCl electrode), an electrochemical reference electrode, a metal wire, a metallized surface of a microfluidic chamber, or preferably at least one on-chip accommodates the field effect transistor, integrated reference electrode. However, it is also suitable stimulating agents that are applied directly to the sample or introduced into it, such as patch clamps or intracellular electrodes.

Since the sample is electrically connected at least partially between the stimulation means and the gate of the field-effect transistor, it depends on the specific properties of the sample in each case to what extent an applied alternating voltage leads to a loading of the gate with a potential. For example, the sample changes the input impedance of the field effect transistor. The charges that cause the potential at the gate need not be concentrated on the gate, but may, for example, also be on the contactor or on the sample contacted with it. The potential present at the gate can be measured directly or indirectly with the aid of the field-effect transistor, preferably via the strength of a current which flows through the source-drain path of the field-effect transistor. This current does not flow through the sample because the source-drain path in a field-effect transistor is isolated from the gate. A change in the potential on the gate then leads to a change in the current higher by the amplification factor of the field effect transistor.

It has been recognized that with such a device, the response of a biological sample to an applied AC voltage can advantageously be measured without current flow through the sample. According to US Patent 5,187,096, this response was measured by the impedance of the sample, with a very small current flowing through it. This stream usually flowed at least partially through the sample. It was recognized that the metrological difficulties in measuring these very small currents were the limiting factor for the signal-to-noise ratio and thus also for the sensitivity in the measurement of very small samples. This was all the more true, the longer the paths were, the current had to go back to the first amplifier. According to the invention, the response of the sample is amplified directly at the place of its formation by the field effect transistor. The sensitivity of the device according to the invention is thus large enough to be able to carry out measurements on individual cells, but also on partial regions of cells or else only cell fragments, cell membranes, artificial model membranes, proteins or biomolecules.

In a particularly advantageous embodiment of the invention, a measuring instrument for measuring the amplitude and phase of a current flowing through the source-drain path of the field effect transistor is provided, which is preferably converted in a first amplifier stage by means of suitable electronics into an output voltage (V _out ). The time course of these quantities includes a convolution of the impedance of the sample with the transfer function of the field effect transistor.

In particular, the frequency response of a current flowing through the source-drain path or a variable derived therefrom in dependence on the frequency of the alternating voltage is understood as the transfer function of the field-effect transistor in the sense of this invention.

If this transfer function is known, for example, from a measurement without sample leaves invert the convolution and determine the impedance of the sample by unfolding. The impedance is in biology, a common measure of, so that the possibility of the device to determine this, comparability Messergebm sse ^'increased significantly with the results of other tests.

In a further particularly advantageous embodiment of the invention, the measuring instrument is frequency-selective. In the simplest case, then only the current can be measured, which has the same frequency as the AC voltage with which the sample is applied. This reduces the influence of external disturbances. However, it is also possible with a frequency-selective measuring instrument, also simultaneously, to measure at frequencies other than the frequency of the applied alternating voltage. In particular, it is possible and useful to measure on a discrete or continuous spectrum of frequencies. In particular, it is then possible to track slower biological effects that alter several frequency components of the current.

An example of such slow biological effects are extracellular stresses that may occur with or without external stimulation on biological cells. These voltages also apply a potential to the gate of the field effect transistor. The extracellular voltages change very slowly compared to the typical frequency ranges used for impedance analysis. They therefore cause in the current flowing through the source-drain path, a proportion that can be considered approximately as a DC component. With the device according to the invention and in particular with the frequency-selective measuring instrument, it is possible to measure the electrical activity of the sample and its response to the alternating voltage at the same time and using one and the same field effect transistor. It is also possible to use one and the same instrument, such as a lock-in amplifier, for the simultaneous measurement of amplitude and phase at different measurement frequencies. In particular, a lock-in amplifier is suitable for simultaneously characterizing a signal component with a specific measurement frequency and a signal component which can be regarded approximately as a direct current component compared to this frequency. However, a passive low-pass filter may also be provided in order to separate off such a DC-like component of the signal.

A further advantageous embodiment of the arrangement additionally comprises excitation means for applying a further electrical voltage to the sample. In the context of this invention, excipients are understood to mean those agents which can apply a sufficiently high voltage to the sample in such a way that a biological reaction in the sample is thereby produced.

As such a means, for example, a patch-clamp arrangement or an intracellular electrode is suitable. With these excitation means, the measurable with the device electrical activity of the sample can be stimulated independently of a current inventive measurement of other biological properties.

Advantageously, means for carrying out further simultaneous measurements, such as amperometry, voltammetry, Coulombmetrie, gravimetry, optometry, Hall measurement, atomic force microscopy (AFM), light microscopy, temperature jump method, calorimetry or 2-, 3- or 4-pole measurement , be provided.

Advantageously, the arrangement comprises at least one further field-effect transistor, which in particular can be constructed identically to the first field-effect transistor. Then this can be used to determine the transfer function of the field effect transistor as such (without sample) and in particular the typical drift of the output signal either for the purpose of subsequent signal processing or to charge from the outset by differential measurement with the measurement signal. This is particularly advantageous when it is intended to determine the impedance of the sample by unfolding. For this purpose, it is particularly advantageous if the further field effect transistor is electrically isolated from the sample. The transfer function of the field effect transistor is dependent on the input impedance of the field effect transistor, for example. Any changes in the impedance of the path between the stimulation means and the gate of the field effect transistor thereby change the input impedance of the field effect transistor.

In a particularly advantageous embodiment of the invention, the device comprises means for contacting a plurality of field effect transistors with the same sample. If these means are arranged, for example, in an array of at least 2 to several thousand field-effect transistors, it is possible, for example, to measure many samples simultaneously. However, it is also possible to perform spatially resolved measurements on an extended sample. For example, the response of different functional areas of a cell to the AC voltage can be studied simultaneously. This is due to the fact that field effect make transistors that are significantly smaller than a cell. Typical sizes of biological cells are 2 mm diameter (oocytes) up to 1 μm (bacteria). Nanowires as the smallest conceivable field effect transistors typically have diameters up to 10 nm.

In a particularly advantageous embodiment of the invention, the stimulation means comprise a liquid reservoir. Such a reservoir can be produced, for example, by adhering glass rings to a chip containing the field-effect transistor in a moisture-tight manner. However, it is also possible to insert milled or drilled holes into the chip encapsulation in a moisture-tight manner, so that a small culture dish with volumes of preferably greater than 0.1 .mu.l, more preferably greater than 10 .mu.l and most preferably greater than 100 .mu.l is formed. The reservoir should be large enough to completely wet the sample, the field effect transistor, and the reference electrode serving as the stimulation means.

In a further advantageous embodiment of the invention, the liquid reservoir is closed. It may have an inlet or a drain (microfluidics). The volume should in this case be at least so large that the sample and the reference electrode are wetted. This makes it possible, for example, to supply cell poisons in increasing gradients to the sample. Likewise, it is advantageously possible to switch back and forth between different substances within the shortest or respectively defined periods or to supply sequences of different fluids to the sample. The use of microfluidic systems further reduces the volumes required for measurement.

In a further advantageous embodiment of the invention, the contacting means comprise a liquid reservoir, which may in particular be identical to the reservoir which is already part of the stimulation means. Contacting via a liquid is easy to handle, controllable via the chemical composition of the liquid and can be dissolved again without residue.

One of the stimulation or contacting means comprising liquid reservoir is advantageously filled with an electrolyte.

The method according to the invention for measuring biological properties of a sample provides for applying an alternating voltage to the sample. The sample exchanges charges with the gate of a field effect transistor (FET). One of the potential at the gate of the Field effect transistor-dependent primary physical quantity is measured. In this case, a change in the potential at the gate leads to a change in the primary physical measured variable which is higher by one amplification factor. In particular, a current flowing through the source-drain path of the field-effect transistor can be measured as the primary physical measured variable, wherein the amplification factor is the usual amplification factor of the field-effect transistor. The amplification factor is influenced, for example, by the sample changing the input impedance of the field effect transistor.

The frequency of the alternating voltage applied to the sample is preferably between 0.1 Hz and 1 GHz, more preferably between 1 Hz and 100 MHz and most preferably between 1 Hz and 10 MHz. At these frequencies, the expected measurement effects are greatest.

The amplitude of the alternating voltage applied to the sample is preferably between 0.001 mV and 10 V, more preferably between 0.1 mV and 1 V and most preferably between 1 mV and 100 mV. The amplitude should be so large that sufficient transmission and amplification is ensured for the field effect transistor used in each case. An upper limit is reached in case of an influence up to the destruction of the sample by an alternating voltage with too high an amplitude.

The amplitude of the alternating voltage is advantageously kept constant during the measurement. However, it is also possible to vary the amplitude approximately at a constant frequency of the alternating voltage and thus to operate in terms of amplitude spectroscopy.

It has been recognized that this method can measure the response of a biological sample to an applied AC voltage without current flow through the sample. According to the cited prior art, this response was measured by the impedance of the sample, with a very small current flowing at least partially through the sample. It was recognized that the metrological difficulties in measuring these very small currents were the limiting factor for the signal-to-noise ratio and thus also for the sensitivity in the measurement of very small samples. This was all the more true, the longer the paths were, the current had to go back to the first amplifier. According to the invention, the response of the sample is amplified directly at the place of its formation by the field effect transistor. The sensitivity is thus large enough to take measurements on biological Samples such as single cells, subregions of cells, cell fragments, cell membranes, artificial model membranes, proteins or biomolecules to carry out.

In a particularly advantageous embodiment of the invention, the sample is subjected to alternating voltages of different frequencies. The response of a sample to an AC voltage is generally frequency dependent. From this frequency dependence biological properties of the sample can be derived.

In a particularly advantageous embodiment of the invention, amplitude and phase of the primary physical quantity are measured. The time course of these variables includes a convolution of further characteristics of the sample with the transfer function of the field effect transistor.

Advantageously, the time course of the amplitude and phase of the primary physical measured variable is measured between 1 and 100, preferably between 3 and 30 and particularly preferably between 5 and 10 oscillations of the applied alternating voltage. This time periodically represents a compromise between the necessary measuring time and the information content for the subsequent evaluation, for example for determining the impedance.

Advantageously, the time characteristic of the primary physical measured quantity and the transfer function of the field effect transistor are processed to determine further characteristics of the sample. For example, the time characteristic of the primary physical measured variable may represent a convolution of such a characteristic with the transfer function of the field effect transistor. If the transfer function of the field effect transistor is known, for example, by a prior measurement without a sample, the characteristic sought can then be obtained by a development, ie an inversion of the folding process, from the recorded time course. However, during the measurement, the transfer function can also be at least partially offset with the time characteristic of the primary physical quantity, for example by differentially measuring it with a second field effect transistor not directly contacted with the sample, which is preferably arranged adjacent to the first field effect transistor, for example in the same array. However, the parameter can also be determined, for example, by a mathematical model of the time characteristic of the primary physical quantity containing the transfer function of the field effect transistor, which contains the parameter sought as a parameter, while varying this Parameters is fitted to the recorded time course. These measures for processing the time course of the primary physical quantity and the transfer function have the effect that the transfer function is virtually corrected out of the time course.

In particular, the impedance of the sample can be determined from the primary physical measured variable, for example by the above-described calculation of the time characteristic of the primary measured variable with the transfer function of the field effect transistor. The determination of the impedance with the method according to the invention significantly increases the comparability of the measurement results with the results of other experiments.

In a further advantageous embodiment of the invention, a further secondary physical measured variable is measured. This measured variable advantageously represents a measure of an electrical activity of the sample, which in turn advantageously allows conclusions to be drawn about the biological activity. The electrical activity may be, for example, an extracellular voltage that is applied either spontaneously or in response to the application of the AC voltage to the sample. The electrical activity of the sample and its response to the AC voltage are advantageously measured with one and the same field effect transistor, which avoids errors due to deviations in the properties of different transistors.

Advantageously, the sample is subjected to a stimulation of electrical activity with a further electrical voltage, for example by a further external voltage source, such as by contacting by patch-clamp technique or intracellular electrodes. However, the further electrical voltage can also be advantageously applied together with the AC voltage, for example, by the AC voltage is modulated to a DC voltage as further electrical voltage.

The secondary physical measured variable may be, for example, a low-frequency component of the current flowing through the source-drain path of the field-effect transistor in comparison to the frequency of the alternating voltage. This can be considered approximately as DC component.

Advantageously, the primary and secondary physical quantities can be measured simultaneously. This is particularly advantageous when the primary physical quantity of the current flowing through the source-drain path of the field effect transistor and the secondary physical quantity is a low-frequency, approximately considered as a direct current component of this stream. Then advantageously a lock-in amplifier can be selected as a measuring instrument.

In general, it is advantageous to measure the current flowing through the source-drain path of the field-effect transistor, or generally the primary physical measured variable, at frequencies other than the frequency of the alternating voltage. In particular, it is advantageous to record a frequency spectrum of the current, or in general the primary physical measured variable. From this, the current at the frequency of the AC voltage can then be extracted as the primary physical measurand. As a secondary physical measure, low-frequency components can be extracted. Many slow biological effects, such as extracellular activities, change the current at several frequencies that are low compared to the frequency of the AC voltage. They are modulated to these frequencies, analogous to the signals in the telecommunications, which are modulated on carrier frequencies.

In a further advantageous embodiment of the invention, a sample is selected which comprises a plurality of interacting biological samples, such as several biomolecules or a cell and a substance acting on this cell, which substance may be for example a protein or a chemical agent. Then, advantageously, the biological effect of this literary action can be studied. For example, initially only one biological sample may be present and the other sample added during the measurement. For example, biomolecular binding reactions between an antigen and the associated antibody can be studied. Likewise, cell membranes can be examined before, during and after the addition of biological or artificial membrane-permeable proteins, such as antibiotics.

For manipulating the sample, in particular for contacting it with the contacting and / or stimulation means, it is possible, for example, to use micromechanical, optical and electrokinetic methods. Other possible measurement methods would be z. As amperometry, voltammetry, Coulombmetrie, gravimetry, optometry, Hall measurement, atomic force microscopy (AFM), Lichtmicrospkopie, temperature jump method, calorimetry or 2-, 3- or 4-pole measurement. Advantageously, the sample is applied to a support electrically connected to the gate. This support may advantageously consist of a material which is more resistant to the substances contained in the sample than the gate. Then, a sample may be chosen that would chemically modify the gate itself, for example. In addition, the carrier can be prepared in any way with the sample and connected to the gate just before the measurement. The connection of the carrier to the gate can advantageously be made detachable. Then, for example, more samples can be stored on carriers than field effect transistors are available.

For example, cells, biomolecules, proteins or parts thereof may be applied in a suspension which may comprise a nutrient medium. The components to be measured then settle on the ground by their mass in the solution and adhere to the surface of the carrier. Already this process of adhesion can be analyzed after the electrical connection of the carrier to the gate.

In a particularly advantageous embodiment, a carrier is selected with adhesives that support the adhesion of the sample. As an adhesive, for example, linker molecules are suitable which react chemically with the carrier and the sample. However, proteins such as fibronectin, polylysine, laminin or other proteins are also suitable. The choice of a carrier with adhesive is particularly advantageous when the sample contains cell membranes or artificial lipid membrane systems. In this case, the membranes can be transferred to the support by various methods, for example by self-assembled monolayer formation, by layered membrane deposition or as a Langmuir film.

In a further particularly advantageous embodiment, a carrier is selected on which the adhesive is laterally structured. Then, for example, the migration of cells along the lateral structures, which may include, for example, lines or nodes, can be observed.

If the sample is capable of replication, it can be incubated before measurement to increase the number of cells and thus the signal strength. In this case, a sufficient for cell growth level of humidity and carbon dioxide is presented. The sample can also be incubated during the measurement in order to use the method according to the invention Study process of cell division.

Measurements have shown that, for example, the following biological properties of samples can be measured with the device according to the invention and the method according to the invention, the device advantageously being used to carry out the method:

- electrical properties of the cell;

- effects of biological, chemical and physical factors, such as toxic substances, on a cell;

- Electrical sealing properties between the cell and the surface of the gate or the reference electrode

- micromotion of cells;

- signal transmission from and to and between cells;

Changing the chemical composition of a measuring solution in the liquid reservoir or between the cell and the surface of the gates or the reference electrode;

- cell adhesion;

- cell motility;

- cell migration;

- cell vitality.

In the case of a suitable evaluation, it is also possible to carry out the investigation of the following properties and processes on individual cells which can hitherto only be detected on cell networks according to the prior art:

- Distance between a cell adhered to the surface of a field effect transistor or the reference electrode and the respective surface;

- apoptosis;

- mutagenic potential of known and unknown substances;

- signal transduction;

- toxicology;

- cell metabolism;

Cell behavior under flow in fluidic systems; - Electroporation of the cells.

In a suitable evaluation, the investigation of the following detectable properties and processes on cell fragments, cell membranes, artificial model membranes, proteins or biomolecules appears to be feasible with the invention, which could not hitherto be detected according to the prior art:

- Formation of cell membranes or artificial model membranes;

- spreading and fusion of vesicles;

- Domain separation in artificial model membranes;

- Fluctuations in cell membranes or artificial model membranes;

- activity of ion channels;

- pharmacology of ion channels;

- Reconstitution of ion channels or proteins in cell membranes or artificial model membranes;

- formation of pores in ion channels, cell membranes or artificial model membranes;

- electroporation of cell membranes or artificial model membranes;

- adhesion of biomolecules to the surface;

- binding assays of biomolecules;

In this case, all measurements can be performed spatially and temporally resolved and temporal changes of the respective measured variable can be determined.

Special description part

The subject matter of the invention will be explained in more detail below with reference to figures, without the subject matter of the invention being restricted thereby. It is shown:

Figure 1: Block diagram of a Ausführangsbeispiels of the device according to the invention.

FIG. 2 shows two measurements carried out with the device from FIG. 1 with and without a biological sample.

FIG. 3: Change of the measuring signal as a function of the salt concentration in the liquid reservoir 3 a. FIG. 4: Change in the detection sensitivity for the sample as a function of the salt concentration in the liquid reservoir 3 a.

Figure 5: Normalized transfer functions of two field effect transistors 2, of which only one is overgrown with a cell.

FIG. 6: Detection of cell detachment with the method according to the invention.

FIG. 7: Difference between the time courses of the normalized transfer function with or without a sample.

Figure 1 shows the block diagram of an embodiment of the device according to the invention in operation. The device is realized in this embodiment as a sensor chip. A cell 1 is contacted with the gate of a field effect transistor 2. The field effect transistor 2 comprises a source terminal 2a, a drain terminal 2b and a gate 2c. Above the cell is a liquid reservoir 3a, which is arranged as a small culture dish above the gate surface. A bath electrode 3 is immersed in this liquid reservoir 3a. The bath electrode 3 is acted upon by a voltage and frequency generator 4 with an AC voltage tunable frequency. The liquid reservoir 3a, the bath electrode 3 and the voltage and frequency generator 4 together form a unit that represents the stimulation means. The contacting means also comprise a liquid film between the gate of the field effect transistor and the cell 1. A readout and amplifier electronics 5 acts on the source-drain path of the field effect transistor 2 with a voltage and measures the current flowing through this path. Since in the field effect transistor the source-drain path is isolated from the gate, this current does not flow through the sample. The electronics 5 internally convert the current into a voltage so that it ultimately performs a voltage measurement. The measured voltage, which is a measure of the response of the sample to the AC voltage supplied by the voltage and frequency generator 4, and the AC voltage can be processed and displayed by an evaluation unit 6.

Embodiment 1: Influence of the Presence of a Biological Sample on the Frequency Dependence of the Normalized Transfer Function

FIG. 2 shows by way of example two measurements carried out with the apparatus shown in FIG. 1 in a logarithmic representation. In FIG. 2, in each case the normalized transfer function V _S in / V ₀ Ut is plotted against the frequency of the applied alternating voltage, wherein V _S in the voltage applied to the bath electrode 3 AC voltage and V _{out is} the output voltage supplied by the readout and amplifier electronics 5. The field effect transistor was first cleaned before the measurement and coated with the protein poly-L-lysine as a sample. Thereafter, a liquid reservoir was filled with an aqueous electrolyte solution (standard electrophysiological solution: 5 mM KCl, 140 mM NaCl, 10 mM HEPES, 5 mM glucose, pH 7.4 adjusted with NaOH).

The transfer function of the sample field effect transistor system was at different frequencies of f _s ; _n = 1 Hz - 750 kHz and an amplitude of V _S j _n = 20 mV measured (Figure 2, curve (D) due to the total impedance of the system reference electrode sample with contacting means, field effect transistor and first amplifier stage, the stimulation signal is only up to frequencies. amplified by about f _s j _n = 90 kHz. for higher frequencies, especially the field effect transistor will not increase to the overall signal gain of the system part and thus much lower output signals V _out is measured. this is only true for this currently realized embodiment of the measuring system and does not provide fundamental Limitation of the scope of the invention.

The curve © in FIG. 2 shows by way of example the measurement of the normalized transfer function V _s i _n / V _0Ut after a renewed cleaning of the surface and thus after the removal of the biological sample (protein) from the surface. The basic course of the transfer function of the AC signal in the field effect transistor is comparable to that in the presence of the protein on the field effect transistor (curve (D in Figure 2), but only for frequencies above f _S j _n = 300 kHz, a decreasing gain of V _sin .

The measurement with sample (protein) shown in FIG. 2 was repeated with altered salt concentrations in the electrolyte solution. Figure 3 shows the results of these measurements. The salt concentration decreases from curves (D to ®): (D - undiluted © - 1: 1 diluted (D - diluted 1:10 - diluted 1: 20 (D - diluted 1: 100 © - diluted 1: 200 © - 1: 1000 diluted ® - diluted 1: 2000.

The diluent used was distilled water in each case.

It becomes clear that this changes the course of the normalized transfer function V _sm / V _0Ut as a function of the frequency. In this case, the resistance of the aqueous electrolyte solution R _EL and the capacitance of the track of the field effect transistor C _{LB form} an effective low pass with the cutoff frequency / π> = 1 / (2π x REL XC _L B). It can be seen that with progressively lower concentration of the electrolyte solution the amplification of the transmission of the applied AC signal with the poly-L-lysine-coated field effect transistor becomes smaller at progressively lower frequencies.

After cleaning the field effect transistor (removal of the sample) shows a qualitatively comparable dependence of the normalized transfer function of the frequency. Analogously to FIG. 2, however, the presence or absence of the sample decides on the cut-off frequency up to which unimpeded transmission can take place. FIG. 4 shows the difference Δ (V _S i _n / Vout) of the respective transfer functions in the presence and absence of the poly-L-lysine for different salt concentrations (of Φ-decreasing salt concentration of the electrolyte solution). The difference in magnitude, and thus the sensitivity of the measuring arrangement for detecting the presence of the sample, increases with decreasing salt concentration.

It will be appreciated that the present invention is useful for detecting biomolecules such as proteins or DNA. It was also clearly demonstrated that the present invention is suitable for detecting the adhesion of biomolecules to the field effect transistors. By a differential measurement even a quantitative statement regarding the amount of bound biomolecules is possible.

Exemplary Embodiment 2: Differential Measurement for Investigating the Properties of a Single Cell.

FIG. 5 shows an example of a differential measurement series of the normalized transfer function of two different, equally coated (poly-L-lysine) field-effect transistors on the same sensor chip with the device according to the invention according to FIG The difference between the two field effect transistors is that one cell (HEK293) has grown on one of the field effect transistors (©), while there is no cell on the other field effect transistor (©). This chip was stored for incubation after application of the cell suspension for a period of three days in an incubator under constant CO ₂ atmosphere and constant temperature.

The normalized transfer function of the single-cell field effect transistor system was measured at different frequencies of f _sin = 1 Hz - 750 kHz and an amplitude measured by V _S jn = 20 mV. For the field effect transistor without cell there is an unimpeded overall signal amplification of the system, and the applied AC voltage signals thereby experience a higher gain up to frequencies of about f _s j _μ = 70 kHz; for higher frequencies, a considerably lower amplification of the transmitted alternating voltage signals (curve ©) takes place. For the field effect transistor having the adherent cell, a significantly different course of Übertragimgsfunktion be observed (curve ^(D): In principle, the cell coverage of the field effect transistor leads to a different transfer function of the AC voltage signal to the field effect transistor In all cases, the field effect transistor will no longer at substantially lower frequencies. in the overall signal amplification of the system, and the applied AC voltage is no longer amplified (here: for frequencies above f _S j _n = 10 kHz).

Due to the measured transfer function, it is possible by a suitable evaluation to determine the electrical sealing properties between the cell and the field effect transistor. For this exemplary cell, a seal resistance of about 1 MΩ resulted.

After the measurement, the sensor chip was cleaned, removing both the cell and the coatings. Subsequently, a complete measurement of the frequency response of the normalized transfer function was performed again. After cleaning, the transfer functions of the field effect transistors previously shown by way of example no longer differed on the same sensor chip. In this respect, it is clearly shown that the present invention is suitable for measuring the transfer function as well as changes in the transfer function due to the presence of individual cells on the field effect transistors. Exemplary Embodiment 3: Measurement of the Time Course of a Cell Detachment.

FIG. 6 shows by way of example a time-dependent measurement of the change in the transfer function of the two field effect transistors shown in FIG. 5 with and without a cell on the field effect transistor. Plotted is the normalized transfer function V _s j _n / V _0Ut against the time t. As a constant frequency of the AC voltage f _S i _n = 200 kHz was chosen. This is the frequency at which the maximum difference in the transfer function was detected between the two field effect transistors (see FIG. 5). The amplitude of the AC voltage was V _S i "= 20 mV. The graph shows only slight differences between the transmission function long-time signals of the field effect transistors with cell (curve Φ) and without cell (curve ©) up to 240 s.

After 240 seconds, 100 μl trypsin was added to the culture dish on the sensor chip (concentration: arrow marks the addition). In this embodiment, the culture dish was designed so large that thereby the gates of both field effect transistors were wetted with the trypsin. As a result of this enzyme, all membrane-bound proteins of the cells - including those responsible for the adhesion of the cells on the surface - are enzymatically degraded. After some time, the cells dissolve from the surface. Visually, this can be observed with increasing cell turnover.

In the measurement, the addition of trypsin has a significant effect: The normalized transfer function of the AC voltage increases significantly for the field effect transistor with the adhered cell and finally reaches a steady state value, which is about 80% higher than at the beginning of the measurement. In contrast, the transfer function for the field effect transistor without cell changes only slightly during the addition of trypsin.

It has been clearly demonstrated that with the present invention, the change in the adhesion of a single cell to a field effect transistor is significantly detectable. In this respect, the present invention is suitable for the study of both the site-and time-resolved cell adhesion, as well as the site-and time-resolved cell detachment. Thus, it is possible to register the detachment of a cell, for whatever reason. This can be used, for example, in toxicology or to demonstrate the presence of others Cell detachment triggering substances are used.

Exemplary embodiment 4: Detection of micromovements of a cell.

FIG. 7 shows the result of a measurement carried out analogously to FIG. In contrast to FIG. 5, the normalized transfer function was measured in a time-dependent manner analogous to FIG. In contrast to FIG. 6, the system did not intervene during the measurement. The upper part of Figure 7 shows the normalized transfer function of the field effect transistor with cell, the lower part of Figure 7 that of the field effect transistor without a cell. The transfer functions are plotted against time t. As a constant frequency of the AC voltage f _s j _n = 200 kHz is selected. This is the frequency at which the maximum difference in the transfer function (see FIG. 4) has been established between the two field-effect transistors. The amplitude of the AC voltage was V _sin = 20 mV.

Figure 7 shows the only slightly visible in Figure 6 differences between two transfer functions in magnification. While only small fluctuations are to be registered for the field effect transistor without a cell (below), significant changes in the transfer function can already be observed for the field effect transistor with cell. These changes in the transfer function can be correlated with local micro-movements of the adherent cell.

In this respect, it is clearly demonstrated that with the present invention, the change in the adhesion of a single cell due to micro-movements of the cell is significantly detectable. Therefore, the present invention is useful for the rapid and efficient measurement of the toxicity of unknown substances, as such micro-movements of the cell as well as cell motility is a parameter strongly linked to cell vitality, which is significantly changed by toxic substances.

The embodiments shown here do not limit the invention to biological samples. The basic idea, to use a field effect transistor for the currentless measurement of an AC resistance, can be realized on any system which can be charged with an AC voltage and which can exchange charges with the gate of a field effect transistor.

Claims

P a n t a n s p r e c h e

I. Device for measuring biological and electronic properties of a sample, comprising a field effect transistor (2) with contacting means for contacting the sample with the gate of the field effect transistor, characterized by stimulation means (3, 3a, 4) for applying an alternating voltage to the sample (1).

2. Apparatus according to claim 1 or 2, characterized by a measuring instrument (5) for

Measurement of amplitude and phase of a current flowing through the source-drain path of the field effect transistor.

3. Apparatus according to claim 2, characterized by a frequency-selective measuring instrument (5).

4. Device according to one of claims 1 to 3, characterized by excitation means for acting on the sample (1) with a further electrical voltage.

5. Apparatus according to claim 4, characterized by a patch-clamp configuration as an excitation means.

6. Device according to one of claims 4 to 5, characterized by an intracellular electrode as an excitation means.

7. Device according to one of claims 1 to 6, comprising at least one further field effect transistor.

8th . Apparatus according to claim 7, characterized in that the further field effect transistor is electrically isolated from the sample.

9. Device according to one of claims 1 to 8, characterized by means for contacting a plurality of field effect transistors (2) with the same sample (1).

10. Device according to one of claims 1 to 9, characterized by stimulation means (3, 3a) comprising a liquid reservoir (3a).

II. Device according to one of claims 1 to 10, characterized by contacting comprising a liquid reservoir (3 a). 2. Device according to one of claims 1 to 11, characterized by a liquid reservoir (3a) having a volume greater than 0.1 ul, in particular greater than 100 ul. 3. Device according to one of claims 1 to 12, characterized by a closed liquid reservoir (3a). 4. Device according to one of claims 1 to 13, characterized by a liquid reservoir (3a) with an inlet and / or a drain.

5. Method for measuring biological properties of a sample with the steps:

- The sample (1) is applied with an AC voltage;

- The sample exchanges charges with the gate of a field effect transistor (2);

- A dependent of the potential at the gate of the field effect transistor (2) primary physical quantity is measured.

6. A method according to claim 15, characterized in that an alternating voltage having a frequency between 0.1 Hz and 1 GHz, in particular between 1 Hz and 10 MHz is selected. 7. Method according to one of claims 15 to 16, characterized in that an alternating voltage having an amplitude between 0.001 mV and 10 V, in particular between 1 mV and 100 mV, is selected. 8th . Method according to one of claims 15 to 17, characterized in that the amplitude of the alternating voltage is kept constant during the measurement.

9. Method according to one of claims 15 to 18, characterized in that the sample (1) is subjected to alternating voltages of different frequencies.

o. Method according to one of claims 15 to 19, characterized in that amplitude and phase of the primary physical quantity are measured.

1 . A method according to claim 20, characterized in that the time course of amplitude and phase of the primary physical measured variable over between 1 and 100, in particular over between 5 and 10 oscillations of the applied AC voltage is measured.

22. Method according to one of Claims 15 to 21, characterized in that a current flowing through the source-drain path of the field-effect transistor (2) is measured as the primary physical measured variable.

23. Method according to one of claims 15 to 22, characterized in that the

Time course of the primary physical quantity and a transfer function of the field effect transistor (2) are processed to determine other characteristics of the sample.

24. Method according to one of claims 15 to 23, characterized in that the impedance of the sample (1) is determined from the primary physical measured variable.

25. Method according to one of claims 15 to 24, characterized in that a further secondary physical measured variable is measured.

26. A method according to claim 25, characterized in that a secondary physical quantity is selected, which is a measure of an electrical activity of the sample (1).

27. Method according to one of claims 15 to 26, characterized in that the sample (1) is applied to stimulate electrical activity with a further electrical voltage.

28. Method according to one of Claims 15 to 27, characterized in that a portion of the current flowing through the source-drain path of the field-effect transistor (2) in comparison with the frequency of the alternating voltage is selected as the secondary physical measured variable.

29. The method according to any one of claims 15 to 28, characterized in that primary and secondary physical measurement are measured simultaneously.

3 o. Method according to one of claims 15 to 29, characterized in that a

Sample (1) comprising at least two interacting biological samples.

31. Method according to one of claims 15 to 30, characterized in that the sample (1) is applied to a carrier electrically connected to the gate.

A method according to claim 31, characterized in that a carrier is selected with adhesives that support the adhesion of the sample (1).

A method according to claim 32, characterized in that a carrier is selected, on which the adhesive is laterally structured.

Method according to one of claims 15 to 33, characterized in that the sample (1) is incubated.

Use of a device according to one of Claims 1 to 14 for carrying out the method according to one of Claims 15 to 34.